Fig 1: The B7-H3/KIF15 axis confers radioresistance in vitro and in vivo.a Viability was assessed in B7-H3-overexpressing HCT116 and RKO cells treated with KIF15 siRNA or SB743921 prior to treatment with 4 Gy X-ray irradiation. b Colony formation was assessed in B7-H3-overexpressing HCT116 and RKO cells treated with KIF15 siRNA or SB743921 prior to treatment with 4 Gy X-ray irradiation. c Apoptosis was measured using Annexin V/7-AAD double staining in B7-H3-CRC cells treated with KIF15 siRNA or SB743921 prior to treatment with 4 Gy X-ray irradiation. The data are shown as the mean ± SD of three independent experiments. d Cell cycle progression was measured in B7-H3-CRC cells treated with KIF15 siRNA or SB743921 prior to treatment with 4 Gy X-ray irradiation. Values are expressed as the mean ± SD. e The treatment of each group is shown. HCT116 cells were inoculated under the skin of nude mice. At Day 6 after translation, 2.5 mg/kg SB743921 or DMSO was intraperitoneally injected into nude mice. On Day 12, the xenograft mice were irradiated locally with 10 Gy X-ray. Tumor size was measured at 2-day intervals. f Each group was composed of 5 female nude mice. Representative images of tumors formed by EV + DMSO or B7-H3 + DMSO cells with or without SB743921 treatment. g The growth curves of tumors formed by the indicated EV + DMSO or B7-H3 + DMSO cells with or without SB743921 treatment. The data are presented as the mean ± SEM (n = 5 mice per group). h The weights of tumors formed by the indicated EV + DMSO or B7-H3 + DMSO cells with or without SB743921 treatment. The data are presented as the mean ± SEM (n = 5 mice per group). i Ki67-positive cell numbers in tumor tissues of the nude mouse xenograft model with the indicated treatments. j TUNEL-positive cell numbers in tumor tissues of the nude mouse xenograft model with the indicated treatments. The data are presented as the mean ± SEM (n = 5 mice per group). **P < 0.01, *P < 0.05.
Fig 2: Correlation analysis between B7-H3 and KIF15 in CRC tissue samples.a One representative image of the IHC analysis of B7-H3 and KIF15 protein expression in CRC (n = 123) tissue sections at different stages (scale bar, 50 µm). b B7-H3 and KIF15 protein expression based on the staining index in nonmalignant adjacent tissues (NATs) and CRC specimens. c B7-H3 and KIF15 protein expression based on the staining index in CRC specimens at different clinical stages. Values are expressed as the mean ± SEM. d Correlation analysis of the staining index of the expression levels of B7-H3 and KIF15 proteins in human CRC specimens (n = 123). **P < 0.01, *P < 0.05.
Fig 3: KIF15 promotes CRPC in vitro and in vivo. (A, B) Cell proliferation of LNCaP cells with FBS or CSS treatment assessed by MTS assays. LNCaP cells were cultured in FBS medium (A) or CSS medium for 48 hours (B), these cells were transfected with corresponding siRNA and subjected to MTS assays. Vec, vector. ***P <0.001; ****P <0.0001. (C, D) Cell proliferation of C4-2B cells with FBS or CSS treatment assessed by MTS assays. C4-2B cells were cultured in FBS medium (C) or CSS medium for 48 hours (D), these cells were transfected with corresponding siRNA and subjected to MTS assays. NC, negative control; *P <0.05; **P < 0.01; ***P <0.001. (E) Cell proliferation of PC3 cells assessed by MTS assays. PC3 cells were transiently transfected with corresponding siRNA, the cells were subjected to MTS assays. **P < 0.01; ***P <0.001. (F–H) Xenograft tumor growth after KIF15 depletion. C4-2B cells with stable KIF15 knockdown or its control were injected subcutaneously into nude mice (6 mice per group). Tumor size was measured twice every week (F). At the endpoint, tumors isolated from euthanized mice were photographed (G) and weighed (H). **P < 0.01; ****P <0.0001. (I) Representative images of Ki67 IHC staining of xenograft tumor derived from C4-2B NC/shKIF15 cells.
Fig 4: Knockdown of KIF15 repressed the growth of breast cancer cells. (A-B) Knockdown efficiency of KIF15 was evaluated in MDA-MB-231 and SKBR3 cells through RT-qPCR and western blot. (C) Cell viability was measured by CCK-8 when KIF15 was inhibited. (D) Wound healing assay was utilized to measure cell migration capability after silencing KIF15. (E) Transwell experiment was conducted to test cell invasion after KIF15 depletion. (F) Flow cytometry was carried out to evaluate the influence of silencing KIF15 on cell cycle. Data displayed in all bar graphs were obtained from three or more independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 indicated data were statistically significant.
Fig 5: STLC resistance of both U-R and H-R cells is dependent on KIF15/kinesin-12.(A) Experimental procedure. (B) Evaluation of knockdown efficiency by immunoblotting. (C) The effect of KIF15 depletion on STLC resistance of U-R and H-R cells was examined by siRNA-mediated gene knockdown. Relative cell viability for a given STLC concentration is shown as mean ± SD (n = 3). KIF15 depletion substantially reduced the STLC resistance of U-R and H-R cells.
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