UBC9 is degraded by autophagy in epithelial cells.

<p><b>(A)</b> Immunolocalization of UBC9 in ultrathin sections of HKs transduced with empty or HPV16 E6/E7 vectors. Original view (left) and higher magnification (right) of boxed regions. Gold particles are selectively enriched in autophagic structures highlighted by arrows. Scale bar = 1 μm. <b>(B)</b> Top: Representative WB of HaCaT cells treated with the indicated autophagic activators (left) and inhibitors (right). Activation of autophagy was monitored by the conversion of LC3 (LC3-I) to the lipidated LC3 (LC3-II) form, a marker of autophagosome production induced by autophagic stimuli [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006262#ppat.1006262.ref041" target="_blank">41</a>]. LC3-II accumulation was used to verify autophagic impairment [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006262#ppat.1006262.ref041" target="_blank">41</a>]. Bottom: normalized UBC9 expression. Data are expressed as fold over untreated cells. Bars represent means ± SEM of n = 4 different biological replicates. ns: not significant (Kruskal–Wallis one-way ANOVA with Dunn’s post hoc test) compared to vehicle control groups. (<b>C)</b> Representative WB analysis of U-2 OS or MCF7 cells treated with chloroquine. n = 3 different biological replicates. <b>(D)</b> Representative WB analysis of MCF7 cells transduced with scramble or ATG5 shRNA. The observed ATG5 band represents the ATG5-ATG12 conjugated form. LC3-I accumulation is reported to evidence autophagic deficiencies promoted by shATG5. n = 3 replicates of a single transduction.