Figure 3. ROCK1 activity is essential for metabolic stress-induced autophagy.

(a) The effect of ROCK1 depletion on autophagy marker, LC3II. MEF ROCK1^flox/flox cells were generated and treated with Con or Ad-Cre for 72h. Cells were then incubated in nutrient rich (+Glucose) or HBSS media for 2h. Cell extracts were resolved by SDS-PAGE and ROCK1 knockout was confirmed by western blotting (left panel). The same blot was used for LC3 and β-actin expression. Cells from the same experiment were fixed with chilled acetone and immunofluroscence was performed against endogenous LC3 (red). Blue represents nuclear dapi staining. (b) EJ cells (left) with a stable knockdown using shCont. or shROCK1#1 were incubated in control (+ glucose) or nutrient-free (HBSS) medium (− glucose) for 24 h and lysed. Lysates were subjected to western blotting with antibodies indicated. HeLa cells (right) with a transient knockdown using siCont. and siROCK1#2 for 30 h were further incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and analyzed for ROCK1, LC3 and β-actin. Relative ratios of LC3II/LC3I are shown. (c) HeLa cells were treated with H₂O or 10 µM ROCK1 inhibitor, Y27632, for 8 h. Cells were then cultured in high glucose (0 h) or starved for in HBSS nutrient-free media for 6h. 1h prior to harvest, cells were incubated in DMSO (control) or 0.1 µM bafilomycin-A1. Whole cell lysates were resolved by SDS-PAGE and analyzed by western blotting with indicated antibodies. (d) HeLa cells incubated with H₂O or 10 µM Y27632 for 8 h were washed with PBS and cultured in control (+ Glu) or HBSS media (− Glu) for an additional 8 h. Cells were fixed in acetone and immunofluorescence was performed against endogenous LC3 antibody (green). Scale = 20 µm. Graph represents % of LC3 punctae/cell (mean±SD), n=25 cells; (Student t-test) **, p= 0.00041 (e) Transmission electron micrographs of EJ cells with stable shCont. or shROCK1#1 knockdown, cultured in control or HBSS medium for 16 h. Arrow bars indicate autophagic vacuoles. Scale = 500 nm.