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: 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.

Fig 4: Expression of chemoresistance-related genes in mouse tumors: (A) Western blot analysis of HOXB9, ERCC-1, MRP-2 and XIAP in isolated tumors from mice. 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 < 0.05, *** p < 0.001, SKOV3-injected vs. HOXB9 OE/SKOV3-injected group); (B) Immunohistochemical staining analysis of HOXB9, ERCC-1, MRP-2 and XIAP in the tumors of SKOV3 or HOXB9 OE/SKOV3 cells (magnification: 400×; scale bar = 20 μm). Each experiment was independently performed in triplicate. ERCC-1, excision repair cross-complementation-1; MRP-2, multidrug resistance-associated protein-2; XIAP, X-linked inhibitor of apoptosis protein.

Fig 5: Effect of HOXB9 overexpression on apoptosis and chemoresistance-related genes: cell viability of SKOV3 and HOXB9 OE/SKOV3 cells following cisplatin treatment (A) without (F value = 2.603) or (B) with siRNA_HOXB9 treatment (F value = 0.05973). The cells were treated with siHOXB9 100 nM or cisplatin 50 μM for 48 h (N = 4); (C) Apoptosis (F value = 1.026, N = 4) and (D) Caspase 3 activity (F value = 1.619, N = 4) analysis of SKOV3 and HOXB9 OE/SKOV3 cells following cisplatin treatment with or without siRNA_HOXB9 treatment. Each experiment was independently performed in quadruplicate. Results are expressed as mean ± standard deviation. The two-tailed Student t test was used to analyze the differences between the SKOV3 and the others group. (*p < 0.05, ** p < 0.001, *** p < 0.0001); (E) Immunoblot analysis of HOXB9, ERCC-1, MRP-2, XIAP, Bcl-2, Bax, caspase-3, cleaved caspase-3, PARP1, and cleaved PARP in the three cells with or without treatments of siRNA_HOXB9 or cisplatin 50 uM (Supplementary Figure S1: quantification graphs for the protein expression levels. # Control, *p < 0.05, ** p < 0.001, *** p < 0.0001). Immunoblot experiment was independently performed in triplicate. Bax, BCL2 associated X; Bcl2, B-cell lymphoma 2; ERCC-1, excision repair cross-complementation-1; MRP-2, multidrug resistance-associated protein-2; PARP, poly (ADP-ribose) polymerase; XIAP, X-linked inhibitor of apoptosis protein.