PMC

(A–B) IB of lysates from paired ccRCC patient non-tumor (N) and tumor (T) tissues.
(C) Representative ZHX2 immunohistochemistry staining for ccRCC patient tissues.
(D-E) Representative H&E, ZHX2 immunohistochemistry staining of tumor (T) and non-tumor (N) tissues (D) and quantification of ZHX2 nuclear/cytoplasmic staining ratio (E) from ccRCC TMA slides. Error bars represent SEM (unpaired t-test).

(A) Schematic representation of VHL substrate screen.
(B-C) Binding assays of ³⁵S-Methionine labelled in vitro translated cDNA pools (B) or ZHX2 (C) and GST-VBC in the presence of wildtype (WT) or prolyl hydroxylated (p-OH) HIF peptide.
(D) ZHX2/HIF2α binding to GST-VBC in the presence of prolyl hydroxylase inhibitors.
(E-H) Immunoblots (IB) of whole cell extracts (WCE) and immunoprecipitations (IP) of lysates from 786-O cells infected with lentivirus encoding either control vector (Ctrl) or hemagglutinin (HA) tagged VHL and treated as indicated for 8 h.
(I) IB of lysates from UMRC2, UMRC6, or RCC4 cells transfected with indicated plasmids.
(J-K) IB of WCE and IP of RCC4 cells transfected with indicated plasmids followed by densitometry analysis of Flag-VHL (J) or ubiquitination assays in UMRC2 cells transfected with indicated plasmids (K).

(A-B) qRT-PCR quantification of mRNA of NF-κB target genes (A, N=3) or IB of cell fractions (B) from 786-O cells infected with ZHX2 shRNAs (43, 45) or Ctrl.
(C) IB of WCE and IP of 786-O cells infected with either Ctrl or HA-VHL.
(D) Venn diagram showing ChIP-Seq binding peak overlap between ZHX2 and NF-κB–p65. ZHX2 ChIP-seq experiments were performed in duplicate and intersected.
(E) ChIP-seq signal intensity in the 3 kb surrounding the midpoint of unique ZHX2 (green), unique NF-κB–p65 (yellow), and common (purple) sites.
(F) Heatmap for genes downregulated due to ZHX2 and p65 silencing (adj. P < 0.05) are shown.
(G) Heatmap for activated genes that were strongly bound by both ZHX2 and NF-κB–p65 and were significantly associated with ccRCC prognosis (q < 0.01). The log2 Cox Hazard Ratio was colored red (higher expression associated with poorer prognosis).
(H) ChIP-qPCR of NF-κB–p65 binding at IL6 and IKBKE promoters following silencing of indicated genes (N=3).
Error bars represent SEM, ***P<0.001 (unpaired t-test).

(A-F) IB of cell lysates (A, C), cell proliferation (B, D) and soft agar growth (E, F) of 786-O and UMRC2 cells infected with lentivirus encoding control (Ctrl) or ZHX2 shRNAs (43, 45) (N=3). See for soft agar quantitation results.
(G-I) IB of cell lysates (G) and representative soft agar growth assays (H) and their quantification (I) of UMRC2 cells transfected with ZHX2 sh45-resistant HA-ZHX2 or control (Ctrl) vector, followed by ZHX2 sh45 or control (Ctrl) shRNA infection (N=3).
(J-M) Representative bioluminescence imagings of 1 and 7 weeks post-implantation (J) and quantification of bioluminescence imaging (K) from 786-O cells luciferase stable cells infected with either ZHX2 sh45 or control (Ctrl) shRNA, or imagings of 0 week and 6 weeks post-doxycycline treatment (L) and quantification of imaging (M) from 786-O luciferase stable cells infected with lentivirus encoding either Teton-ZHX2 sh45 or Teton-control (Teton-Ctrl) shRNA injected orthotopically into the renal sub-capsule of NOD scid gamma (NSG) mice as indicated. The Mann-Whitney test was used to calculate the p values.
Error bars represent SEM, ***P<0.001 (unpaired t-test) in panel B, D and I.