HPV‐E7/RB axis regulates ceramide‐dependent mitophagy via E2F5 activation HPV‐E7/RB axis regulates ceramide‐dependent mitophagy via E2F5 activation AUM‐SCC‐47.

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HPV‐E7/RB axis regulates ceramide‐dependent mitophagy via E2F5 activation HPV‐E7/RB axis regulates ceramide‐dependent mitophagy via E2F5 activation AUM‐SCC‐47 [HPV(+)] cell death was measured using trypan blue exclusion assay (trypan blue‐positive cells) following shRNA‐mediated knockdown of E2F1, E2F4, or E2F5 in response to C18‐pyr‐cer (20 μM, 48 h) compared to Scr‐shRNA‐transfected and/or vehicle‐treated controls. Efficiency of shRNA‐mediated knockdown of E2F1, E2F4, or E2F5 mRNAs was measured by RT–PCR (normalized to actin mRNA) compared to Scr‐shRNA‐transfected controls (right panel). Data are means ± SD from three independent experiments, analyzed by unpaired Student's t‐test (n = 3, left panel *P = 0.00025, **P = 0.00018, ***P = 5.4 × 10−5; right panel *P = 0.0156, **P = 0.0147. ***P = 0.00118).BEffects of siRNA‐mediated knockdown of HPVE6/E7 on endogenous RB‐E2F5 association in UM‐SCC‐47 cells were measured by PLA using labeled anti‐RB and anti‐E2F5 antibodies using immunofluorescence (scale bars represent 100 μm). Quantification of PLA signals in images was performed as described by the manufacturer using the PLA software. Data are means ± SD from three independent experiments, analyzed by unpaired Student's t‐test (n = 3, *P = 0.040).CEffects of shRNA‐mediated E2F5 knockdown on mitophagy in response to vehicle versus C18‐pyr‐cer (20 μM, 1 h) were measured by live cell imaging confocal micrographs of UM‐SCC‐47 cells stained with LTG and MTR (scale bars represent 100 μm). Images were quantified using ImageJ, and data are means ± SD from three independent experiments, analyzed by unpaired Student's t‐test (*P = 0.023).D–ECell death (trypan blue‐positive cells) or mitophagy was measured in response to ectopic expression of E2F5 in the presence/absence of C18‐pyr‐cer (20 μM, 48 h) by trypan blue exclusion assay (D) or by live cell imaging (1 h) confocal micrographs of UM‐SCC‐22A cells stained with LTG and MTR (E). Vector‐transfected cells were used as controls. Images were quantified using ImageJ (scale bars represent 100 μm), and data are means ± SD from three independent experiments, analyzed by unpaired Student's t‐test (n = 3, *P = 0.0084, **P = 0.0012). Protein abundance of E2F5 after transient transfections was measured by Western blotting. Actin was used as a loading control (D, lower panel). Source data are available online for this figure. Raquela J Thomas et al. EMBO Mol Med. 2017;emmm.201607088 © as stated in the article, figure or figure legend