Mouse mesangial cells (SV40 MES13) purchased from ATCC (Virginia, United States) were maintained in a 3:1 solution of Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Paisley, UK) and F-12 (Invitrogen). The medium was supplemented with 5% fetal bovine serum (FBS), 14 mM HEPES, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin (Invitrogen). Cultured cells were maintained at standard cell culture condition at 37°C with 95% O2 and 5% CO2.
Calcium channel inhibitors
Mibefradil (Sigma Aldrich, Dorset, UK) and verapamil (Sigma Aldrich) were made from 10 mM stock solutions in distilled water and stored at 4°C, and every two months, fresh stock solutions were prepared. TH1177 was a kind gift from Dr Lloyd Gray, University of Virginia, Charlottesville, Va., USA. TH1177 was prepared from a 10 mM stock solution in 100% ethanol and stored at -20 °C
Serum, PDGF and TGF-β1 stimulation
MES13 cells were seeded at 3x105 cells in 35 mm dishes with 1% FBS for 48 h. At 48 h, cells were cultured in varied conditions: 0% FBS, 20% FBS, or 20 ng/ml PDGF-BB and 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN) and vehicle control [TGF- β1 buffer-4mM HCl plus 0.1% BSA, pH3] for 24, 48 and 72 h time points. Cells were harvested for RNA extraction at the end of each time point. The medium was removed and washed with PBS and 350 µl of RLT buffer with beta-mercaptoethanol was used to lyse the cells. The RNA extraction was carried out using the RNeasy Mini Kit (Qiagen Ltd, Crawley, UK) by following the manufacturer’s instructions.
Reverse transcriptase- polymerase chain reaction (RT-PCR)
cDNA was synthesised from 2 µg of RNA using high capacity RNA to cDNA synthesis kit (Applied biosystems, Massachusetts, USA) in the 20 µl reaction mix. RT-PCR amplification was carried out from 5 µl of cDNA using Dream Taq polymerase (Fischer Scientific, Loughborough, UK) using a thermocycler. The PCR amplification was carried out with an initial denaturation of 95°C for 3 min, 35 cycles of denaturation 95°C for 30 s, annealing Tm-5 for 30 s, extension 72°C for 1 min and final extension 72°C for 5 min. The RT-PCR primers are listed in Suppl. Table S1.
plenti-CRISPR-cas9-gRNA for CaV3.1/CaV3.2 was purchased from GenScirpt, New Jersey, USA. The plasmid contained a plentiCRISPRv2.0 backbone cloned with cas9, ampicillin, bleomycin, puromycin selection markers, and 20 nucleotides of the gRNA sequence complementary to exon 4 of Cacna1G and exon 6 of Cacna1H gene under the control of U6 promoter (Suppl. Fig. S2a-b).
Transfection, antibiotic and clonal selection
plenti-CRISPR-cas9-gRNA for CaV3.1/CaV3.2 plasmids were used to generate stable knockouts which were transfected into MES13 cells using lipofectamine 3000 (Thermo fisher Scientific, Massachusetts, USA). Briefly, 6x105 cells were seeded in 6 well plates overnight in the antibiotic-free growth medium. The following day, for SKO generation, 5 µg of plenti-CRISPR-cas9-gRNA for CaV3.1 or CaV3.2 plasmids and for DKO generation, 2.5ug each of plenti-CRISPR-cas9-gRNA for CaV3.1 and CaV3.2 were transfected by lipofection, and the medium was changed after 6 h. Post 72 h of transfection, complete growth medium with 2 µg/ml of puromycin was added for antibiotic selection. The cells resistant to puromycin post 72 h antibiotic selection were trypsinised and sub-cultured to the new six-well plate and the second round of antibiotic selection was carried out using puromycin to minimise the contamination of untransfected cells. Although the cells were sorted with a selection marker, there remained a possibility of a mixed population of cells with a variable length of base-pair deletion of ~1-19 nucleotides. Hence, the knockout cells were subjected to single-cell clonal selection by serial dilution starting with 500 cells in 96-well plates. The SKO clones were picked, expanded and confirmed at the genomic level by Sanger sequencing and confirmed at the translational level by Western blot. This second round of puromycin selection resulted in too few cells to achieve single cell clonal selection for the DKO clones and expansion for Western blotting, hence DKO cells from the first round of antibiotic selection went through clonal selection in the conditional medium. The survived clones were sent for Sanger sequencing alone.
Genomic DNA extraction, PCR and Sanger sequencing
MES13 control cells and CaV3.1, CaV3.2 SKO and DKO clones were subjected to genomic DNA extraction by using the QIAamp DNA extraction kit (Qiagen Ltd, Crawley, UK), following the manufacturer’s instructions. The genomic DNA was quantified in a Nanodrop, and 100 ng of DNA was amplified by using Dream Taq polymerase and primers spanning the gRNA sequence region in 20 µl reaction mix. The primer list is detailed in Suppl. Table S1. The PCR products were cleaned using the ExoSAP-IT Express PCR product clean-up kit (Thermo Fisher Scientific). The PCR product was preceded to Sanger sequencing by GATC Biotech, Ebersberg, Germany.
MES13 cells and SKO clones of CaV3.1 and CaV3.2 treated with or without calcium channel blockers (mibefradil and TH1177) were subjected to a microculture tetrazolium (MTS) assay (Promega, Southampton, UK) to measure the cell number. Briefly, MES13 cells, CaV3.1 and CaV3.2 SKO clones were subjected to serum deprivation in 1% FBS for 48 h. Cells were seeded into 96-well plates at a density of 5000 cells per well and incubated with varied concentration of mibefradil and TH1177 (0, 5, 10 and 20 µM) in 5%FBS. Absorbance at 492 nm was measured at 48, and 72 h in a microplate reader.
Signal transduction evaluation
MES13 and SKO clones of CaV3.1 and CaV3.2 were seeded at 6X105 cells per well in 6 well plates and left overnight. The cells were serum deprived to 1% FBS for 48 h. At 48 h, the cells were stimulated for 30 min with either: 5% FBS, 20 ng/ml PDGF +1% FBS or 10 ng/ml TGF-β1 + 1% FBS.
Similarly, MES13 cells alone were seeded at 6X105 cells per well in 6 well plates and left overnight. The cells were serum deprived to 1% FBS for 48 h. At 44 h, the cells were treated with 5 µM mibefradil +1% FBS and at 48 h, the cells were stimulated with 5% FBS, 20 ng/ml PDGF+1%FBS and 10 ng/ml of TGF- β1+1%FBS for 30 min.
The medium was removed immediately after 30 min stimulation, washed with PBS, and the cells were lysed with RIPA buffer with protease and phosphatase inhibitor (Sigma). The lysate was scraped off from the plates and incubated on ice for 30 min with intermittent vortexing. The lysates were centrifuged for 14,000 rpm at 4°C for 20 min. The supernatant from the lysate was transferred to new labelled tubes and stored at -80°C. The experiments were repeated three times.
The protein concentration was quantified by BCA assay (ThermoFisher Scientific), and 20-40 µg of protein was separated in 8% PAGE gel. The proteins were transferred to a nitrocellulose membrane at 35 V for 3 h for CaV3.1 and CaV3.2 and 100 V for 1 h for pERK1/2. The protein transfer was confirmed by Ponceau staining, and the blots were washed and incubated with 5% non-fat milk (Sigma) for 1 h. The blots were incubated with primary antibodies: CaV3.1 (1:1000) (Alomone, Jerusalem, Israel) and CaV3.2 (1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4°C. The following day, the blots were washed thrice with 1x TBS +0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1x TBS-T and incubated with ECL substrate (Fisher scientific) for 5 min and exposed to X-ray film. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quantified using Image Studio Lite software.
All data were tested for normality and no difference in the variances between groups was detected using the Shapiro-Wilk test. Parametric variables were analysed using a One-Way Analysis of Variance (for multiple group comparisons) with a post hoc Bonferroni test or Two-Way Analysis of Variance with post hoc Tukey test. p< 0.05 is considered as statistical significance. Statistical analyses were performed using Prism software version 8.0 (Graph-Pad Software, San Diego, CA).