Fig. 3

Fig. 3 The expression of a constitutively active form of YAP inhibits autophagy flux during shear stress.

a Representative images of GFP, LC3-I, LC3-II and Actin proteins levels in KECs expressing a constitutively active (5SA) or inactive (5SA/S94A) form of GFP-YAP, then subjected to flow (shear, 24 h) or not (static), in the presence or absence of chloroquine (CQ), by western blot analysis. b The ratio of LC3-II to Actin was determined by densitometry, relative to panel a. Data show the mean ± s.e.m.; n = 3 independent experiments, two-sided t-test. c, d Representative images (c) and quantification (d) of LC3 puncta in KECs expressing a constitutively active (5SA) or inactive (5SA/S94A) form of GFP-YAP, then subjected to shear stress during 24 h in the presence or absence of chloroquine (CQ). Data show the mean ± s.e.m.; n = 27 individual data points from 3 independent experiments, two-sided t-test. Scale bars, 10 µm. e, f Representative images (e) and quantification (f) of LDs (Bodipy) in KECs expressing a constitutively active (5SA) or inactive (5SA/S94A) form of YAP, then subjected to shear stress during 24 h. Data show the mean ± s.e.m.; n = 30 individual data points from 3 independent experiments, two-sided t-test. Scale bars, 10 µm. g Expression of Rubicon in KECs subjected to flow (shear) or not (static) during 24 h. mRNA levels were quantified by real-time RT-qPCR, normalized to β-actin and are presented as fold increases. h Expression of Rubicon in KECs expressing a constitutively active (5SA) or inactive (5SA/S94A) form of YAP, then subjected to flow (shear) or not (static) during 24 h. mRNA levels were quantified by real-time RT-qPCR, normalized to β-actin and are presented as fold increases. i, j Relative enrichment of YAP (ChIP GFP-YAP, % input) on the CTGF (i) and RUBICON (j) genes in KECs expressing a constitutively active (5SA) or inactive (5SA/S94A) form of GFP-YAP. The P-value represent the statistical difference between the 5SA and 5SA/S94A condition at the binding sequence. g–j Data show the mean ± s.e.m.; n = 3 independent experiments, two-sided t-test. Source data are provided as a Source Data file.