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. 2017 Oct 16;114(44):E9271–E9279. doi: 10.1073/pnas.1703921114

Fig. 4.

Fig. 4.

CCL5 is the major chemokine involved in the infiltration of NK cells into BECN1− B16-F10 tumor. (A, Upper) Expression of Beclin1 protein in Ctrl, BECN1−, and BECN1−/CCL5− cells. Actin was used as loading control. (Lower) Quantification of CCL5 secreted into the supernatants of the cells described in the Upper panel. Data represent the average ± SEM of three independent experiments. ***P < 0.001 (two-tailed t test). (B) Tumor cells described above were engrafted subcutaneously in C57BL/6 mice (n = 8 per group). Tumor volume was measured at the indicated days and the average ± SEM was reported. *P < 0.05; **P < 0.01 (two-tailed Student’s t test). (C) Immunohistochemical staining of NK cells (Upper) or CCL5 (Lower) performed on indicated tumor sections using anti–Asialo-GM1 and CCL5 antibodies, respectively. (Scale bars, 200 µm.) (D) Quantification of NK cells infiltrating Ctrl, BECN1− and BECN1−/CCL5− tumors reported as a percentage (%) of the total number of cells for each tumor. Data represent the average ± SEM of two sections from two independent tumors. *P < 0.05; **P < 0.01; ns, not-significant (two-tailed Student’s t test). (E) BECN1− B16-F10 melanoma cells were infected with control (Ctrl) or JNK1/2 (JNK) shRNA. The expressions of JNK, c-Jun, and Beclin1 were assessed. Vinculin was used as loading control. The panel corresponding to the expression of Beclin1 was generated from the same blot but consolidated from noncontiguous loading. (F) The expression of CCL5 mRNA by real-time RT-PCR in cells described in E. Data represent the average ± SEM of three independent experiments. ***P < 0.005 (two tailed Student’s t test). (G) BECN1− and BECN1−/JNK− tumor cells described in E were engrafted subcutaneously in C57BL/6 mice (n = 5 or 6 per group). Tumor volume was measured at the indicated days and the average ± SEM was reported. *P < 0.05; **P < 0.01 (two-tailed Student’s t test). (H) The weight of BECN1− and BECN1−/JNK− tumors described in G at day 18. *P < 0.05 (two-tailed Student’s t test). (I) Immunohistochemical staining of CCL5 performed on BECN1− and BECN1−/JNK− tumor sections using anti-CCL5 antibody. (Scale bars, 100 µm.) (J) Quantification of NK cells infiltrating BECN1− and BECN1−/JNK− tumors reported as a percentage (%) of the total number of cells for each tumor. Data represent the average ± SEM of two sections from one independent tumor. *P < 0.05 (two-tailed Student’s t test).