Fig. 5
LATS1/2 loss promotes tumor immune evasion by downregulating MHC-I expression. (A) The concentration of TNF-α in the supernatant of PBMCs and EC cells co-culture system. Data are shown as means ± SD (n = 3). (B) The concentration of Granzyme B in the supernatant of PBMCs and EC cells co-culture system. Data are shown as means ± SD (n = 3). (C) CCK-8 cell proliferation analysis of parental and LATS1/2-KO KLE cells co-cultured with PBMCs for 48 h. Data are shown as means ± SD (n = 3). (D) Parental and LATS1/2-KO KLE cells or exogenous HLA-A reconstituted LATS1/2-KO KLE cells were co-cultured with PBMCs for 24 h. Flow cytometric analysis was performed to determine the apoptotic rates of KLE cells. Data are shown as means ± SD (n = 3). (E) Western blotting of the indicated proteins in the WCLs from parental, LATS1-KO, LATS2-KO or LATS1/2-KO CT26 cells. (F) RT-qPCR measurement of the mRNA expression of mouse MHC-I in parental, LATS1-KO, LATS2-KO or LATS1/2-KO CT26 cells. Data are shown as means ± SD (n = 3). (G-I) Parental or LATS1/2-KO CT26 cells were injected subcutaneously into the flank of BALB/c or BALB/cnu/nu mice. Tumor growth was measured every 3 days for 21 days. 5 mice per experimental group. Tumors in each group at day 21 were harvested and photographed (G), tumor volume (H) and tumor weight (I) at each time point was documented. Data are shown as means ± SD (n = 5). (J-M) Immunostaining of CD8 and Granzyme B in the CT26 tumor mass in BALB/c mice (J). DAPI: nuclear counterstaining (J). Scale bar, 100 μm. The intensity of CD8 (K) and Granzyme B (L) were quantified using Image J. The ratio of Granzyme B+/CD8+ T cells was shown in (M). Data are shown as means ± SD (n = 5). 1 tissue slide per tumor, 5 mice per group. Unit = 1.9 × 106 µm2. P values are calculated using the Multiple t-tests in (A, B, C, D and F), Two-way ANOVA test in (H), One-way ANOVA test in (I) and Student’s t test in (K, L and M). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significant