Fig 1: Proliferation and apoptosis adaptor protein 15 (PEA15) is accountable for poor prognosis for hepatocellular carcinoma (HCC) and necessary for the proliferation and migration of HCC cells. (A) Box plot of PEA15 protein expression in the 3 subtypes of HCC and surrounding normal tissue (N, Student’s t test). The horizontal black line within each box indicates the mean, the boundaries of each box indicate the 25th to 75th percentiles, and the whiskers above and below each box indicate the 10th and 90th percentiles. (B) Kaplan–Meier plot of overall survival (OS) in HCC patients in the MD Anderson Cancer Center (MDACC) cohort. The patients were stratified according to the expression of PEA15 protein (median cutoff; log-rank test). (C and D) Cell viability assay results for HCC cells with depletion or overexpression of PEA15. Depletion of PEA15 with shRNA specific to PEA15 (shPEA15-1 and shPEA15-2) significantly reduced the proliferation of mesenchymal cells (C). Expression of exogenous PEA15 significantly increased the proliferation of epithelial cells (D) (Student’s t test). (E and F) Cell migration assay results for HCC cells with depletion or overexpression of PEA15. Depletion of PEA15 with shPEA15 significantly reduced the migration of HLF cells (E). Expression of exogenous PEA15 significantly increased the migration of HepG2 cells (F). The whiskers indicate standard error. **P < 0.01, ***P < 0.001. MES, mesenchymal subtype; MA, metabolically active subtype; GS, genome-stable subtype; N, adjacent normal liver tissues; shGFP, shRNA specific to green fluorescent protein; shPEA15, short hairpin RNA for PEA15; PEA15-OE, PEA15 overexpression.
Fig 2: Therapeutic inhibition of proliferation and apoptosis adaptor protein 15 (PEA15) decreases tumor burden in hepatocellular carcinoma (HCC). (A) Cell viability assay results for HLF cells upon indicated antisense oligonucleotide (ASO) treatments (5 μM). HLF cells were treated with the indicated ASOs under free-transfection reagents. Expression of PEA15 was measured by Western blotting (WB) after 72 h of ASO treatment. (B) Weights of xenografted HLF tumors in mice. At 6 weeks after xenografting, the mice were euthanized, and their tumors were weighed (n = 8 to 9 per group). All data are presented as means ± standard error of the mean (SEM) (Student’s t test). (C) Growth of xenografted HLF tumors upon treatment of the ASOs. HLF cells were xenografted subcutaneously in the flanks of mice. After 14 d, HLF tumor-bearing mice were treated with the indicated ASO or vehicle control (50 mg/kg) 3 times a week, and the tumor volumes were measured at the indicated time points for 6 weeks. (D) Representative images of xenografted HLF tumors from each group at week 6. (E) WB analysis of the xenografted HLF tumors treated with control and PEA15-ASOs. ***P < 0.001 (Student’s t test).
Fig 3: Proliferation and apoptosis adaptor protein 15 (PEA15) regulates hypoxia-inducible factor 1A (HIF1A) by down-regulating von Hippel–Lindau tumor suppressor (VHL) in hepatocellular carcinoma (HCC) cells. (A) Expression of 524 genes was significantly altered after depletion of PEA15 in FOCUS cells (P < 0.001). PEA15 was depleted 3 times. Data are presented in matrix format; each row represents an individual gene, and each column represents one sample. Each cell in the matrix represents the expression level of a gene feature in an individual tissue sample. Red and green indicate relatively high and low expression levels, respectively, as indicated in the log2-transformed scale bar. (B) Network analysis of PEA15-dependent genes revealed that many genes are downstream targets of HIF1A. The genes were color-coded according to the ratio of the expression of genes in cells treated with shGFP (control) to the expression of genes in cells treated with shPEA15. Red represents higher expression, and green represents lower expression in PEA15-active (control shGFP) cells. (C) Western blotting (WB) of PEA15 and HIF1A expression in PEA15-depleted (treated with shPEA15#1, shPEA15#2, and shPEA15#3) and control (treated with shGFP) HLF and SNU-449 cells. (D) WB of PEA15 and HIF1A expression in HepG2 cells with ectopic PEA15 overexpression. The numbers in parentheses indicate replicated experiments. (E) Schematic of HIF1A regulation by PHD, factor inhibiting HIF-1 (FIH1), and VHL. (F) WB of VHL, prolyl hydroxylase domain containing protein 2 (PHD2), and FIH1 expression in HepG2 and PLC/PRF/5 cells with ectopic PEA15 expression. PEA15 substantially reduced VHL expression in both cell lines. (G) WB of VHL and HIF1A expression in PEA15-depleted SNU-449 cells. VHL stability was substantially increased by silencing PEA15 expression. (H) WB of VHL and PEA15 expression after treatment with the proteinase inhibitor MG132 and the lysosomal inhibitor chloroquine (CQ) in HepG2 and PLC/PRF/5 cells. VHL degradation was reversed by MG132 but remained unaffected by CQ. (I) WB of endogenous VHL expression after treatment with cycloheximide (CHX) in PLC/PRF/5 cells with or without PEA15 expression. shPEA15, short hairpin RNA for PEA15; PEA15-OE, PEA15 overexpression.
Fig 4: Schematic diagram of proliferation and apoptosis adaptor protein 15 (PEA15)-mediated regulation of von Hippel–Lindau tumor suppressor (VHL) and hypoxia-inducible factor 1A (HIF1A) stability in hepatocellular carcinoma (HCC) cells. PEA15 binds to VHL to inhibit the interaction between VHL and HIF1A and disrupt the stability of the VCB–CUL2 complex. B, elongin B; C, elongin C; VCB, VHL–elongin C–elongin B; HRE, hypoxia response element.
Supplier Page from Abcam for Recombinant Human PEA15 protein (Tag Free)