Fig. 5
YTHDF1-promoted YAP mRNA translation is regulated by m6A modification and interaction with eIF3a. (a) The mRNA and protein levels of YTHDF1 were analyzed by RT-PCR and western blot assays in paired fresh NSCLC tumor cancer tissues (Tumor) and matched adjacent normal tissues (Normal) (left panel, n = 10; right panel, n = 30). (b) The expression of YTHDF1 was analyzed by IHC assay in the different grades of lung cancer tissues. (c) High expression of YTHDF1 is associated with worse prognosis for NSCLC patients (P = 0.018). (d, e) A549 cells were transfected with indicated genes of YTHDF1. (d) The migration growth was analyzed by scratch assay. (e) The expressions of E-cadherin and Vimentin were analyzed by western blot assay. (f) The xenografted tumors (left panel) and the metastasizing lung tumors (right panel) originated from A549 cells with stable expression of indicated genes constructed by subcutaneous injection. (g) The protein levels of YAP, CTGF and Cyr61 were analyzed in A549 cells determined by WB assay. (h) Co-IPs performed using lysates collected from A549 cells with immunoprecipitation by either YTHDF1 or eIF3a antibodies. (i-k) The protein level of YAP was analyzed by ELISA assay in puromycin treated A549 cells with transfection with indicated genes. (l-p) A549 cell were transfected with indicated genes of YTHDF1 and YAP. (l) The protein levels of YTHDF1, YAP, CTGF and Cyr61 were analyzed by western blot assay. (m) The size and number of colons were analyzed by colony formation assay. (n) The cellular invasion and migration growth were analyzed by transwell assay. (o) The expressions of E-cadherin and Vimentin were analyzed by qPCR assay. (p) The relative of cleaved Caspas-3 was analyzed by western blot assay. Results were presented as mean ± SD of three independent experiments. *P < 0.05 or **P < 0.01 indicates a significant difference between the indicated groups. ns, not significant