Fig 2: Inhibition of cisplatin-induced apoptotic cell death by HOXB9 overexpression: The SKOV3 and HOXB9 OE/SKOV3 cells were treated with the indicated doses of cisplatin for 48 h, and then, (A) cell viability and (B) cell proliferation were measured by a Presto blue assay and a CCK-8 assay, respectively; (C) Western blot analysis of HOXB9, MMP-2, Bcl-2, cleaved caspase-3, cleaved PARP and α-tubulin in SKOV3 and HOXB9 OE/SKOV3 cells following cisplatin treatment; (D) Apoptosis and (E) Caspase 3 activity analysis of SKOV3 and HOXB9 OE/SKOV3 cells following cisplatin treatment. Each experiment was independently performed in triplicate: results are expressed as mean ± standard deviation. The two-tailed Student t test was used to analyze the differences between the groups. (** p < 0.01, *** p < 0.001). Bcl-2, B-cell lymphoma-2; MMP-2, matrix metalloproteinase-2.

Fig 3: Promotion of tumor growth by HOXB9 overexpression in mouse xenograft models: (A) Growth rate of tumors in SKOV3- or HOXB9 OE/SKOV3-injected mouse models. The tumor volume is measured for 4 weeks after the implantation of SKOV3 and HOXB9 OE/SKOV3 cells. The two-tailed Student t test was used to analyze the differences between the groups (* p < 0.05, ** p < 0.01, F value = 3.369, SKOV3-injected vs. HOXB9 OE/SKOV3-injected group, N = 6); (B) Mean tumor weight (# control, * p < 0.05, F value = 1.080); (C) Tumor volume (F value = 1.685) of isolated tumors from mice (# control, *** p < 0.001, SKOV3-injected vs. HOXB9 OE/SKOV3-injected group); (D) Western blot analysis of HOXB9, MMP-2, MMP-9, E-Cadherin, Bcl-2, Survivin, Bax, Cleaved Caspase-9, Cleaved Caspase-3, and Cleaved PARP protein expression levels in isolated the tumors (SKOV3- or HOXB9 OE/SKOV3-injected tumor) (* p < 0.05); (E) Gross tumors of the representative mice of each group before harvest and immunohistochemical staining analysis of HOXB9 in the tumors of SKOV3 or HOXB9 OE/SKOV3 cells (magnification: 400×; scale bar = 20 μm). Each experiment was independently performed in triplicate. Bax, BCL2-associated X protein; Bcl-2, B-cell lymphoma 2; MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; PARP, Poly (ADP-ribose) polymerase.

Fig 4: Effect of HOXB9 overexpression in ovarian cancer cells: The SKOV3 and HOXB9 OE/SKOV3 cells were cultured for 1–5 days in 96-well plate, and then, (A) cell viability and (B) cell proliferation were determined by Presto blue assay and CCK-8 assay, respectively. Each experiment was independently performed in triplicate and data are presented as mean (SD). The two-tailed Student t test was used to analyze the differences between Day 1 and 3, 4 and 5. (** p < 0.01, *** p < 0.001). (C) RT-PCR analysis of the EMT (E-Cadherin, N-Cadherin, Vimentin), angiogenesis (VEGF, IGF), cancer stemness (Nanog, Oct4, Sox2) and apoptosis-related gene expression (Bax, Bcl-2) in SKOV3 and HOXB9 OE/SKOV3 cells. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; EMT, epithelial-mesenchymal transition; IGF, insulin-like growth factor; Oct-4, octamer-binding transcription factor 4; Sox-2, sex-determining region Y box transcription factor-2; VEGF, vascular endothelial growth factor.

Fig 5: Induction of cisplatin-resistant tumors by HOXB9 overexpression in mouse xenograft models: mice were injected with cisplatin 10 mg/kg (intraperitoneal) every 2 days. (A) Growth rate of tumors or (B) body weight of mice group with or without cisplatin in SKOV3- and HOXB9 OE/SKOV3-injected mouse model; (C) Tumor weight; (D) Volume of isolated tumors from mice (SKOV3-injected group vs. SKOV3-injected group with cisplatin, HOXB9 OE/SKOV3-injected group vs. HOXB9 OE/SKOV3-injected group with cisplatin, N = 6). The tumor volume is measured for 4 weeks after the implantation of SKOV3 and HOXB9 OE/SKOV3 cells. Each experiment was independently performed in triplicate. The two-tailed Student t test was used to analyze the differences between the groups (* p < 0.05, ** p < 0.01). N.S: Not Significant.