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. 2017 Jun 12;9(8):1030–1051. doi: 10.15252/emmm.201607088

Figure 7. E2F5/Drp1 complex enhances HPV‐E7/ceramide‐dependent lethal mitophagy.

Figure 7

  • A
    UM‐SCC47 cells were treated with cisplatin or C18‐pyr‐cer, and Western blotting was performed with cell lysates in the absence of reducing agents in the lysis buffer to detect monomeric and dimeric Drp1 protein abundance compared to vehicle‐treated controls. Actin was used as a loading control. Images represent three independent studies.
  • B
    Cell death was assessed by trypan blue exclusion assay in UM‐SCC47 cells expressing dominant‐negative Drp1 mutant (K38A‐DRP1) and treated with 40 μM cisplatin (left panel) or 20 μM C18‐ pyr‐cer (right panel) for 48 h compared to vector‐transfected controls. Data are means ± SD from three independent experiments, analyzed by two‐way ANOVA (left panel, n = 3, *P = 0.0048; right panel, n = 3, *P = 0.0015).
  • C
    Effects of E2F5 knockdown on Drp1 oligomerization were measured by Western blotting compared to Scr‐shRNA‐transfected cells in the absence/presence (−/+) of C18‐pyr‐cer. Actin was used as a loading control. Data represent three independent experiments.
  • D, E
    Association between E2F5 and Drp1 in response to ceramide stress (C18‐pyr‐cer at 10 μM for 1 h) was measured in UM‐SCC‐47 cells using PLA (D) or immunoprecipitation followed by Western blotting (E) with anti‐Drp1 or anti‐E2F5 antibodies (IgG was used as a control). Data are means ± SD from three independent experiments, analyzed by unpaired Student's t‐test (n = 3, *P = 0.037). In (D), PLA images were quantified using the PLA software as described by the manufacturer and analyzed by unpaired Student's t‐test (n = 3, *P = 1.3 × 10−5) and scale bars represent 100 μm.
  • F
    Effect of siRNA‐mediated knockdown of HPVE6/E7 on Drp1‐E2F5 association in the absence/presence of ceramide stress (C18‐pyr‐cer, 10 μM, 1 h) was measured by PLA using fluorescently labeled anti‐Drp1 and anti‐E2F5 antibodies in HPV(+) UPI‐SCC‐90 cells. PLA signals were quantified as described by the manufacturer using the PLA quantification software. Data are means ± SD from three independent experiments, analyzed by unpaired Student's t‐test (n = 3,*P = 0.010).
  • G
    Effects of siRNA‐mediated HPV‐E6/E7 knockdown on pRB, HPV‐E7, and p53 protein abundance were confirmed by Western blotting compared to Scr‐siRNA‐transfected controls in the absence/presence (−/+) of C18‐pyr‐cer. Total RB was used as a loading control (Rb band, upper panel).

Source data are available online for this figure.