Fig. 6.

UCHL3 increases AhR protein stability in a DUB activity-dependent manner. a UCHL3 stable knockdown by shRNA#1 and shRNA#2 in H358 cells induced changes in AhR and c-Myc protein expression that were rescued by transient overexpression of UCHL3 but not UCHL3-C95S and UCHL3-D33A. b The MTS assay was used to assess cell viability in A549 cells stably overexpressing UCHL3 and UCHL3 point mutants(n = 5). Data are shown as the mean ± SD; ****p < 0.0001. c Colony formation assays were performed to detect the colony formation ability of A549 cells stably overexpressing UCHL3 and UCHL3 point mutants (n = 3); representative images are shown in the Supplementary section. Data are shown as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. d Flow cytometry analysis showing side populations among A549 cells stably overexpressing UCHL3 and UCHL3 point mutants, with the results shown as a bar graph (n = 3). Data are shown as the mean ± SD; ***p < 0.001, ****p < 0.0001. e A549 cells stably overexpressing UCHL3 and UCHL3 point mutants were seeded in ultralow attachment dishes to allow tumor sphere formation, and the results are shown as a bar graph (n=5, scale bar = 100 μm). Data are shown as the mean ± SD; ****p < 0.0001. f UCHL3 decreased AhR ubiquitination in HEK293T cells. AhR-Flag and Ub-His were coexpressed with vector, UCHL3, UCHL3-C95S, UCHL3-D33A or UCHL3-G96D in HEK293T cells. Cell lysates were harvested after 72 h, AhR proteins were immunoprecipitated with anti-AhR antibody, and polyubiquitinated AhR proteins were detected by WB using anti-Ub antibody. Data in all bar graphs were assessed by one-way ANOVA with multiple comparisons