Fig. 2

LATS1/2 loss leads to MHC-I downregulation in EC cells independent of the Hippo-YAP pathway. (A) Western blotting of the indicated proteins in whole cell lysates (WCLs) from KLE cells with LATS1/2 single or double KO by CRISPR-Cas9 methods. Parental KLE cells were used as a control. (B) Volcano plot of the differentially expressed genes in parental and LATS1/2-KO KLE cells. (C) KEGG pathway analysis of the differentially expressed genes in parental and LATS1/2-KO KLE cells. (D) Heatmap depicting the expression of the top 50 differentially expressed genes in parental and LATS1/2-KO KLE cells. (E) GSEA of the antigen processing and presentation gene signature in parental and LATS1/2-KO KLE cells. The hallmark antigen processing and presentation gene set (Standard name: JECHLINGER_EPITHELIAL_TO_MESENCHYMAL_TRANSITION _UP) was obtained from the Molecular Signatures Database (MsigDB).(F) RT-qPCR measurement of the mRNA expression of HLA-ABC in parental, LATS1-KO, LATS2-KO and LATS1/2-KO KLE cells. Data are shown as means ± SD (n = 3). (G) Western blotting of the indicated proteins in the WCLs from parental, LATS1-KO, LATS2-KO or LATS1/2-KO KLE cells. (H) Representative IF images of parental and LATS1/2-KO KLE cells stained with HLA-ABC (red) and DAPI (blue). Quantification of MHC-I intensity is shown on the right. Scale bar, 20 μm. Data are shown as means ± SD (n = 3).(I) MHC-I surface expression on Parental and LATS1/2-KO KLE cells detected with flow cytometry. Flow cytometric analysis was performed to determine the MFI of MHC-I. Data are shown as means ± SD (n = 3). (J) Exogenous LATS1 was reintroduced into LATS1/2-KO KLE cells to generate LATS1 Tet-On inducible cells. The cell lines were treated with (DMSO) or doxycycline (DOX) (10 ng/ml) for 24 h, and the WCLs were prepared for Western blotting. Parental KLE cells were used as the control. (K) Western blotting of the indicated proteins in the WCLs from KLE cells treated with TDI-011536 (3 µM) for 24 h. (L) Western blotting of the indicated proteins in the WCLs from parental and LATS1/2-KO KLE cells treated with DMSO or verteporfin (0.5 µM) for 24 h. (M, N) Western blotting of the indicated proteins in the WCLs from parental and LATS1/2-KO KLE cells stably overexpressing sh ctrl, sh YAP (M) or sh ctrl, sh TEADs (N). P values are calculated using the Multiple t-tests in (F, I) and Student’s t test in (H). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001