Fig. 3.
YTHDF2 regulates the lncFAL expression levels in a m6A-dependent manner. (A) Top sequence motif of m6A methylation sites of pre-lncFAL identified using online SRAMP tools. (B) Expression of YTHDF2 in HCC and NCL tissues from the TCGA-LIHC dataset. (C) Western blot analysis of YTHDF2 protein and qRT–PCR analysis of lncFAL and pre-lncFAL expression in PLC5 cells transfected with HA-control vector, HA-YTHDF2 expression vector, and HA-YTHDF2ΔYTH expression vector. β-Actin and GAPDH were used as internal controls. (D) Western blot analysis of PLXNB2 protein and qRT–PCR analysis of lncFAL expression in YTHDF2-overexpressing PLC5 and Huh7 cells transfected with CTL-si and PLXNB2-si, respectively. β-Actin and GAPDH were used as internal controls. (E) Western blot analysis of YTHDF2 protein and qRT–PCR analysis of lncFAL expression in PLC5 and Huh7 cells transfected with CTL-si and YTHDF2-si, respectively. β-Actin and GAPDH were used as internal controls. (F) qRT–PCR analysis of lncFAL and pre-lncFAL expression in PLC5 cells transfected with the indicated overexpression vector and then treated with ActD (5 μg/mL) for the indicated times. GAPDH was used as an internal control. (G) qRT–PCR analysis of lncFAL expression in PLC5 cells transfected with the indicated overexpression vectors an treated or not treated with DMSO, DEPC, or RNasin (5 U/μL) treatment following ActD (5 μg/mL) treatment for the indicated times. GAPDH was used as an internal control. (H) Western blot analysis of the indicated proteins and qRT–PCR analysis of lncFAL expression in PLC5 cells transfected with the indicated overexpression vectors and siRNAs and then treated with ActD (5 μg/mL) for the indicated times. β-Actin and GAPDH were used as internal controls. (I) The interaction between YTHDF2 and lncFAL was assessed by RIP assays followed by qRT–PCR. IgG was used as a negative control. (J) MeRIP-qPCR analysis of pre-lncFAL in PLC5 and Huh7 cells transfected with CTL-si and YTHDF2-si, respectively. IgG was used as a negative control. (K) MeRIP-qPCR analysis of the indicated segments of pre-lncFAL in PLC5 cells. IgG was used as a negative control. (L) qRT–PCR analysis of lncFAL expression in PLC5 cells transfected with the indicated overexpression vectors. All the above experiments were independently performed in triplicate (N ≥ 3). The data in B-L are presented as the means ± SDs. The statistical analyses in B-L were performed by the two-tailed unpaired Student's t-test.