Fig. 6

FEZF1-AS1epigenetically silenced P21 transcription through LSD1-Mediated H3K4me2 demethylation. a RIP experiments were performed using the LSD1, EZH2, SUZ12, WDR5, coREST antibodies for immunoprecipitation. Specific primers for FEZF1-AS1 were used to detect FEZF1-AS1. b Biotinylated FEZF1-AS1 or antisense RNA was incubated with total-cell extracts (SGC7901 cells), targeted with streptavidin beads and washed; the associated proteins were resolved in a gel. Western blotting analysis of the specific association of LSD1 as well as UPF1 with FEZF1-AS1. A nonspecific protein (GAPDH) is shown as a control. c, d Expression of P21 in AGS, SGC-7901 and MGC-803 cells with si-LSD1 or pcDNA-FEZF1-AS1was detected by qRT–PCR and Western blotting. e Expression of P21 in AGS and SGC-7901 cells with inhibtor of LSD1 was detected by qRT–PCR. f ChIP analyses in AGS, SGC-7901and MGC-803cells with si-FEZF1-AS1 or pcDNA-FEZF1-AS1 were performed on the P21 promoter regions using anti-H3K4me1, H3K4me2 and LSD1 antibodies. Enrichment was determined relative to the input controls. All experiments were performed in triplicate with three technical replicates.*P < 0.05 and **P < 0.01