Figure 5
From: Enhanced insulin receptor, but not PI3K, signalling protects podocytes from ER stress
Knockdown of PTEN in podocytes increases ER stress. (a) Representative western blot with densitometric quantification normalised to β-actin demonstrating near-complete knockdown of PTEN using shRNA compared with wt cells and cells treated with scrambled (scr) shRNA, (n = 3). Unpaired t test with Welch’s correction (wt vs PTEN shRNA **p = 0.0022). (Full blot shown in Supplementary Fig. 3). (b) Insulin sensitivity monitored by pAkt immunostaining in cell nuclei following 10 minutes of 10−10-10−6 M insulin stimulation (n = 4). Data expressed as the percentage of cells positive for pAkt in the nucleus. Two-way ANOVA, effect of PTEN p < 0.0001****, insulin ****p < 0.0001, but no significant interaction between the effect of PTEN knockdown and insulin concentration. A Bonferroni post-hoc comparison revealed no significant difference between wt and PTEN kd cells at each insulin concentration. (c) ATF6-driven luciferase activity for wt and PTEN kd podocytes treated with diabetic media relative to normal growth media. Unpaired t test with Welch’s correction (n = 3), not significant, ‘ns’. (d) ERSE-driven luciferase activity for wt and PTEN kd podocytes treated with diabetic media relative to normal growth media. Unpaired t test with Welch’s correction (n = 3), **p < 0.01 (n = 3). (e) WT and PTEN kd podocytes treated with 10−5-10−3 M Palmitate for 24 hr were fixed and stained with DAPI and for CHOP (n = 3). Two-way ANOVA p < 0.0001 for cell type (****) and for palmitate concentration (****), with no significant interaction between cell type and palmitate concentration. A Bonferroni post-hoc comparison revealed statistical significance at 10−5 M (*) and 10−3.5 M (**) palmitate. (f) WT and PTEN kd cells were differentiated in the presence of either normal growth media or diabetic (‘D’) media for 14 days before being fixed and immunostained for CHOP and with DAPI (n = 3). Paired t test: wt and wt + D, p = 0.0091 (**); PTEN kd and PTEN kd + D, p = 0.4737 (ns). (g) PTEN kd podocytes treated with 10−5-10−3 M Palmitate were additionally treated with 10 µM MEK (PD 184352) and 30 µM ERK (FR 180204) inhibitors (‘i’) for 24 hr. Two-way ANOVA, wt vs PTEN kd *p = 0.0166; PTEN kd vs PTEN kd + MEK inhibitor ** p = 0.0012; PTEN kd vs PTEN kd + ERK inhibitor ****p < 0.0001; wt vs PTEN kd + MEK inhibitor not significant; wt vs PTEN kd + ERK inhibitor **p = 0.0065; PTEN kd + MEK inhibitor vs + ERK inhibitor *p = 0.0264. Effect of palmitate ****p < 0.0001 in all comparisons. A Bonferroni post-hoc comparison for PTEN kd cells -/ + ERK inhibitor revealed statistical significance at 10−5 M (**) and 10−4-10−3.5 M palmitate (*). Inset Bar graph of 103.5 M palmitate values with two-way ANOVA statistics from whole palmitate ranges shown. (h) Caspase 3/7 activation in wt and PTEN kd podocytes following 24 hr palmitate treatment (n = 3). Two-way ANOVA, effect of PTEN kd *p = 0.0208, effect of palmitate ****p < 0.0001, with a significant interaction between PTEN kd and palmitate dose **p = 0.009. A Bonferroni post-hoc comparison for wt and PTEN kd revealed statistical significance at 10−3 M palmitate (****).
