Fig 1: HOXB5 transcription activates ANGPT2. (A) JASPAR-predicted HOXB5 binding motif. (B) HOXB5 was upregulated in patients with esophageal cancer. *P<0.05. (C) HOXB5 upregulation was not significantly associated with low overall survival rate of patients with esophageal cancer. (D) Relative mRNA expression of HOXB5 was detected using RT-qPCR. (E) Protein expression of HOXB5 was detected using western blot analysis. ***P<0.001 vs. HEEC. Relative (F) mRNA and (G) protein expression levels of HOXB5 in HOXB5-overexpressing EC-9706 cells were detected using RT-qPCR and western blot analysis. ***P<0.001 vs. pcDNA3.1. (H) Relative luciferase activity was detected using luciferase reporter assay. ***P<0.001 vs. ANGPT2-WT + pcDNA3.1. (I) Relative enrichment of ANGPT2 was detected using chromatin immunoprecipitation. ***P<0.001 vs. input; ###P<0.001 vs. IgG. HOXB5, homeobox B5; ANGPT2, angiopoietin-2; RT-qPCR, reverse transcription-quantitative PCR; ESCA, esophageal carcinoma; WT, wild type; MUT, mutated; TPM, transcripts per million.
Fig 2: Overexpression of transcription factor HOXB5 reverses the inhibitory effects of ANGPT2 silencing on the metastasis and angiogenesis of EC-9706 esophageal cancer cells. (A) Wound healing assay was used to detect (B) cell migration (scale bar, 100 µm). (C) Transwell assay was used to determine (D) cell invasion (scale bar, 50 µm). (E) Expression levels of epithelial-mesenchymal transition-associated proteins were detected using western blot analysis. (F) Angiogenic effect measured using tube formation analysis (scale bar, 250 µm). ***P<0.001 vs. shRNA-NC, #P<0.05, ##P<0.01 and ###P<0.001 vs. shRNA-ANGPT2 + pcDNA3.1. HOXB5, homeobox B5; ANGPT2, angiopoietin-2; shRNA, short harpin RNA; NC, negative control.
Fig 3: Overexpression of transcription factor HOXB5 reverses the inhibitory effects of ANGPT2 silencing on the ERK/AKT signaling pathway. Expression levels of signaling pathway-associated proteins were detected using western blot analysis. ***P<0.001 vs. shRNA-NC. ###P<0.001 vs. shRNA-ANGPT2 + pcDNA3.1. HOXB5, homeobox B5; ANGPT2, angiopoietin-2; sh, short hairpin; NC, negative control; p-, phosphorylated.
Fig 4: Overexpression of transcription factor HOXB5 reverses the inhibitory effects of ANGPT2 silencing on proliferation of EC-9706 esophageal cancer cells. (A) Relative mRNA and protein expression levels of ANGPT2 were detected using reverse transcription-quantitative PCR and western blot analysis. (B) Viability of EC-9706 cells was detected using Cell Counting Kit-8. **P<0.01 and ***P<0.001 vs. shRNA-NC. ##P<0.01 and ###P<0.001 vs. shRNA-ANGPT2 + pcDNA3.1. (C) Proliferation was detected using EdU staining (scale bar, 50 µm). (D) Colony forming ability was detected using colony formation assay (scale bar, 34 mm). HOXB5, homeobox B5; ANGPT2, angiopoietin-2; shRNA, short harpin RNA; OD, optical density; NC, negative control.
Supplier Page from Abcam for Anti-HOXB5 antibody