Fig. 6

Deficiency in HectH9 or HK2 inhibits CSC self-renewal via ROS production. a Representative image of flow cytometry analysis for ROS production in PC-3 cells with GFP or HectH9 knockdown. Green arrow indicates higher ROX level in HectH9-knockdown PC-3 cells. b Quantitative results for ROS production in PC-3 cells with GFP or HectH9 knockdown. Data were collected from three independent experiments. c Populations of CD44⁺/ALDH⁺ cells were determined by flow cytometry analysis in PC-3 cells with GFP or HectH9 knockdown (n = 3). d and e Tumor sphere formation assays in PC-3 (d) and HeLa (e) cells with Luciferase, HectH9 or HK2 knockdown (n = 3). Scale bar represents 100 μm. f Tumor sphere formation assay in HeLa cells infected with lentiviruses containing shRNA targeting the 3’-UTR of HectH9, followed by restoration with vector control, catalytically active form (WT) or defective mutant (C4341A) of HA-HectH9 (n = 3). Scale bar represents 100 μm. g Tumor sphere formation assay in HeLa cells infected with viruses expressing vector control or HK2 overexpression, followed by Luciferase or HectH9 knockdown (n = 3). Scale bar represents 100 μm. h Tumor sphere formation assay in PC-3 cells with Luciferase or HectH9 knockdown in the absence and presence of NAC (n = 3). Scale bar represents 100 μm. i Tumor sphere formation assay in PC-3 cells incubated with vehicle, 2-DG alone or 2-DG and BI8626 in combination for 12 days (n = 3). j and k Cell growth inhibition assay in PC-3 cells with Luciferase or HectH9 knockdown in the absence and presence of doxorubicin for 96 h (j) or paclitaxel for 48 h (k) (n = 3). Cell numbers were counted by using a hemocytometer. Results are presented as mean value ± SD; *p < 0.05, **p < 0.01, by Student’s t-test. Experiments were performed at least twice in triplicates