Fig. 3

Sumoylated RhoB is required for UV or MMS-induced autophagy. a Knockout of RhoB decreases UV or MMS treatment-induced mRFP-LC3 puncta formation. RhoB^+/+ or RhoB^−/− cells stably expressing mCherry red fluorescent protein fused LC3 (mRFP-LC3) were subjected to fluorescence microscopy 4 h after UV (80 Jm⁻²), 6 h MMS (0.5 mM), 8 h CPT (10 μM), or 8 h DOX (0.5 μM) treatment. Scale bar, 10 μm. b Knockout of RhoB inhibits UV or MMS-induced augment of LC3-II. RhoB^+/+ or RhoB^−/− cells pretreated 1 h with or without 50 μM chloroquine (CQ) were subjected to immunoblotting 4 h after UV (80 Jm⁻²) or 6 h with MMS (0.5 mM) treatment. c Reintroduction of RhoB WT but not 4KR restores UV or MMS-induced mRFP-LC3 aggregation. RhoB^−/− cells stably expressing mRFP-LC3 were transduced with lentivirus encoding HA-RhoB WT or 4KR. Cells were subjected to immunofluorescence assay 4 h after UV (80 Jm⁻²) or 6 h after MMS (0.5 mM) treatment. The dot lines indicate cells expressing RhoB WT or 4KR. Scale bar, 10 μm. d Reintroduction of RhoB WT but not 4KR restores UV or MMS-induced augment of LC3-II. RhoB^−/− cells transduced with HA-RhoB WT or 4KR were treated as in panel b. e Knockout of RhoB decreases the autophagic flux promoted by UV or MMS. RhoB^+/+ or RhoB^−/− cells stably expressed mRFP-GFP-LC3 were subjected to fluorescence microscopy after 8 h UV (80 Jm⁻²) or 10 h MMS (0.5 mM) treatment. Scale bar, 10 μm. f Reintroduction of RhoB WT but not 4KR promotes the autophagic flux upon UV or MMS treatment. RhoB^−/− cells stably expressing mRFP-GFP-LC3 were transduced with HA-RhoB WT or 4KR. Cells were treated and subjected to immunofluorescence assay as in panel e. The dot lines indicate cells expressing RhoB WT or 4KR. Scale bar, 10 μm. g Knockout of RhoB attenuates UV or MMS-induced autophagosomes and autolysosomes formation. RhoB^+/+ or RhoB^−/− cells were assessed by electron microscopy 6 h after UV (80 Jm⁻²) or 8 h after MMS (0.5 mM) treatment. The magnified images are the areas indicated by the squares. Red arrow indicates autophagosome/autolysosome with mitochondria