Fig. 5: HECTD3 positively regulates RNF168 activity by promoting autophagy.

A Hectd3 depletion decreased rapamycin induced autophagy. Hectd3 WT and KO MEF cells were treated with 5–10 μM rapamycin for 4 h. The protein levels of LC3-II were measured by WB. B Quantitation of GFP-LC3 puncta in Hectd3 + /+ and Hectd3 -/- MEFs treated with rapamycin. All data are represented as the mean ± SEM. *p < 0.05. Student’s t test was used for the statistical analysis. C Hectd3 knockout decreased rapamycin-induced autophagy. GFP-LC3 was transfected into MEF cells for 48 h. The cells were treated with rapamycin (5 μM) for 4 h. Puncta formation indicated autophagy. D HECTD3 knockout leads to p62 accumulation in nucleus after IR. HECTD3 (Con) and HECTD3 (KO) HCC1806 and HCC1937 cells were irradiated with 10 Gy and released for 1 h. WB was performed to determine p62 protein levels in the nucleus and cytoplasm. E HECTD3 knockout decreased RNF168 protein levels. HECTD3 (Con) and HECTD3 (KO) HCC1806 and HCC1937 cells were irradiated with 10 Gy and RNF168 was detected by WB. F, G HECTD3 knockdown reduced IR-induced RNF168 foci after IR treatment in HCC1806 and HCC1927 cells. Cells transfected with HECTD3Con or HECTD3 KO were treated with IR (10 Gy) after 1 h, the cells were immunostained with anti-RNF168 (red), anti-γH2AX (green) and DAPI (blue) and analyzed using a confocal microscopy. The cells containing > 5 RNF168 foci were recorded as RNF168/γH2AX positive cells, whose percentage was averaged from at least 100 cells. The data are displayed as mean plus SD of three counts, and statistical significance was calculated and represented as the P-value. *p < 0.05 and ns, not significant. Scale bars, 20 μm. H HECTD3 increases histones ubiquitination upon IR. HECTD3 (Con) and HECTD3 (KO) HCC1937 cells were stably expressed Flag-Ub, then were irradiated with 10 Gy and subjected to acid extraction of chromatin 1 h after IR.