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. 2021 Nov 15;10:e70412. doi: 10.7554/eLife.70412

Figure 4. ZHX2 regulates hypoxia-inducible factor (HIF) signaling in triple-negative breast cancer (TNBC).

(A, B) Venn diagrams showing the overlap in downregulated (A) or upregulated (B) differentially expressed genes (DEGs) between ZHX2 shRNA 43 and 45. (C) Pathway analysis of the significantly decreased pathways in ZHX2 depleted MDA-MB-231 cells. (D, E) Venn diagrams showing overlap in DEGs (D) and downregulated DEGs (E) between ZHX2 depletion and HIF double knockout (DKO) (GSE108833). (F, G) Immunoprecipitations (IP) and immunoblots with indicated antibodies of MDA-MB-231 cells overexpress either HA-ZHX2 (F) or HA-HIF1α (G). IP was performed with HA beads. (H) IPs of MDA-MB-231 cells treated with DMOG for 8 hr. (I, J) Immunoblots of cell lysates from MDA-MB-231 cells (I) and MDA-MB-468 cells (J) infected with lentivirus encoding ZHX2 shRNAs or Ctrl, followed by treating with normoxia or hypoxia (1% O2). (K–M) Immunoblots (K) and IPs (L) of cell lysates, qRT-PCR of mRNA (M) from MDA-MB-231 cells infected with lentivirus encoding control vector (EV) or ZHX2. (N) Immunoblots of cell lysates from MDA-MB-231 cells infected with lentivirus encoding ZHX2 shRNAs or Ctrl treated with MG132 overnight under hypoxia (1% O2). (O) IPs of cell lysates from MDA-MB-231 cells infected with lentivirus encoding control vector (EV) or HA-ZHX2. Error bars represent mean ± standard error of the mean (SEM), unpaired t-test. n.s., not significant.

Figure 4—source data 1. Uncropped western blot images for Figure 4.

Figure 4.

Figure 4—figure supplement 1. ZHX2 does not affect p65 translocation in triple-negative breast cancer (TNBC).

Figure 4—figure supplement 1.

(A, B) Immunoprecipitations and immunoblots of MDA-MB-231 and 468 cell lysates by ZHX2 antibody (A) or p65 antibody (B). (C) Immunoblot of cell lysates from inducible ZHX2 knockdown MDA-MB-231 cells treated with or without doxycycline (Dox). (D–E) Immunoblot of cell fractions from MDA-MB-231 (D) or MDA-MB-468 (E) infected with ZHX2 shRNA (Ctrl, sh43, or sh45).
Figure 4—figure supplement 1—source data 1. Uncropped western blot images for Figure 4—figure supplement 1.
Figure 4—figure supplement 2. ZHX2 regulates hypoxia-inducible factor (HIF)1 signaling in triple-negative breast cancer (TNBC).

Figure 4—figure supplement 2.

(A) Gene set enrichment analysis (GSEA) of the significantly decreased pathways in ZHX2 depleted MDA-MB-231 cells. (B, C) Plot (B) and related heatmap (C) from GSEA analysis suggest hypoxia pathway is significantly downregulated in ZHX2 depleted MDA-MB-231 cells. (D) qRT-PCR quantification of relative mRNA expression of hypoxia target genes from MDA-MB-231 cells infected with ZHX2 shRNA 43, 45, or Ctrl under normoxia or hypoxia conditions. (E) Hypoxia response element (HRE) double luciferase gene assay of MDA-MB-231 cells infected with ZHX2 sh45, sh45-resistant (ZHX2res), or Ctrl under normoxia or hypoxia condition. (F) HRE double luciferase gene assay of HEK293T cells infected with different amount of HA-ZHX2 (100, 200, and 400 ng), HRE-Luci, and pCMV-Renilla. (G) Immunoprecipitations and immunoblots with indicated antibodies of MDA-MB-231 cells overexpressing HA-ZHX2 and HA-HIF1α. (H) Immunoblot of MDA-MB-231 cells infected with lentivirus encoding either HIF1A shRNA 3809, 3810, or Ctrl. Error bars represent mean ± standard error of the mean (SEM), unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4—figure supplement 2—source data 1. Uncropped western blot images for Figure 4—figure supplement 2.