Abstract

Type II phosphatidylinositol 4-kinase β (PtdIns 4-kinase II β) is an enigma among the phosphatidylinositol 4-kinase family. The role of PtdIns 4-kinase II β in MCF-7 cells was addressed with the help of short hairpin RNA (shRNA). PtdIns 4-kinase II β shRNA transfection increased pan-caspase activity and induced apoptosis in cancerous MCF-7 cells. Non-cancerous MCF-10A cells were resistant to PtdIns 4-kinase II β shRNA-induced apoptosis. Caspase 8 and 9 inhibitors rescued MCF-7 cells from apoptosis. Shotgun proteomic studies with Flag-tagged PtdIns 4-kinase II β immunoprecipitates showed tumor suppressor prostate apoptosis response-4 (Par-4) as one of the interacting proteins in HEK293 cells. In reciprocal experiments, Par-4 antibodies co-precipitated PtdIns 4-kinase II β from MCF-7 cells. Deletion of membrane localization motif (ΔCCPCC) or a mutation in ATP-binding region (D304A) of PtdIns 4-kinase II β did not affect its interaction with Par-4. Pull-down assays with GST-PtdIns 4-kinase II β-truncated mutants showed that the region between 101 and 215 amino acid residues is essential for interaction with Par-4. At molecular level, PtdIns 4-kinase II β shRNA transfection increased Par-4 stability, its nuclear localization and inhibition of NF-κB binding to target DNA. Knocking down of Par-4 with siRNA (small interfering RNA) rescued MCF-7 cells from PtdIns 4-kinase II β shRNA-induced apoptosis. These results suggest that PtdIns 4-kinase II β may be a novel regulator of Par-4 through protein–protein interactions. These studies have potential implications in cancer therapy.

Introduction

Type II phosphatidylinositol 4-kinases II (PtdIns 4-kinases II) feed into two important signaling pathways mediated by phosphatidylinositol 3-kinases and phospholipase C. These signaling pathways play a role in cell proliferation [13]. Apart from these signaling pathways, PtdIns 4-kinases II were also shown to have independent roles in vesicular trafficking [4]. Two isoforms of PtdIns 4-kinases II (α and β) that differ in their first 100 amino acids and subcellular localizations have been described [5]. The α isoform is predominantly localized to Golgi and endocytic vesicles, and is suggested to participate in endocytosis, Wnt signaling, and probably apoptosis [610]. The physiological significance of β isoform is largely unknown. The β isoform has a high degree of internally disorganized regions at N-terminal end with multiple protein–protein-interacting domains (proline-rich regions including PXXP regions and Src homology 3-binding domains). In addition, it has membrane localization domain (169CCPCC174), atypical leucine zipper-like region (179LIPNQGYLSEAGAYLVDNKLHL201), and a potential SUMOylation site (403FKTD407), suggesting that it may have pleiotropic roles in cell signaling. Consistent with this hypothesis, the β isoform has both cytosolic and plasma membrane distribution and, upon growth factor, stimulation part of the cytosolic enzyme translocates to the plasma membrane [11,12]. Molecular mechanisms that regulate these translocations are not fully understood. However, a role for protein tyrosyl phosphorylation has been suggested [13]. Alternately, PtdIns 4-kinase II β may be localized to the cytosol through its interaction with HSP90 [14]. Knockdown of PtdIns 4-kinase II β with short hairpin RNA (shRNA) in human T-cell leukemia (Jurkat) cells has been correlated with reduced intracellular calcium release and reduced cell adhesion to extracellular matrix-coated surfaces [15]. PtdIns 4-kinase II β siRNAs (small interfering RNAs) were shown to increase metastatic potential in hepatocellular carcinoma [16]. In a recent study, it was shown that depletion of PtdIns 4-kinase II β increases the metastatic potential in cancer cells through increased invadopodia formation [17]. Inhibition of type II PtdIns 4-kinase(s) with pharmacological agents showed a reduction in the release of inflammatory mediators in RBL-2H3 cells [18,19]. PtdIns 4-kinase II β is shown to interact with adaptor protein AP1 and regulate endosomal TGN sorting and pectoral fin development in zebrafish [20]. These scattered studies indicate that PtdIns 4-kinase II β may affect signaling pathways that lead to cell proliferation, motility, development, and inflammation through possible interactions with other proteins.

Par-4 gene was identified as an inducible gene during ionomycin-induced apoptosis in prostate cancer cells [21]. Later, Par-4 expression was shown to be down-regulated in several cancerous tissues, suggesting that it is a tumor suppressor gene [22]. Human Par-4 is a 38 kDa protein with two nuclear localization signals: one SAC (selective for apoptosis induction in cancer cell) domain and a leucine-rich region at C-terminus. The leucine-rich region is not required for its apoptotic function, but it acts as an interacting domain for other proteins [23,24]. The function of Par 4 is regulated by atypical protein kinase C, PKA, and Akt through phosphorylations [25,26]. Phosphorylation by PKA promoted Par-4 nuclear localization and apoptosis. Phosphorylation of Par-4 at Ser230 by Akt prevented its nuclear localization and inhibited apoptosis. In addition, Par-4 has large internally unstructured regions that can interact with other proteins. Par-4 induces apoptosis in cancer cells through extrinsic (Fas and Fas ligand) and intrinsic pathways [27]. At the molecular level, Par-4 is suggested to inhibit nuclear factor kappa β (NF-κB) functions in the nucleus and down-regulation of pro-survival genes [28]. Par-4 is also regulated by ubiquitination and proteasomal pathways, suggesting that it is regulated at multiple levels [29].

In the present study, the role of PtdIns 4-kinase II β in MCF-7 cells was addressed with the help of shRNA. The results suggest a novel role for PtdIns 4-kinase II β in Par-4 regulation and apoptosis.

Materials and methods

Cell culture and transfection

MCF-7 cells and MCF-10A cells were obtained from National Center for Cell Science, Pune, India. MCF-7 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM), and MCF-10A cells were cultured in DMEM/Ham's F12 medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2 in a humidified incubator. MCF-10A cells were supplemented with epidermal growth factor (20 ng/ml), insulin (10 µg/ml) and hydrocortisone (500 ng/ml) as per the provider's recommendation. All growth factors were from Sigma. Lipofectamine 3000 was from Invitrogen and used as per manufacturer's protocol.

Plasmids and antibodies

PtdIns 4-kinase II β shRNA sequences were cloned in pmu6 vector as described earlier [15]. Two non-overlapping sequences were used for silencing the PtdIns 4-kinase II β. For exogenic expression, HA-tagged PtdIns 4-kinase II β in pEGFN1 plasmid and control plasmids are a generous gift from Dr Tamas Balla. HA-tagged PtdIns 4-kinase II β (wild type), HA-tagged PtdIns 4-kinase II β ΔCCPCC mutant, and HA-tagged PtdIns 4-kinase II β D304A mutant were synthesized and cloned in pcDNA3.1(+) by Genscript, U.S.A. GST-PtdIns 4-kinase II β was cloned in pGEX-4T3 vector and its deletion mutants were cloned using polymerase chain reaction protocols. For PCRs, primers were obtained from Sigma–Aldrich. PtdIns 4-kinase II α shRNA and Myc-PtdIns 4-kinase II α were generous gifts from Prof. K.V. Kandror [30]. Par-4 siRNA (sc-36190) and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-HA antibodies (sc-7392) and mouse monoclonal anti-myc antibodies (sc-9E10) are from Santa Cruz Biotechnology, anti-Rabbit polyclonal Par-4 antibodies (ab5787) and anti-Rabbit polyclonal PtdIns 4-kinase II β (ab37812) are from Abcam, and rabbit polyclonal anti-β-actin antibodies (#4967) are from Cell Signaling Technology.

Cell proliferation assay

MCF-7 cells and MCF-10A cells were cultured overnight in six-well plates in triplicates. These cells were transfected with either PtdIns 4-kinase II β shRNA or scrambled shRNA. Cells were allowed to grow for 48 h. At indicated time points, cells were harvested. ‘0’ h represents the counting of cells soon after transfection. An aliquot of cells was used for counting and remaining cells were used for immunoblotting with anti-PtdIns 4-kinase II β antibodies. Viable cells were counted by the trypan blue dye exclusion method.

Apoptotic assay

MCF-7 cells were transfected with either PtdIns 4-kinase II β shRNA plasmid or scrambled shRNA plasmids. All cells were cultured for 36 h. At the end of incubation, cells were stained with propidium iodide and were analyzed on a fluorescent activated cell sorter as described earlier [31].

Caspase assay

MCF-7 cells were transfected with either scrambled shRNA or PtdIns 4-kinase II β shRNA plasmids. Cells were cultured for 36 h. At the end of incubation, cells were lysed in 0.1 ml of lysis buffer (25 mM Tris pH 8.0, 150 mM NaCl and 0.5% Triton X-100). Pan-caspase assays were carried out in a volume 0.1 ml buffer [100 mM HEPES (pH 7.5), 20% glycerol, 5 mM DTT, 0.5 mM EDTA] with 100 µM peptide substrate. The reaction was initiated by the addition of MCF-7 cell lysate (20 µg) and incubated at 37°C for 24 h. Absorbance was measured at 405 nm.

Treatment with caspase inhibitors

All caspase inhibitors were from Calbiochem and used as per the manufacturer's recommendation. MCF-7 cells were pre-incubated with inhibitors of caspase 8 (50 µM) and caspase 9 (10 µM) for 1 h, followed by PtdIns 4-kinase II β shRNA transfection. Cells were incubated for 36 h and processed for apoptotic assay using flow cytometry.

Quantitative real-time polymerase chain reaction

Total RNA was isolated using the High Pure RNA isolation kit following the manufacturer's (Roche) protocol. About 1 µg of RNA was converted into cDNA using the Transcriptor First Strand cDNA Synthesis Kit according to the manufacturer's (Roche) protocol. Quantitative polymerase chain reaction (qPCR) was performed on the Rotor Gene 3000 machine (Corbett Research, U.S.A.). PCR mix was prepared by mixing 2 µl of cDNA, 10 µl of 2× SYBR green PCR mix (Roche), and 10 pmol of specific gene primers, and the volume was made up to 20 µl. PCR was performed as follows: initial denaturation was done at 95°C for 5 min, with annealing at 58°C for 15 s and elongation at 72°C for 20 s. Subsequent denaturation was done at 95°C for 15 s. PCR was carried out for 40 cycles. The threshold value was determined and used for measuring the expression levels of genes by REST-384 version 2 software. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used for normalization and relative changes in the gene expression with respect to control cells were plotted.

Clonogenic assay

MCF-7 cells and MCF-10A cells were transfected with PtdIns 4-kinase II β shRNA or scrambled shRNA plasmids. After 12 h of transfection, 1 × 103 cells were replated in six-well plates. MCF-7 cells and MCF-10A cells were incubated for 12 and 18 days, respectively, to allow visible colony formation. The plates were stained with crystal violet and colonies were counted.

Shotgun proteomic assay

HEK293 cells were transfected with human Flag-tagged PtdIns 4-kinase II β. After 24 h of transfection, cells were washed with PBS and lysed in 1 ml of lysis buffer (50 mM HEPES-KOH, pH 8.0, 100 mM KCl, 2 mM EDTA, 0.1% Nonidet P-40 and 10% glycerol) supplemented with 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail. Cell lysis was carried out by two freeze–thaw cycles between liquid nitrogen and 37°C water bath. The lysate was clarified by centrifuging at 13 000×g at 4°C for 15 min. Clarified lysate was incubated with 50 µl of anti-FLAG agarose gel (Sigma) in a tube rotator overnight at 4°C. Nonspecific binding was removed by washing three times with lysis buffer and three times with wash buffer (50 mM NH4HCO3, pH 8.0, 75 mM KCl) and bound proteins were eluted with 100 µl of elution buffer (0.5 M NH4OH, pH 11.0). In solution tryptic digestion: NH4OH eluates were lyophilized and digested with trypsin (250 ng) in 50 mM NH4HCO3 at 37°C overnight. After digestion, formic acid was added to a final concentration of 2%. Tryptic digest was lyophilized and resuspended in 7 ml of HPLC reverse-phase buffer (2% acetonitrile and 0.1% formic acid). Mass spectrometry and data analysis: Mass spectrometry was performed as described [32]. Raw files were converted to the mz XML format and searched with the Comet algorithm against the human UniProt proteomes database. The files were searched with semi-tryptic enzyme constraint, 10 ppm precursor tolerance, 0.02 fragment bin tolerance, appended decoy entries, oxidized methionine variable modification, and carbamidomethyl cysteine static modification. The searched results were subsequently processed using PeptideProphet and ProteinProphet for peptide and protein statistics.

Immunoprecipitation and PtdIns 4-kinase assay

MCF-7 cells (1 × 106 cells) were transfected with scrambled shRNA, PtdIns 4-kinase II β shRNA and PtdIns 4-kinase II α shRNA plasmids. Post-transfection cells were lysed in 1 ml of lysis buffer (25 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 mM benzamidine hydrochloride, and 1 mM sodium orthovanadate). For experiments with Par-4 siRNA, MCF-7 cells were transfected either with scrambled or Par-4 siRNA as per the manufacturer's instructions. Anti-Par-4 antibody (2 µg) was added to cell lysates and incubated at 4°C overnight. Protein A agarose beads were added to the lysate to precipitate the immune complex. The beads were washed thrice with 1 ml of lysis buffer. PtdIns 4-kinase activity in Par-4 immunoprecipitates was assayed as described earlier [12]. Briefly, the reaction was carried in 50 µl of PtdIns 4-kinase assay buffer [50 mM Tris–HCl (pH 7.6), 10 mM MgCl2, 0.025 mM EGTA, 0.1 mM sodium orthovanadate], 20 µg/ml phosphatidylinositol, [γ-32P] ATP 100 µM (specific activity 200–300 cpm/pmol), and 0.3% Triton X-100. The reaction was initiated with the addition of immunoprecipitates. The reaction was incubated at room temperature (∼25°C) for 10 min and stopped with 12 N HCl (50 µl). Phospholipids were extracted with 500 µl of chloroform : methanol : water (15 : 15 : 5). The aqueous phase was removed and the organic phase was washed with 100 µl methanol : 1 N HCl. The organic phase was spotted on TLC plates (silica gel-coated) activated with 2% sodium potassium tartrate in 50% ethanol. The chromatogram was developed in chloroform : methanol : ammonium hydroxide : water (90 : 90 : 7 : 20). The chromatogram was exposed to X-ray film to identify labeled phospholipids. Radioactivity in lipid spots was counted in a scintillation counter. For experiments with inhibitors, adenosine was added to the final concentration of 50 µM in the reaction buffer.

GST mutants and pull-down assay

Wild-type PtdIns 4-kinase II β cDNA sequences were used to create deletion mutants with the help of polymerase chain reactions. These deletion mutants were arbitrarily named as D1, D2, T1 and T2 mutants and were cloned in pGEX-4T3 vector with GST tag. D1 mutant has first 100 amino acids (1–100 amino acids) and D2 mutant has amino acids from 101 to 481, T1 mutant has amino acids from 101 to 215 amino acids, and T2 mutant has amino acids from 216 to 481 amino acids. All plasmids were expressed in Escherichia coli BL-21(DE3) cells. For induction of GST-fusion proteins, 0.2 mM IPTG was added to the mid-log cultures and incubated at room temperature for 4 h. Cells were lysed and proteins were solubilized with 1.5% N-lauroylsarcosine. The final concentration of sarcosyl was adjusted to 0.75% with lysis buffer. Lysate was centrifuged at 23 000×g for 20 min. Solubilized proteins were incubated with Glutathione-Sepharose beads for 4 h. The beads were washed three times with STE buffer (25 mM Tris, 150 mM NaCl, 5 mM EDTA, and 1× protease inhibitors). MCF-7 cell lysates were incubated with these beads overnight. After incubation, beads were washed with lysis buffer and bound proteins were analyzed on SDS–PAGE followed by immunoblotting.

Immunostaining and confocal microscopy

MCF-7 cells were grown on glass coverslips and transfected with PtdIns 4-kinase II β shRNA plasmids. Post-transfection, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 for 5 min. Cells were incubated with blocking buffer (5% BSA in phosphate-buffered saline) at room temperature for 1 h. Cells were incubated with Par-4 c-terminus antibodies (1 : 300, from Abcam) at 4°C overnight. Cells were washed thrice with wash buffer (1% BSA in PBS) and incubated with TRITC-conjugated secondary antibodies in dark for 1 h. Cells were washed and slides were prepared with mounting media (90% glycerol, 2.5% DABCO, and 1 µg/ml Hoechst 33258 in PBS). Cells were observed under a confocal microscope (Zeiss Axio-Observer Z1 microscope).

Cycloheximide treatment

MCF-7 cells were transfected with PtdIns 4-kinase II β shRNA or scrambled shRNA plasmids and incubated for 24 h. Post-transfection, cells were treated with cycloheximide (CHX) (50 µg/ml) for 0, 45, 90, 135 and 180 min. Cells were lysed and Par-4 protein levels were analyzed on immunoblots. Similarly, HA-PtdIns 4-kinase II β (wild type), HA-PtdIns 4-kinase II β ΔCCPCC, and HA-PtdIns 4-kinase II β (D304A) plasmids were transfected into MCF-7 cells. After 36 h of transfection, cells were treated with CHX. Cells were lysed, and HA-PtdIns 4-kinase II β and Par-4 levels were analyzed on immunoblots with anti-HA (Santa Cruz Biotechnology sc-805) and anti-Par-4 antibodies.

Electrophoretic mobility shift assay

Nuclear extracts for the electrophoretic mobility shift assay were isolated as described earlier [33]. Briefly, MCF-7 cells were transfected with PtdIns 4-kinase II β shRNA and incubated for 18 h. At the end of the incubation, cells were suspended in 0.2 ml of cytoplasmic extraction buffer (10 mM HEPES, pH 7.4, 10 mM KCl, 10 mM EDTA, and 5 mM EGTA) with intermittent vortexing on ice for 1 h. The cytoplasmic extract was collected by centrifugation at 15 000×g for 30 min. The nuclear extract was obtained by suspending nuclear pellet in 30 µl of nuclear extraction buffer (5 mM HEPES, pH 7.4, 1.5 mM KCl, 4.6 M NaCl, 10 mM EDTA, 5 mM EGTA and 20% glycerol) on ice with intermittently vortexing for 3 h. The nuclear extract was collected by centrifugation at 15 000×g for 30 min. For NF-κB-binding studies, 8 µg of nuclear extract was added to 32P (16 fmol) end-labeled 45-mer of double-stranded NF-κB target oligo-deoxy nucleotides (5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG3′) and 0.5 µg of poly (2′-deoxyinosinic-2′-deoxycytidylic acid) in binding buffer (25 mM HEPES, pH 7.9, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, and 5% glycerol). The above mixture was incubated at 37°C for 1 h. Native polyacrylamide gel (6.6%) electrophoresis was performed to separate the free oligonucleotides from the protein-bound oligonucleotides in running buffer (50 mM Tris, pH 8.5, 200 mM glycine, and 1 mM EDTA). The gel was dried and exposed to a Molecular-Dynamics PhosphorImager Screen. The screen was scanned using a phosphorImager scanner (Amersham).

Results

PtdIns 4-kinase II β shRNA induces apoptosis in MCF-7 cells

PtdIns 4-kinase II β shRNA sequences cloned in the pmu6 vector were described earlier [15]. Two non-overlapping sequences were used for silencing PtdIns 4-kinase II β. Both sequences were shown to down-regulate PtdIns 4-kinase II β protein in Jurkat cells. Quantitative real-time PCR assays showed two-fold down-regulation of PtdIns 4-kinase II β mRNA in MCF-7 cells within 24 h. These shRNAs did not affect PtdIns 4-kinase II α mRNA levels (Figure 1A). Time course experiments with PtdIns 4-kinase II β shRNA showed approximately eight-fold reduction in PtdIns 4-kinase II β mRNA levels in 36 h (Supplementary Figure S1). Reduction in mRNA levels was supported by down-regulation of exogenously expressed PtdIns 4-kinase II β protein levels (Figure 1B).).

Down-regulation of PtdIns 4-kinase II β with shRNA in MCF-7 cells.

Figure 1.
Down-regulation of PtdIns 4-kinase II β with shRNA in MCF-7 cells.

(A) MCF-7 cells were transfected with PtdIns 4-kinase II β shRNA and scrambled shRNA plasmids and cultured for 24 h. Relative expressions of PtdIns 4-kinase II α and β were quantified using qPCR. GAPDH was used for normalization. Data shown are the mean ± SD of three independent experiments. (B) PtdIns 4-kinase II β shRNA reduces exogenously expressed HA-PtdIns 4-kinase II β protein levels. MCF-7 cells were transfected with empty vector and scrambled shRNA plasmid or HA-tagged PtdIns 4-kinase II β in pEGFP-N1 and scrambled shRNA plasmid or empty vector and PtdIns 4-kinase II β shRNA plasmid or HA-tagged PtdIns 4-kinase II β in pEGFP-N1 and PtdIns 4-kinase II β shRNA plasmid. Cells were cultured for 36 h and cell lysates were analyzed with anti-HA antibodies. Β-actin was used as loading control. Empty vector denotes HA-tagged pEGFP-N1 without PtdIns 4-kinase II β insert.

Figure 1.
Down-regulation of PtdIns 4-kinase II β with shRNA in MCF-7 cells.

(A) MCF-7 cells were transfected with PtdIns 4-kinase II β shRNA and scrambled shRNA plasmids and cultured for 24 h. Relative expressions of PtdIns 4-kinase II α and β were quantified using qPCR. GAPDH was used for normalization. Data shown are the mean ± SD of three independent experiments. (B) PtdIns 4-kinase II β shRNA reduces exogenously expressed HA-PtdIns 4-kinase II β protein levels. MCF-7 cells were transfected with empty vector and scrambled shRNA plasmid or HA-tagged PtdIns 4-kinase II β in pEGFP-N1 and scrambled shRNA plasmid or empty vector and PtdIns 4-kinase II β shRNA plasmid or HA-tagged PtdIns 4-kinase II β in pEGFP-N1 and PtdIns 4-kinase II β shRNA plasmid. Cells were cultured for 36 h and cell lysates were analyzed with anti-HA antibodies. Β-actin was used as loading control. Empty vector denotes HA-tagged pEGFP-N1 without PtdIns 4-kinase II β insert.

Transient transfection of PtdIns 4-kinase II β shRNA showed a reduction in viable cell numbers in cancerous MCF-7 cells but not in non-cancerous MCF-10A cells. Both cell lines showed reduction in endogenous PtdIns 4-kinase II β protein levels upon shRNA transfection (Supplementary Figure S2). The decrease in MCF-7 cell numbers is as high as 73% in comparison with scrambled shRNA plasmid transfectants (Figure 2A). These cells showed a significant increase in pan-caspase activity in in vitro (Figure 2B). Flow cytometry studies showed increased apoptosis in MCF-7 cells (Figure 2C and Supplementary Figure S3). In addition to MCF-7 cells, transfection of PtdIns 4-kinase II β shRNA induced apoptosis in MDA-MB-231 cells and glioblastoma (U-87 MG) cells to varying levels (Supplementary Figure S4). Caspase 8 and caspase 9 inhibitors rescued MCF-7 cells from PtdIns 4-kinase II β shRNA-induced apoptosis with varying efficiencies. Caspase 9 inhibitor was more effective in rescuing the cells from apoptosis (Figure 2D). Post-transfection with shRNA, clonogenic assays showed reduction in the colony-forming ability of MCF-7 cells but not in MCF-10A cells (Figure 2E,F). These results suggest that PtdIns 4-kinase II β may have different roles in cancerous cells probably due to interaction with cancer cell proteome.

PtdIns 4-kinase II β shRNA induces apoptosis in MCF-7 cells but not in MCF-10A cells.

Figure 2.
PtdIns 4-kinase II β shRNA induces apoptosis in MCF-7 cells but not in MCF-10A cells.

(A) MCF-7 cells were transfected with either scrambled shRNA (⬤-⬤) or PtdIns 4-kinase II β shRNA (○-○). MCF-10A cells were transfected with either scrambled shRNA (▾-▾) or PtdIns 4-kinase II β shRNA (▿-▿). Viable cells were counted at different time intervals. This experiment was repeated two times with quadruplicates, and the represented results are average of these two experiments. Each point represents mean ± SD. (B) PtdIns 4-kinase II β shRNA transfection increased pan-caspase activity in MCF-7 cells. MCF-7 cells were transfected with either PtdIns 4-kinase II β shRNA or scrambled shRNA. All cells were cultured for 36 h. Cells were lysed and 20 µg of protein was assayed for pan-caspase activity with the synthetic peptide as a substrate. A detailed procedure was described in Materials and Methods. The data indicate mean ± SD of three independent experiments (**P < 0.001). (C) MCF-7 cells transfected with either scrambled shRNA or PtdIns 4-kinase II β shRNA plasmids and cultured for 36 h. At the end of incubation, cells were stained with propidium iodide and were analyzed on a fluorescence activated cell sorter. The data indicate mean ± SD of three independent experiments (**P < 0.001). (D) Effect of caspase 8 and caspase 9 inhibitors on PtdIns 4-kinase II β shRNA-induced apoptosis in MCF-7 cells. The data indicate mean ± SD of three independent experiments (**P < 0.001). (E) PtdIns 4-kinase II β shRNA transfection reduced colony-forming ability in MCF-7 cells but not in MCF-10A cells. (F) Quantification of the data from clonogenic plates. The data indicate the mean ± SD of two independent experiments (**P < 0.001).

Figure 2.
PtdIns 4-kinase II β shRNA induces apoptosis in MCF-7 cells but not in MCF-10A cells.

(A) MCF-7 cells were transfected with either scrambled shRNA (⬤-⬤) or PtdIns 4-kinase II β shRNA (○-○). MCF-10A cells were transfected with either scrambled shRNA (▾-▾) or PtdIns 4-kinase II β shRNA (▿-▿). Viable cells were counted at different time intervals. This experiment was repeated two times with quadruplicates, and the represented results are average of these two experiments. Each point represents mean ± SD. (B) PtdIns 4-kinase II β shRNA transfection increased pan-caspase activity in MCF-7 cells. MCF-7 cells were transfected with either PtdIns 4-kinase II β shRNA or scrambled shRNA. All cells were cultured for 36 h. Cells were lysed and 20 µg of protein was assayed for pan-caspase activity with the synthetic peptide as a substrate. A detailed procedure was described in Materials and Methods. The data indicate mean ± SD of three independent experiments (**P < 0.001). (C) MCF-7 cells transfected with either scrambled shRNA or PtdIns 4-kinase II β shRNA plasmids and cultured for 36 h. At the end of incubation, cells were stained with propidium iodide and were analyzed on a fluorescence activated cell sorter. The data indicate mean ± SD of three independent experiments (**P < 0.001). (D) Effect of caspase 8 and caspase 9 inhibitors on PtdIns 4-kinase II β shRNA-induced apoptosis in MCF-7 cells. The data indicate mean ± SD of three independent experiments (**P < 0.001). (E) PtdIns 4-kinase II β shRNA transfection reduced colony-forming ability in MCF-7 cells but not in MCF-10A cells. (F) Quantification of the data from clonogenic plates. The data indicate the mean ± SD of two independent experiments (**P < 0.001).

PtdIns 4-kinase II β associates with Par-4 protein in MCF-7 cells

In parallel experiments, proteins that may potentially interact with PtdIns 4-kinase II β were studied by shotgun proteomic techniques. HEK293 cells were transfected with Flag-tagged PtdIns 4-kinase II β . Anti-flag antibody immunoprecipitates were trypsinized and analyzed by LC–MS. The LC–MS data showed a large number of proteins belonging to actin regulators, proton pump ATPase subunits, Par-4, Rab-GTPases, 26S proteasomal subunits, and ER proteins in anti-flag immunoprecipitates (Supplementary Table S1). The association of tumor suppressor protein Par-4 with PtdIns 4-kinase II β was investigated further. Seven unique peptides of Par-4, covering 37.1% sequence, were identified from two independent experiments (Table 1).

Table 1
Par-4 peptides found in Flag-PtdIns 4-kinase II β immunoprecipitates

HEK293 cells were transfected with FLAG-tagged PtdIns 4-kinase II β. Anti-FLAG immunoprecipitates were subjected to shotgun proteomic analysis. Human Par-4-specific peptides identified in anti-Flag immunoprecipitates were shown below.

KREDAITQQNTIQNEAVNLLDPGSSYLLQEPPR 
DLDDIEDENEQLK 
DLDDIEDENEQLKQENK 
STTSVSEEDVSSR 
TSSGLGGSTTDFLEEWK 
RSEDEPPAASASAAPPPQRDEEEPDGVPEK 
DANVSGTLVSSSTLEK 
KREDAITQQNTIQNEAVNLLDPGSSYLLQEPPR 
DLDDIEDENEQLK 
DLDDIEDENEQLKQENK 
STTSVSEEDVSSR 
TSSGLGGSTTDFLEEWK 
RSEDEPPAASASAAPPPQRDEEEPDGVPEK 
DANVSGTLVSSSTLEK 

PtdIns 4-kinase II β interaction with Par-4 was examined in MCF-7 cells under normal cell culture conditions. Par-4 antibodies co-precipitated PtdIns 4-kinase activity from MCF-7 cells. The enzyme activity was inhibited by adenosine, suggesting that it belongs to PtdIns 4-kinase II family (Figure 3A). Transfection with PtdIns 4-kinase II β shRNA abolished Par-4-associated PtdIns 4-kinase activity. However, transfection with PtdIns 4-kinase II α shRNA did not affect Par-4-associated PtdIns 4-kinase activity (Figure 3B).

Par-4 antibodies co-precipitate PtdIns 4-kinase II β activity.

Figure 3.
Par-4 antibodies co-precipitate PtdIns 4-kinase II β activity.

(A) Par-4 immunoprecipitates from MCF-7 cells were assayed for PtdIns 4-kinase activity in the presence or absence of adenosine (50 µM). (B) Knockdown of PtdIns 4-kinase II β but not of PtdIns 4-kinase II α abolished PtdIns 4-kinase activity in Par-4 immunoprecipitates.

Figure 3.
Par-4 antibodies co-precipitate PtdIns 4-kinase II β activity.

(A) Par-4 immunoprecipitates from MCF-7 cells were assayed for PtdIns 4-kinase activity in the presence or absence of adenosine (50 µM). (B) Knockdown of PtdIns 4-kinase II β but not of PtdIns 4-kinase II α abolished PtdIns 4-kinase activity in Par-4 immunoprecipitates.

MCF-7 cells transfected with either HA-PtdIns 4-kinase II β or Myc-PtdIns 4-kinase II α were analyzed for co-precipitation of Par-4 with anti-HA or anti-Myc antibodies. Par-4 was detected only in anti-HA immunoprecipitates (Figure 4A). In a reciprocal experiment, Par-4 immunoprecipitates were analyzed on immunoblots with anti-HA and anti-Myc antibodies (Figure 4B). These results provide evidence for PtdIns 4-kinase II β-specific association with Par-4. Deletion of CCPCC domain or a mutation in the catalytic domain did not affect PtdIns 4-kinase II β interaction with Par-4 (Figure 4C). These results suggest that both membrane localization domain and catalytic activity are not required for its association with Par-4 and indicate a role for protein–protein-interacting regions on PtdIns 4-kinase II β.

PtdIns 4-kinase II isoform-specific association with Par-4.

Figure 4.
PtdIns 4-kinase II isoform-specific association with Par-4.

(A) MCF-7 cells were transfected with either HA-PtdIns 4-kinase II β or Myc-PtdIns 4-kinase II α plasmids. PtdIns 4-kinase II β and PtdIns 4-kinase II α were immunoprecipitated with mouse anti-HA and mouse anti-Myc monoclonal antibodies. These immunoprecipitates were analyzed for the presence of Par-4 on immunoblots. MCF-7 cell lysate was used as positive control for Par-4. (B) In reciprocal experiments, Par-4 immunoprecipitates from these transfectants were analyzed with anti-HA and anti-Myc on immunoblots. Cell lysates from MCF-7 cells transfected with either HA-PtdIns 4-kinase II β or Myc-PtdIns 4-kinase II α plasmids were used as positive control for the expression of tagged proteins. (C) MCF-7 cells were transfected with HA-tagged PtdIns 4-kinase II β wild-type or ΔCCPCC or D304A mutant plasmids. Anti-HA immunoprecipitates were analyzed for the presence of Par-4.

Figure 4.
PtdIns 4-kinase II isoform-specific association with Par-4.

(A) MCF-7 cells were transfected with either HA-PtdIns 4-kinase II β or Myc-PtdIns 4-kinase II α plasmids. PtdIns 4-kinase II β and PtdIns 4-kinase II α were immunoprecipitated with mouse anti-HA and mouse anti-Myc monoclonal antibodies. These immunoprecipitates were analyzed for the presence of Par-4 on immunoblots. MCF-7 cell lysate was used as positive control for Par-4. (B) In reciprocal experiments, Par-4 immunoprecipitates from these transfectants were analyzed with anti-HA and anti-Myc on immunoblots. Cell lysates from MCF-7 cells transfected with either HA-PtdIns 4-kinase II β or Myc-PtdIns 4-kinase II α plasmids were used as positive control for the expression of tagged proteins. (C) MCF-7 cells were transfected with HA-tagged PtdIns 4-kinase II β wild-type or ΔCCPCC or D304A mutant plasmids. Anti-HA immunoprecipitates were analyzed for the presence of Par-4.

PtdIns 4-kinase II β has many protein–protein-interacting motifs: PXXP motifs at its N-terminus, a leucine-rich region at middle and potential SUMOylation motif at its carboxy terminus. To map interacting regions, a series of PtdIns 4-kinase II β deletion mutants were cloned as GST-fusion proteins (Figure 5A). These mutant proteins were assayed for their ability to interact with Par-4 from MCF-7 cells. The results showed that the region between 101–215 amino acids was required for PtdIns 4-kinase II β and Par-4 interactions (Figure 5B,C). This region has atypical leucine heptad on PtdIns 4-kinase II β, suggesting a possible role for hydrophobic interactions similar to leucine zipper motifs.

Mapping of PtdIns 4-kinase II β-interacting regions with Par-4.

Figure 5.
Mapping of PtdIns 4-kinase II β-interacting regions with Par-4.

(A) Schematic representation of PtdIns 4-kinase II β structure and its truncated proteins. GST-PtdIns 4-kinase II β and its truncated mutants were cloned in pGEX-4T3 vectors and expressed in E. coli. D1 mutant has first 100 amino acids (1–100 amino acids) and D2 mutant has amino acids from 101 to 481. The D2 mutant was further truncated, T1 mutant has amino acids from 101 to 215 amino acids and T2 mutant has amino acids from 216 to 481 amino acids. (B,C) Wild-type GST-PtdIns 4-kinase and its deletion mutants were adsorbed onto Glutathione-Sepharose beads. Glutathione-bound PtdIns 4- kinase fragments were used for Par-4 pull-down assays. The presence of Par-4 was detected on immunoblots.

Figure 5.
Mapping of PtdIns 4-kinase II β-interacting regions with Par-4.

(A) Schematic representation of PtdIns 4-kinase II β structure and its truncated proteins. GST-PtdIns 4-kinase II β and its truncated mutants were cloned in pGEX-4T3 vectors and expressed in E. coli. D1 mutant has first 100 amino acids (1–100 amino acids) and D2 mutant has amino acids from 101 to 481. The D2 mutant was further truncated, T1 mutant has amino acids from 101 to 215 amino acids and T2 mutant has amino acids from 216 to 481 amino acids. (B,C) Wild-type GST-PtdIns 4-kinase and its deletion mutants were adsorbed onto Glutathione-Sepharose beads. Glutathione-bound PtdIns 4- kinase fragments were used for Par-4 pull-down assays. The presence of Par-4 was detected on immunoblots.

Silencing of PtdIns-4-kinase II β stabilizes Par-4 and promotes its nuclear localization

Par-4 is a tumor suppressor protein and its increased levels are directly correlated with apoptosis in cancer cells. The effect of PtdIns 4-kinase II β shRNA transfection on Par-4 stability was explored. MCF-7 cells transfected with scrambled shRNA showed a rapid turnover of Par-4. Within 45–90 min, Par-4 protein levels were not detectable on immunoblots. However, Par-4 protein levels were stable up to 135 min in PtdIns 4-kinase II β shRNA-transfected cells (Figure 6A). Overexpression of HA-tagged PtdIns 4-kinase II β and its mutants did not alter Par-4 stability significantly (Figure 6B). Concomitantly, a qualitative increase in Par-4 in nuclear region was observed in PtdIns 4-kinase II β shRNA transfectants (Figure 6C). In the absence of quantification of Par-4 levels in the nucleus, we looked for an alternate way of assaying Par-4 levels in the nucleus. Par-4 levels in the nucleus are strongly correlated with inhibition of NF-κB binding to its target DNA sequences [34]. Transfection of MCF-7 cells with PtdIns 4-kinase II β shRNA showed reduction in NF-κB binding to its target oligo-deoxynucleotide sequences in in vitro. In scrambled shRNA-transfected cells, NF-κB function is unaltered (Figure 6D).

PtdIns 4-kinase II β negatively affects Par-4 levels and signaling.

Figure 6.
PtdIns 4-kinase II β negatively affects Par-4 levels and signaling.

(A) MCF-7 cells were transfected with scrambled shRNA and PtdIns 4-kinase II β shRNA plasmids. Post-transfection, cells were treated with CHX for indicated time points. Par-4 protein levels were analyzed on immunoblots. (B) MCF-7 cells were transfected with HA-PtdIns 4-kinase II β wild-type, ΔCCPCC and D304A mutant plasmids. Post-transfection, cells were incubated with CHX for indicated time points, and protein levels were analyzed on immunoblots with anti-HA and anti-Par-4 antibodies. (C) Immunostaining of MCF-7 cells transfected with scrambled and with PtdIns 4-kinase II β shRNA plasmids with Par-4 C-terminus antibody. TRITC-conjugated secondary antibody was used for visualization in confocal microscopy. (D) MCF-7 cells were transfected with scrambled shRNA and PtdIns 4-kinase II β shRNA plasmids. Nuclear extracts from these cells were assayed for NF-κB binding to its target sequences as described in Materials and Methods.

Figure 6.
PtdIns 4-kinase II β negatively affects Par-4 levels and signaling.

(A) MCF-7 cells were transfected with scrambled shRNA and PtdIns 4-kinase II β shRNA plasmids. Post-transfection, cells were treated with CHX for indicated time points. Par-4 protein levels were analyzed on immunoblots. (B) MCF-7 cells were transfected with HA-PtdIns 4-kinase II β wild-type, ΔCCPCC and D304A mutant plasmids. Post-transfection, cells were incubated with CHX for indicated time points, and protein levels were analyzed on immunoblots with anti-HA and anti-Par-4 antibodies. (C) Immunostaining of MCF-7 cells transfected with scrambled and with PtdIns 4-kinase II β shRNA plasmids with Par-4 C-terminus antibody. TRITC-conjugated secondary antibody was used for visualization in confocal microscopy. (D) MCF-7 cells were transfected with scrambled shRNA and PtdIns 4-kinase II β shRNA plasmids. Nuclear extracts from these cells were assayed for NF-κB binding to its target sequences as described in Materials and Methods.

Par-4 siRNA rescues MCF-7 cells from PtdIns 4-kinase II β shRNA-induced apoptosis

The concomitant nuclear localization of Par-4 and inhibition of NF-κB signaling in PtdIns 4-kinase II β knockdown cells suggests that PtdIns 4-kinase II β may be mediating apoptosis through Par-4. Transfection of MCF-7 cells with Par-4 siRNA showed a two-fold increase in cell proliferation. Co-transfection of MCF-7 cells with Par-4 siRNA and PtdIns 4-kinase II β shRNA rescued MCF-7 cells from apoptosis (Table 2).

Table 2
Par-4 siRNA rescues MCF-7 cells from PtdIns 4-kinase II β-induced apoptosis

MCF-7 cells were double-transfected with PtdIns 4-kinase II β shRNA or control shRNA plasmids and Par-4 siRNA or control siRNA in different combinations. Post-transfection, viable cells were counted with haemocytometer using the dye exclusion method and expressed as percentage viable cells relative to cells transfected with control shRNA plasmids and control siRNA.

S. no. Transfection of MCF-7 cells with: Viable cells (%) 
Control shRNA + control siRNA 100 
PtdIns 4-kinase II β shRNA1 + control siRNA 25 
PtdIns 4-kinase II β shRNA2 + control siRNA 34 
Control shRNA + Par-4 siRNA 206 
PtdIns 4-kinase II β shRNA1 + Par-4 siRNA 140 
PtdIns 4-kinase II β shRNA2 + Par-4 siRNA 134 
S. no. Transfection of MCF-7 cells with: Viable cells (%) 
Control shRNA + control siRNA 100 
PtdIns 4-kinase II β shRNA1 + control siRNA 25 
PtdIns 4-kinase II β shRNA2 + control siRNA 34 
Control shRNA + Par-4 siRNA 206 
PtdIns 4-kinase II β shRNA1 + Par-4 siRNA 140 
PtdIns 4-kinase II β shRNA2 + Par-4 siRNA 134 

Discussion

Type II PtdIns 4-kinase(s) are known to be associated with growth factor receptors like EGF receptor (epidermal growth factor receptor), TCR (T-cell receptor)–CD3 complex, FcεR1 and play a role in calcium signaling, cell proliferation, vesicular trafficking, and inflammatory responses [6,12,3537]. In scanty reports, phosphatidylinositol phosphate(s) turnover was shown to be associated with Fas signaling and apoptosis [38]. Most of the pharmaceutical agents are directed against the catalytic activity of PtdIns 4-kinase II α and β isoforms and have limited utility in addressing the role of individual isoforms in signaling networks. An alternate approach is to use RNA interference techniques to understand their physiological roles in cell signaling.

The results presented in the present manuscript showed depletion of PtdIns 4-kinase II β with shRNAs resulted in increased pan-caspase activities and apoptosis in cancerous MCF-7 cells. Both receptor-mediated and intrinsic apoptotic pathways were suggested to be involved. These results indicate that PtdIns 4-kinase II β may modulate caspase activation through a mechanism that is yet to be discovered. Proteomic studies have identified Par-4, an apoptosis inducer as one of the molecular partners of PtdIns 4-kinase II β.

Par-4 induces apoptosis in cancer cells through FAS ligand—Fas signaling and intrinsically by inhibiting NF-κB functions [27,28]. The presence of Par-4 in PtdIns 4-kinase II β immunoprecipitates suggests an endogenous association of their interaction. The absence of PtdIns 4-kinase activity in Par-4 immunoprecipitates from PtdIns 4-kinase II β knockout but not in PtdIns 4-kinase II α knockout cells provided further evidence for isoform-specific association. PtdIns 4-kinase II β knockdown did not significantly affect total PtdIns 4-kinase activity in in vitro (results not shown). These results are in agreement with earlier observation that PtdIns 4-kinase II β has low catalytic activity in cytosolic fractions [39]. Both CCPCC domain and catalytic activity are not required for its association with Par-4. These studies suggested that PtdIns 4-kinase II β and Par-4 association may be mediated through unidentified protein–protein-interacting domains. GST-PtdIns 4-kinase II β fusion protein and its truncated mutants have shown that the region between 101 and 215 amino acids is essential for its interaction with Par-4. This region of PtdIns 4-kinase II β contains atypical leucine heptad (179LIPNQGYLSEAGAYLVDNKLHL201). Par-4 also contains a leucine-rich region at C-terminus, which plays an important role in its regulation. It may be possible that this region forms a leucine zipper-like association between PtdIns 4-kinase II β and Par-4. Silencing of PtdIns 4-kinase II β regulated Par-4 protein levels and its nuclear localization resulting in apoptosis. The mechanism by which PtdIns 4-kinase regulates Par-4 stability and its subcellular localization is not known at present. HSP90 is shown to affect PtdIns 4-kinase II β cytosolic localization and stabilization through physical interaction. Disruption of PtdIns 4-kinase II β interaction with HSP90 promotes PtdIns 4-kinase II β degradation through the proteasomal pathway [14]. In our proteomic data, proteasomal subunits, such as PSMA1, PSMA2, PSMA4, PSMA6, PSMA7, PSMB1, PSMB4, PSMB6, and PSMD14, are shown to be co-precipitated with Flag-PtdIns 4-kinase II β (Supplementary Table S1). Interaction with PtdIns 4-kinase II β may lead to Par-4 rapid degradation in the cytoplasm through proteasomal pathways. Alternately, the potential SUMOylation motif (403FKTD407) in PtdIns 4-kinase II β may play a role in subcellular localization of PtdIns 4-kinase II β–Par-4 complex. This hypothesis needs further studies for validation.

Par-4 is also known to affect redox signaling. The ability of caspase 9 inhibitors to rescue cells from apoptosis induced by PtdIns 4-kinase II β shRNA suggests that PtdIns 4-kinase II β may also influence redox signaling and endoplasmic reticulum stress. These things will have potential implications for radiation therapy. The role of PtdIns 4-kinase II β in redox signaling and endoplasmic reticulum stress in cancer cells is being studied (Manuscript in preparation). This may be the first report to suggest that PtdIns 4-kinase II β functions as a regulatory protein in apoptosis induction in cancer cells.

A hypothetical model is proposed to explain the role of PtdIns 4-kinase II β in apoptosis induction in cancer cells (Figure 7). Disruption of PtdIns 4-kinase II β-Par-4 complex may be an alternate approach to activate Par-4 in cancer cells. In non-cancerous cells such as MCF-10A cells, where Par-4 is low/absent, knocking down of PtdIns 4-kinase II β has no effect (Supplementary Figure S5). Par-4 is being investigated for potential therapeutic interventions in cancer management [40]. Combinatorial effects of silencing of PtdIns 4-kinase II β with other pharmaceutical agents are an interesting topic to pursue in future.

Cartoon picture of Par-4 regulation by PtdIns 4-kinase II β.

Figure 7.
Cartoon picture of Par-4 regulation by PtdIns 4-kinase II β.

In the presence of PtdIns 4-kinase II β, Par-4 forms a complex with lipid kinase in cytoplasm leading to its (Par-4) rapid degradation and promotes pro-survival signaling. In the absence of PtdIns 4-kinase II β, Par-4 protein levels increase, translocate to the nucleus, inhibit NF-κB-induced pro-survival pathways, and induce apoptosis.

Figure 7.
Cartoon picture of Par-4 regulation by PtdIns 4-kinase II β.

In the presence of PtdIns 4-kinase II β, Par-4 forms a complex with lipid kinase in cytoplasm leading to its (Par-4) rapid degradation and promotes pro-survival signaling. In the absence of PtdIns 4-kinase II β, Par-4 protein levels increase, translocate to the nucleus, inhibit NF-κB-induced pro-survival pathways, and induce apoptosis.

Abbreviations

     
  • CHX

    cycloheximide

  •  
  • DMEM

    Dulbecco's Modified Eagle's Medium

  •  
  • GAPDH

    glyceraldehyde 3-phosphate dehydrogenase

  •  
  • NF-κB

    nuclear factor kappa B

  •  
  • Par-4

    prostate apoptosis response-4

  •  
  • PtdIns 4-kinase II β

    type II phosphatidylinositol 4-kinase β

  •  
  • qPCR

    quantitative polymerase chain reaction

  •  
  • shRNA

    short hairpin RNA

  •  
  • siRNA

    small interfering RNA

Author Contribution

S.C. designed and carried out experiments with PtdIns 4-kinase II β shRNA, immunoprecipitation of Par-4, PtdIns 4-kinase assays, confocal microscopy and Par-4 siRNA rescue experiments. V.J. was involved in the construction of GST-PtdIns 4-kinase II β fusion proteins and pull-down assays with GST-PtdIns 4-kinase proteins. N.B. was involved with the construction of FLAG-PtdIns 4-kinase II β and proteomic studies with HEK293 cells. M.T. and S.K.S. were involved with RT-PCR, EMSA and providing access to their laboratory facilities and resources at Bhabha Atomic Research Center, Mumbai. G.S.M. is involved in conceptualizing the project, getting grants for the project, overall supervision of the project and writing the manuscript.

Funding

This work was supported by the Board of Research in Nuclear Sciences (grant no. 16BRNS007) to Gosukonda Subrahmanyam. Sonica Chaudhry and Vibhor Joshi were supported by the Indian Council of Medical Research Fellowships.

Acknowledgements

The authors are grateful for critical comments from Dr K.B. Sainis and from Nitin Kachariya for their technical assistance in the initial stages of the project.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Supplementary data