Figure 3
From: Enhanced insulin receptor, but not PI3K, signalling protects podocytes from ER stress
Knockdown of PTP1B protects against ER stress. (a) Representative western blot with densitometric quantification normalised to GAPDH demonstrating PTP1B knockdown of 56% in the PTP1B kd cell line compared with wt cells (n = 3). Unpaired t test **p = 0.005. Quantification of PTP1B in cells treated with scrambled (scr) shRNA also shown. (Full blot shown in Supplementary Fig. 3). (b) Insulin sensitivity monitored by pAkt immunostaining in cell nuclei following 10 min 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 PTP1B p < 0.0001****, insulin ***p = 0.0002, but no significant interaction between the effect of PTP1B and insulin concentration. A Bonferroni post-hoc comparison revealed statistical significance at 10−7 M insulin (*). (c) ATF6-driven luciferase activity for wt and PTP1B kd podocytes treated with diabetic media relative to normal growth media. Unpaired t test with Welch’s correction (n = 3), *p < 0.05. (d) ERSE-driven luciferase activity for wt and PTP1B kd podocytes treated with diabetic media relative to normal growth media. Unpaired t test with Welch’s correction (n = 3). (e) CHOP response in wt and PTP1B kd podocytes for cells treated with or without diabetic media (‘D’), (n = 3). Comparison of wt and PTP1B kd, unpaired t test with Welch’s correction; paired t test for comparison of wt and wt + D p = 0.0091 (**), wt + D and PTP1B kd p = 0.0006 (***), PTP1B kd and PTP1B kd + D (ns). (f) Caspase 3/7 activation in wt and PTP1B kd podocytes following 24 hr palmitate treatment (n = 3). Two-way ANOVA, effect of PTP1B kd not significant, effect of palmitate ****p < 0.0001.
