Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1

Figures

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  Fig. 1. Modulation of Mouse mesangial cell proliferation by TTCC blockers. MES13 cells treated with TTCC blockers mibefradil (a), TH1177 (b) from 0–20 µM concentration shows dose-dependent inhibition of mesangial cell proliferation in comparison to vehicle control and L type calcium channel blocker verapamil (c). The data are represented as mean ± SD. n = 4, ***P < 0.001, ****P < 0.0001 is the comparison between the concentration to vehicle control. Two-Way ANOVA with Turkey post hoc correction

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  Fig. 2. Mibefradil inhibits phosphorylation of ERK1/2 in the presence of 5%, FBS, PDGF or TGFβ1. A representative Western blot showing phosphorylated ERK1/2 levels in MES13 cells with and without mibefradil treatment in 1% FBS (a) for 4 h, in all media conditions (with 5% FBS, PDGF or TGFβ1). The full-length images of Western blot are presented in Supplementary figure S5. (b) shows the mean densitometry quantification of pERK1/2 levels (normalised to total ERK1/2) in three independent experiments with MES13 cells treated with mibefradil for 4 h and controls in a range of media conditions (5% FBS, PDGF or TGFβ1). The data are represented as mean ± SD, n = 3, ****p < 0.0001, from a One-Way ANOVA with Bonferroni post hoc correction

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  Fig. 3. TH1177 inhibits phosphorylation of ERK1/2 in the presence of 5%, FBS, PDGF or TGFβ1. a shows a representative Western blot showing phosphorylated ERK1/2 levels in MES13 cells with and without TH1177 treatment for 4 h, in all media conditions (with 5% FBS, PDGF or TGFβ1). The full-length images of Western blot are presented in Supplementary figure S6. b shows the mean densitometry quantification of pERK1/2 levels (normalised to total ERK1/2) in three independent experiments of MES13 cells treated with TH1177 for 4 h and controls in a range of media conditions (5% FBS, PDGF or TGFβ1). The data are represented as mean ± SD, n = 3, * p < 0.05, from a One-Way ANOVA with Bonferroni post hoc correction

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  Fig. 4. CaV3.1 but not CaV3.2 SKO in 5% FBS, PDGF and TGF-β1inhibits phosphorylation of ERK1/2. A representative Western blot showing phosphorylated ERK1/2 levels in MES13 control cells, CaV3.1 SKO clone (a) and CaV3.2 SKO clone (c) in all media conditions (with 5% FBS, PDGF or TGF-β1). The full-length images of Western blot are presented in Supplementary figure S7. b shows the mean densitometry quantification of pERK1/2 levels (normalised to total ERK1/2) in three independent experiments of CaV3.1 SKO clones and control in a range of medium conditions (5%FBS, PDGF or TGF-β1). The data are represented as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, from a One-way Anova with Bonferroni post hoc correction. d shows similar densitometry for CaV3.2 KO cells

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  Fig. 5. Modulation of CaV3.1 SKO, CaV3.2 SKO and MES13 cell proliferation by TTCC blockers. Cell proliferation of the CaV3.1, CaV3.2 knockout cells after treatment with TTCC blockers: mibefradil (a, b) and TH1177 (c, d) were compared to wild type control cells (MES13). Cell number was calculated by using the MTS assay in time points at 48 and 72 h. The data are represented as mean ± SD. Letters ‘abc’, ‘lmn’ and ‘xyz’ are used to denote the significance between drug concentration in control, CaV3.1 and CaV3.2 respectively. The same letter above the graphs indicates no change between the groups. Different letters above the graph indicate the significant difference between the means. The data are represented as mean ± SD, n = 4, **P < 0.01, ***P < 0.001 are for the comparison between KO to its respective control at each concentration. Two-Way ANOVA with Turkey post hoc correction