Fig. 2: Autophagy is activated in a TGFβ-dependent manner in fibrosis.

a–e Activation of autophagy in cultured fibroblasts by TGFβ. a mRNA levels of BECLIN1 and ATG7 (n = 3 samples per group). b Protein levels of ATG7, BECLIN1, and p62. Representative western blots as well as the quantifications (n = 2 biological replicates per group over two independent experiments). c Ratio of LC3 II to LC3 I: Representative western blots and quantification. Bafilomycin and starvation served as control (n = 4 samples per group). d EGFP/RFP-LC3 reporter activity; representative images and quantification (n = 5 biological replicates per group). Horizontal scale bar, 5 µm. e Representative electron microscopy images at 12,000-fold magnification (horizontal scale bars, 2 µm; n = 4 biological replicates) and quantification. Autophagosomes are indicated by arrows. f–h Overexpression of TβRIact in murine skin. f mRNA levels of Beclin1 and Atg7 (n = 8 biological replicates per group). g Representative immunofluorescence staining of BECLIN1, ATG7 or p62 (all green) in combination with DAPI (blue) and vimentin (red; n = 7 biological replicates per group for all experiments). h Representative images and quantification of EGFP/RFP-LC3 reporter activity in the skin (n = 5 biological replicates for both groups). Horizontal scale bars, 50 µm. All data are presented as median ± IQR. p-values were determined by ANOVA one-way with Sidak’s multiple comparisons test (a) or with Tukey’s multiple comparison post hoc test (c–e) and by two-sided Mann–Whitney test (f–h). p-values are indicated in the figure. See source data for more detailed information. Western blot samples in panel c cropped at the black horizontal line were not run on the same gel. Images were cropped at the line only for the purpose of this figure. int.: intensity, fluo.: fluorescence, TβRIact: constitutively active TGFβ receptor type I. BafA1: bafilomycin A1, fluo.: fluorescence.