Fig 2: HOXD9 transcription activates Snail1 expression. (A) Verifying the correlation between HOXD9 and Snail1 mRNA in 100 TNBC specimens. (B) The correlation between HOXD9 and Snail1 mRNA expression was calculated in GEPIA database. (C) HOXD9 knockdown treatment could effectively inhibit Snail1 protein expression in BT549 cells. (D) Western blot analysis of the Snail1 protein expression in HOXD9 knockdown treated BT549 cells. (E) qRT-PCR analysis of the Snail1 mRNA expression in HOXD9 knockdown treated BT549 cells. (F) ChIP experiments was used to verify the binding site between HOXD9 and the promoter region of Snail1 gene. (G) Dual luciferase experiment was employed to verify the binding site between HOXD9 and the wild-type promoter region of Snail1. (H) Western blot analysis of the Snail1 protein expression in miR-205 knockdown treatment in BT-549 cells and miR-205 overexpression treatment in MDA-MB-231 cells. (I) qRT-PCR analysis of the Snail1 mRNA expression in miR-205 knockdown treatment in BT-549 cells and miR-205 overexpression treatment in MDA-MB-231 cells. **P<0.01,***P<0.001.

Fig 3: HOXD9 regulates glioma cells proliferation, apoptosis, glucose uptake and aerobic glycolysis.HOXD9 expression in U87 cells transfected with the vector or HOXD9 overexpression. A The mRNA was quantified. B The protein was quantified. **p < 0.01, ***p < 0.001. C qRT-PCR detected the mRNA expression of PLEKHA4, ***p < 0.001. D Western Blot detected the protein expression of PLEKHA4, ***p < 0.001. E Luciferase reporter assay shows that HOXD9 binds to PLEKHA4, ***p < 0.001. The luciferase reporter plasmid containing WT or MUT-PLEKHA4 was co-transfected into U87 cells with HOXD9 overexpression or vector. F The mRNA, and G, H. The protein expression levels of HOXD9 and PLEKHA4 in U87 cells co-transfected with shPLEKHA4 and HOXD9 overexpression. ***p < 0.001 compared with Vector + shNC, #p < 0.05, ###p < 0.001 compared with Vector + shPLEKHA4 group. I CCK-8 detected the activity of the U87 cells. ***p < 0.001 compared with Vector + shNC group, ###p < 0.001 compared with Vector + shPLEKHA4 group. J, K Flow cytometry detected apoptosis. ***p < 0.001 compared with Vector + shNC group, ###p < 0.001 compared with Vector + shPLEKHA4 group. L Glucose uptake assays, M lactate production, N ATP production were measured, ***p < 0.001 compared with Vector + shNC group, ##p < 0.01, ###p < 0.001 compared with Vector + shPLEKHA4 group. The cell experiments were conducted three independent cultures, with two replicates conducted each time.