Fig. 5: The LEF1/KDM4A complex regulates OSCC carcinogenesis by suppressing LATS2 expression.

Verification of RNA-seq results through qChIP analysis of genes in A Fig. 4D and B Fig. 4F in CAL-27 cells. Results are expressed as fold-change relative to IgG control. GAPDH was used as an internal standard. C Primer pairs including #1 to #10 synthesised to cover the promoter region of LATS2. qChIP-based promoter-walk experiments were performed using CAL-27 cells; the enrichment of LEF1 or KDM4A was mapped to three or two regions of the LATS2 promoter, respectively. D ChIP analysis on CAL-27 cells with antibodies against LEF1 and KDM4A at the LATS2 promoter. E qChIP analysis of LEF1 and KDM4A recruitment to the LATS2 promoter in CAL-27 cells after transfection with control shRNA (shSCR) or shRNAs targeting LEF1 or KDM4A. IgG served as a negative control. F qChIP analysis of H3K9me3 and H3K36me3 enrichment on the LATS2 promoter in CAL-27 cells after transfection with control shRNA (shSCR) or shRNA targeting KDM4A. IgG served as a negative control. G Expression of LATS2 measured using western blotting in LEF1/KDM4A-depleted CAL-27 and SCC-9 cells. GAPDH served as a loading control. H CAL-27 cells treated with siRNAs were assessed by flow cytometry to detect cell cycle distribution, revealing that LATS2 knockdown in CAL-27 cells causes altered cell cycle distribution. I Immunohistochemical staining of LATS2 in normal epithelial tissue and tumours (n = 4). Scale bar: 100 μm or 25 μm. J Analysis of public datasets (TCGA) for LATS2 expression in the normal epithelium and HNSCCs with histological grades I, II, III, and IV (44 normal, 63 grade I, 305 grade II, 125 grade III, and 29 grade IV). Data represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.