Fig 1: Negative modulation of HDGF by RXRa. A Schematic diagram of the putative RXRa binding site in HDGF promoter. B, C RXRa knockdown inhibited HDGF mRNA (B) and protein (C) in MCF7 and MDA-MB-231 cells. D Quantification of HDGF protein in (C). E ChIP-qPCR assay of RXRa binding with the HDGF promoter in MDA-MB-231 cells. F Luciferase assay of RXRa inhibiting HDGF promoter activity in MDA-MB-231 cells. G Effects of 9-cis-retinoic acid (20umol/L) with or without HDGF overexpression co-transfection into MDA-MB-231 cells. H Quantification of HDGF protein in (G). I–K HDGF rescued 9-cis-retinoic acid (20umol/L)-inhibited cell proliferation (I, J) and surviving fractions (K) after irradiation. As shown in B, D, E, F, G, H, I, J, and K, Error bars ± S.D. *P < 0.05. **P < 0.01. ***P < 0.001. Data are representative of three independent experiments
Fig 2: Inhibition of breast cancer radioresistance by HDGF depletion combined with STAT3 activity inhibition. A, C, D Effects of Stattic (20 μmol/L) with or without HDGF knockdown on STAT3 phosphorylation level (A), cell proliferation (C), and surviving fractions (D) after irradiation. B Quantification of protein in (A). E Representative bioluminescence images of Stattic (20 μmol/L) or Stattic (20 μmol/L) combined with HDGF knockdown -transfected MDA-MB-231 cells injected into the mice mammary gland fat pads. Mice were imaged 4 weeks after transplantation. Data were from two independent experiments with 5 mice per group with similar results. F Quantification of bioluminescence activity in (E). G the working model showing that RXRα binds with HDGF promoter and suppresses HDGF transcriptional activity. HDGF could associate with TKT and STAT3, which promotes STAT3 phosphorylation and transcriptional activity, thereby exerts tumor radioresistance in breast cancer. As shown in B, C, D, and F Error bars ± S.D. *P < 0.05. ***P < 0.001. Data are representative of three independent experiments
Fig 3: The interaction of HDGF with STAT3 promotes its transcription activity. A Immunoprecipitation followed by silver staining of the HDGF binding proteins. B, C Immunoprecipitation and western blotting for HDGF association with STAT3 protein in HEK-293 T cells. D HDGF interacts with STAT3 in MDA-MB-231 cells. E Luciferase assay detected STAT3 downstream genes. F HDGF knockdown lowers the mRNA levels of STAT3 downstream genes in MDA-MB-231 cells. G Effects of STAT3–Tyr705 and STAT3–Ser727 phosphorylation levels in HDGF knockdown breast cancer cells. H, Quantification of protein in (G). I Effects of the cellular distribution of the phosphorylated STAT3–Y705 and STAT3–S727 in HDGF knockdown breast cancer cells. J Quantification of protein in (I). K, L Cell proliferation (K) and surviving fractions (L) for MDA-MB-231 cells transfected with wild–type STAT3, the STAT3–S727D mutant or control after irradiation. As shown in E, F, H, J, K, and L, Error bars ± S.D. *P < 0.05. ***P < 0.001. Data are representative of three independent experiments
Fig 4: HDGF-mediated STAT3 phosphorylation in combination with transketolase. A Effects of STAT3–Tyr705 and STAT3–Ser727 phosphorylation levels in TKT knockdown breast cancer cells. B Quantification of protein in (A). C Effects of TKT rescue HDGF knockdown on STAT3–Tyr705 and STAT3–Ser727 phosphorylation levels. D Quantification of protein in (C). E, F TKT overexpression rescue HDGF knockdown-inhibited cell proliferation (E) and surviving fractions (F) after irradiation. G HDGF knockdown inhibits TKT association with STAT3. H TKT knockdown inhibits HDGF association with STAT3. As shown in B, D, E, and F Error bars ± S.D. *P < 0.05. ***P < 0.001. Data are representative of three independent experiments
Fig 5: HDGF overexpression in breast cancer and its negative correlation with the survival prognosis of patients. A HDGF copy number analysis in TCGA breast cancer dataset. Data were downloaded from http://xena.ucsc.edu/public-hubs. B HDGF expression is higher in breast cancer tissues than that in adjacent normal tissues. Expression data of HDGF were downloaded from the REMBRANDT dataset. Data are presented as mean ± SEM.***P < 0.001, by two-tailed t-test. C, D HDGF expression is negatively correlated with patients' disease-free survival (C) and distance metastasis-free survival (D). Data were retrieved from Kaplan–Meier Plotter (http:// kmplot.com/ analysis/index). E HDGF protein is upregulated in breast cancer (MCF7, MDA-MB-231, BT549, and MDA-MB-453) compared with that in the normal MCF10A cells. F Quantification of HDGF protein in E. G, H HDGF is upregulated in breast cancer I.R. cell lines at the mRNA (G) and protein level (H). I Quantification of HDGF protein in (H). As shown in B, F, G, and I, Error bars ± S.D. *P < 0.05. ***P < 0.001. Data are representative of three independent experiments
Supplier Page from Abcam for Anti-HDGF antibody