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. 2023 Apr 19;11:e15172. doi: 10.7717/peerj.15172

Figure 3. Sema4D repression renders B16-F10R cells sensitive to nivolumab treatment.

Figure 3

(A) Effects of nivolumab on cell viability of B16-F10 and B16-F10R. B16-F10 cells were derived into B16-F10 and B16-F10+nivolumab group, B16-F10R cells were derived into B16-F10R and B16-F10R+nivolumab groups, B16-F10 and B16-F10R groups were as control group without treatment, B16-F10+nivolumab and B16-F10R+nivolumab groups were treatment with 50 ng/mL nivolumab and co-cultured with TIL at a 1:5 ratio (Van den Berg et al., 2020). Compared with the B16-F10+nivolumab group, ∗∗ is p < 0.01, n = 3, one-way ANOVA was used to compare the differences. (B) RNA expression of Sema4D overexpression. Cells were derived into B16-F10+LV-Sema4D and B16-F10+LV-NC group, compared with the LV-NC group, ** is p < 0.01, n = 3, t-test was used to compare the differences. (C) Effects of nivolumab on B16-F10 cell viability after Sema4D overexpression. B16-F10 cells were derived into B16-F10, B16-F10+nivolumab, B16-F10+LV-Sema4D and B16-F10+LV-NC group, B16-F10 is control group, another 3 groups were treatment with 50ng/mL nivolumab and co-cultured with TIL at a 1:5 ratio, B16-F10+LV-Sema4D group was also treated with overexpression Sema4D, B16-F10+LV-NC group was also treated with Sema4D negative control. Compared with B16-F10 group, **** is p < 0.0001; Compared with B16-F10+LV-NC group, #### is p < 0.0001, n = 3, one-way ANOVA was used to compare the differences. (D) Effects of nivolumab on B16-F10R cell after Sema4D silencing. B16-F10R cells were derived into B16-F10R, B16-F10R+nivolumab, Sema4D-shRNA and Sema4D-NC group , B16-F10R is control group, another 3 groups were treatment with 50ng/mL nivolumab and co-cultured with TIL at a 1:5 ratio, Sema4D-shRNA group was knockdown Sema4D, and Sema4D-NC group was Sema4D-shRNA negative control group. Compared with Sema4D-shRNA group, * is p < 0.05, ** is p < 0.01, n = 3, one-way ANOVA was used to compare the differences. (E) B16-F10R cell apoptosis rate after treatment with nivolumab. B16-F10R cells were derived into Sema4D-shRNA group and Sema4D-NC group, the treatment is the same as C. Cell apoptosis was detected byPI and Annexin V/Alexa Fluor 647 double staining, Alexa Fluor 647 was a fluorescent dye binding with Annexin V, reflecting early apoptosis of cells. Compared with Sema4D-NC group, ** is p < 0.01, n = 3, t-test was used to compare the differences. (F) Tumor volume of knockdown Sema4D with nivolumab treatment. Balb/C mice were derived into Sema4D-shRNA group (n = 6) and Sema4D-NC group(n = 5), B16-F10R-Sema4D-shRNA cells and B16-F10R-Sema4D-NC cells were intradermal injected into the right side of mice, then intraperitoneally injected nivolumab 10 mg/kg once at day 1, t-test was used to compare the differences. (G) Tumor weight of knockdown Sema4D with nivolumab, t-test was used to compare the differences. Compared with Sema4D-NC group, *** is p < 0.001, **** is p < 0.0001. (H) Mouse melanoma specimen. Major marks represent cm on the scale.