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. 2021 May 1;40(10):e106214. doi: 10.15252/embj.2020106214

Figure 5. BNIP3 stabilizes normoxic HIF‐1α on a post‐translational level in melanoma.

Figure 5

  1. Immunoblot detection of PHD2 and ACTIN protein levels from lysates of normoxic cultures (n = 4) or B16‐F10 tumors [CntlKD (n = 5), BNIP3KD (n = 7), and ATG5KD (n = 6)]. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. In vitro data were analyzed using a one‐sample t‐test against shCntl whereas the in vivo data were analyzed using a one‐way ANOVA with Tukey’s multiple comparisons test.
  2. Immunoblot detection of Hydroxylated HIF‐1α (HIF‐1α‐OH), total HIF‐1α, LDHA, and ACTIN protein levels from normoxic lysates of B16‐F10 cells cultured alone or in the presence of the PHD2 inhibitor IOX2 (100 µM) for 24 h.
  3. Glut1, Pdk1, Mct4, and Vegfa transcript levels from B16‐F10 cells cultured alone or in the presence of the PHD2 inhibitor IOX2 (100 µM) for 24 h (n = 4). Data were analyzed using a one‐sample t‐test against shCntl, except for Pdk1 shBNIP3 IOX2‐ which was analyzed using the non‐parametric Wilcoxon test.
  4. Immunoblot detection of Hydroxylated HIF‐1α (HIF‐1α‐OH), total HIF‐1α, LDHA, and ACTIN protein levels from normoxic lysates of B16‐F10 cells cultured alone or in the presence of the iron chelator DFO (200 µM) for 24 h.
  5. Immunoblot detection of Hydroxylated HIF‐1α (HIF‐1α‐OH), total HIF‐1α, LDHA, PHD2, and ACTIN protein levels from normoxic lysates of B16‐F10 cells transfected in presence of non‐targeting siRNA sequences (Scr) or siRNA against PHD2.

Data information: All quantitative data are mean ± SEM. Densitometric quantifications of protein levels relative to ACTIN for (B, D, E) are shown in Appendix Table S2. *P < 0.05, **P < 0.01, ***P < 0.001 when compared against shCntl. (B, D, E) provide a representative blot from n = 3 biologically independent experiments.