Skip to main content
. 2015 Jan 28;10(12):2193–2207. doi: 10.4161/15548627.2014.981786

Figure 2.

Figure 2.

Autophagy induces myofibroblast differentiation in starved fibroblasts. (A) Western blot showing LC3B-I and -II protein levels in WI-38 fibroblasts at baseline or starved in the presence of 3-methyladenine (1 mM; 3-MA), wortmannin (100 nM; W), LY294002 (5 μM; LY) or vehicle (V) for 4 h. Representative of 4 independent experiments. (B) Western blot showing ACTA2 protein levels in WI-38 fibroblasts at baseline or starved and incubated with the same inhibitors as in A for 4 d. Representative of 4 independent experiments. (C) Evaluation of the myofibroblast markers ACTA2 (red) and stress fiber formation (green) by immunofluorescence microscopy in fibroblasts exposed to SS in the presence of LY or V for 4 d. Cell nuclei are visualized in blue. ACTA2 and stress fiber staining of fibroblasts grown in normal medium or starved for 4 d from the same experiment are shown in Figure 1F. Representative of 3 independent experiments. (D) COL1A1 and COL3A1 mRNA levels evaluated by real time qPCR in WI-38 fibroblasts serum starved for 4 d in the presence of the PtdIns3K inhibitor LY or vehicle. GAPDH was used as the reference gene (***P < 0.001 V vs LY for COL1A1 and **P < 0.01 V vs LY for COL3A1). Collagen mRNA levels of fibroblasts grown in normal medium or starved for 4 d from the same experiment are shown in Figure 1G. Representative of 2 independent experiments performed in triplicate. (E) Left panel: Western blot showing ATG7, SQSTM1 and tubulin (TUBA) protein levels in WI-38 fibroblasts starved for 2 d post-nucleofection with control siRNA (siCTL) or ATG7 siRNA (siATG7). Representative of 3 independent experiments. Right panel: Densitometric analysis of SQSTM1 protein level relative to tubulin (representative of 3 independent experiments, *p = 0.0318) in WI-38 fibroblasts silenced for ATG7 expression (ATG7 silencing is effective at 80.6% ± 6.0%, representative of 3 independent experiments, ***P < 0.0001). (F) Left panel: Western blot showing ATG7, ACTA2, and tubulin (TUBA) protein levels in WI-38 fibroblasts starved for 4 d post-nucleofection with control siRNA (siCTL) or ATG7 siRNA (siATG7). Representative of 4 independent experiments. Right panel: Densitometric analysis of ACTA2 level relative to tubulin (***p = 0.0005 representative of 4 independent experiments) in WI-38 fibroblasts silenced for ATG7 expression (ATG7 silencing is effective at 87.4% ± 4.4%, from 4 independent experiments, ***P < 0.0001).