Fig. 7
OTUD7B maintains NPCs stemness. a Immunoblotting of Sox2 in MEF with OTUD7B knockout and WT control. b The MEF cells were treated with CHX (10 µg/ml), and collected at the indicated times for western blot. Quantification of Sox2 levels relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. **P < 0.01, two-way ANOVA test. c Sox2 ubiquitylation in MEF with OTUD7B knockout and WT control. MEFs were treated with MG132 for 8 h and the lysates were subjected to immunoprecipitation with anti-Sox2 and immunoblotted with anti-Ub. d NPCs were plated in Matrigel-coated six-well plate for neuronal differentiation. For the indicated times, fixed and stained with anti-OTUD7B (green). Nuclei were counterstained with HO (blue). e, f NPCs were infected with lentiviruses expressing OTUD7B shRNA or negative control (NC) shRNA for 126 h. Immunoblotting (e) and immunostaining (f) were used to detect the level of Sox2, TUJ1, or Nestin in NPCs. Scale bar, 50 µm. g NPCs with stable OTUD7B knockdown and Sox2-AA overexpression were generated with lentivirus infection. Immunostaining were performed for Sox2, TUJ1. Scale bar, 50 µm. The ratio of TUJ1-positive or Sox2-positive cells were quantified. Results are shown as mean ± s.d. Each error bar shows the standard deviation of numbers of positive cells in 10 fields of view. *P < 0.05. Student’s t-test. h NPCs with stable OTUD7B overexpression and Sox2 knockdown were generated with lentivirus infection. Cas9-based activator of OTUD7B (SAM-OTUD7B) were used to activate OTUD7B expression in NPCs. Immunostaining were performed for Sox2, TUJ1. Scale bar, 50 µm. The ratio of TUJ1-positive or Sox2-positive cells were quantified. Results are shown as mean ± s.d. Each error bar shows the standard deviation of numbers of positive cells in 10 fields of view. *P < 0.05. Student’s t-test. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
