Fig 2: Sumoylated RhoB is required for UV or MMS-induced mitophagy and cell death. a and b Knockout of RhoB inhibits mitochondria clearance after UV or MMS treatment. RhoB+/+ or RhoB-/- cells were treated as Fig. 3e and subjected to immunofluorescence (a) or immunoblotting (b). Scale bar, 10 µm. c UV or MMS-induced downregulation of Hsp60 is through lysosome pathway. HeLa cells pretreated 1 h with or without 100 µM CQ were treated and processed as in panel b. d Reintroduction of RhoB WT but not 4KR down-regulates Hsp60 after UV or MMS treatment. RhoB-/- cells transduced with HA-RhoB (WT or 4KR) were treated as in panel b. e and f Knockout of RhoB decreases UV or MMS-promoted mitophagic flux. RhoB+/+ and RhoB-/- cells stably expressing mRFP-GFP-tagged signal peptide (residues 101–152) of mitochondrial fission 1 protein (mRFP-GFP-FIS1101-152) were treated and processed as in panel a. Scale bar, 10 µm. The percentage of cells with dominant red fluorescence were quantified and plotted in panel f. Five random areas were counted for each experiment and data of three independent experiments were assessed by one-way ANOVA (F(5,84) = 174.51) followed by LSD post hoc test after arcsine transformation and represented as mean ± SD (f). g and h Sumoylated RhoB is required for UV or MMS-induced apoptosis. RhoB+/+ or RhoB-/- cells (g), or RhoB-/- cells transduced with RhoB WT or 4KR (h) were subjected to flow cytometry assay to determine the apoptosis rate 30 h after treated with UV (80 Jm-2) or MMS (0.5 mM). Data of three independent experiments were assessed by one-way ANOVA (F(5,12) = 2939.3) (g) or (F(8,18) = 4854.2) (h) followed by LSD post hoc test after arcsine transformation and represented as mean ± SD. i Double knockout of ULK1/2 abrogates RhoB-promoted apoptosis in response to UV or MMS treatment. ULK1/2 WT or double knockout (DKO) MEF cells were transduced with RhoB. Cells were subjected to flow cytometry assay to determine the apoptosis rate 24 h after treated with UV (80 Jm-2) or MMS (0.5 mM). Data of three independent experiments were assessed by one-way ANOVA (F(17,36) = 1069.3) followed by LSD post hoc test after arcsine transformation and represented as mean ± SD

Fig 3: Immunoblotting detection and quantitative analysis. (A) Total lysates of control and GLE- or RE- treated cells after 4 h, 14 h, and 24 h of exposure, immunoreacted with anti-p62/SQSTM1, -Beclin-1, -HSP 60, -LC3, and -actin antibodies. The histograms show the densitometric analysis of the obtained bands for (B) LC3-II/LC3-I ratio, (C) Beclin-1, (D) p62/SQSTM1, and (E) HSP 60. Actin was used as the loading control and, in C-E, the relative protein expression, reported as arbitrary units, was calculated as the band density ratio to that of actin. The experiments were performed in triplicate and the data are expressed as means ± standard deviation (n = 3 ± SD). * p = 0.05; ** p = 0.01; *** p = 0.005 vs. control for each time of treatment.