Fig 1: Loss of HOXA9 leads to enhanced glycolysis and tumor growth in vivo. siNC and siHOXA9 oligos were injected into A431 cell xenografts every 3 days. a Loss of HOXA9 promotes subcutaneous tumor growth in a mouse xenograft model. Tumor volumes (mm3) were plotted according to day. Tumor volume statistical data represent the average of four independent experiments ± s.d, respectively. b The mice were sacrificed at the end of the experiment and images taken along with the dissected tumors from five representative mice are shown. White arrows indicate the siNC-treated xenografts whereas black arrows indicate siHOXA9-treated xenografts. Scale bar, 1 cm. c The expression of HOXA9, HIF1A, HK2, GLUT1, and PDK1 was measured in the dissected tumors by qRT-PCR. qRT-PCR statistical data represent the average of four independent experiments ± s.d. d The protein expression of HOXA9, HIF-1α, HK2, GLUT1, and PDK1 was detected in xenografts after siHOXA9 treatment by western blot. e Histopathology analysis (IHC staining) of HOXA9, HIF-1α, HK2, GLUT1, and PDK1 on tumor sections. HOXA9 pre-absorption tests was also performed to validate the specificity of HOXA9 antibody. Scale bar, 100 µm (200×). f Comparison of glucose consumption between siHOXA9-treated and siNC-treated xenograft tumors by microPET/CT imaging of the uptake and retention of 18F-FDG injected via the tail vein. A representative microPET/CT image is shown. g A model of the miR-365-HOXA9-HIF-1α glycolysis-regulatory axis in cSCC development. In cSCC tumors, loss of HOXA9 up-regulates HIF-1α and its downstream glycolytic genes of HK2, GLUT1, and PDK1 in the HIF-1 pathway, which contributes to the enhancement of glycolysis and promotes cSCC progression. In normal skin or HOXA9-treated cSCC, HOXA9 interacts with CRIP2 and epigenetically represses HIF-1α expression, which leads to the replacement of HIF-1α by the HOXA9-CRIP2 complex at the promoter regions and represses the expression of glycolytic genes including HK2, GLUT1, and PDK1, which subsequently contributes to the inhibition of tumor progression. Each experiment was performed in triplicate and data are presented as mean ± s.d. One-Way ANOVA and Dunnett’s multiple comparison test were used to analyze the data (*P < 0.05, **P < 0.01, ***P < 0.001)
Fig 2: HOXA9 represses glycolytic genes by association with CRIP2. a The chromatographic analysis of the protein mixes immunoprecipitated by HOXA9 antibody or control IgG. Arrows indicate the identified CRIP2 peptide peaks in the HOXA9-pulldown sample, which were lacking in control IgG sample. b CRIP2 was verified to be the interacting partner of HOXA9 by Co-IP followed with western blot detection. IP immunoprecipitation, WB western blot. c Co-IP of both HOXA9 and CRIP2 using specific antibodies was performed and followed with western blot detection to confirm their direct interactions. Each experiment was performed at least in triplicate
Fig 3: The glycolysis-inhibitory role of HOXA9 is dependent on CRIP2. a Western blot detection showed that CRIP2 knockdown led to significant upregulation of HIF-1α, HK2, GLUT1, and PDK1, whereas HOXA9 expression was not affected. b–d Depletion of CRIP2 enhanced cell proliferation, migration, and invasiveness of A431 cells by CCK-8 assay, transwell migration assay, and Matrigel invasiveness measurement. e, f ECAR and OCR analysis of A431 cells following depletion of CRIP2 by siRNA as summarized from raw data. g Western blot detection showing the variations of protein expression levels of HOXA9, CRIP2, HIF-1α, HK2, GLUT1, and PDK1 in response to CRIP2 knockdown and/or HOXA9 overexpression. h, i ECAR and OCR analysis of A431 cells depleted of CRIP2 by siRNA and/or overexpressing HOXA9 as summarized from raw data. Each experiment was performed in triplicate and data are presented as mean ± s.d. One-Way ANOVA and Dunnett’s multiple comparison test were used to analyze the data (*P < 0.05, **P < 0.01, ***P < 0.001)
Supplier Page from Abcam for Anti-CRP-2 antibody