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. 2015 Jul 31;16(9):1145–1163. doi: 10.15252/embr.201540759

Figure 6.

Figure 6

Increased ROS production drives HIF stabilization and tumor progression in BNip3 null tumors

  1. Staining of frozen sections of wild-type and BNip3 null tumors at d35 for dihydroethidine (n = 4 for each genotype) and of FFPE sections for 8-hydroxyguanine (8-OHdG) at d80 (n = 4 for each genotype). Scale bar is 50 μm.
  2. Quantification of staining for DHE and 8-OHdG presented in (A).
  3. Flow cytometric analysis of mitochondrial ROS using MitoSOX fluorescent probe (n = 4 for each genotype).
  4. Quantification of total levels of reduced and oxidized glutathione, glycine and cysteine in wild-type and BNip3 null tumor cells (3 biological replicates and 3 experimental replicates).
  5. Effect of BHA diet supplementation on MMTV-PyMT tumor volume in wild-type (n = 12) and BNip3 null (n = 19) mice.
  6. Immunohistochemical staining for Ki67 on wild-type and BNip3 null tumors at d80 having been fed regular chow or BHA-supplemented chow from d65 (n = 4 for each genotype and treatment).
  7. Immunohistochemical staining for Hif-1α on BNip3 null tumors at d80 having been fed regular chow or BHA-supplemented chow from d65 (n = 4 for each treatment). Scale bar is 100 μm.
  8. qPCR for expression of key Hif target genes and other growth-related genes in tumors from wild-type and BNip3 null mice at d80 having been fed regular chow or BHA-supplemented chow from d65 (performed in triplicate).

Data information: Results are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.