Fig. 2

ALKBH5 associates with PVT1 and inhibits its stability. a The RIP assays using METTL3, METTL14, WTAP, FTO and ALKBH5 antibodies were carried out. b MG63 and U2OS cell lysates were incubated with biotin-labeled PVT1 or antisense PVT1; after pull-down, ALKBH5 was detected by western blot. c qRT-PCR analysis of ALKBH5 mRNA levels in six OS cell lines and a normal osteoblast cell line Nhost. d MG63 and U2OS cells were infected with lentivirus expressing scramble control shRNA (shNC) or ALKBH5 shRNAs (shALKBH5). The knockdown efficacy was validated by qRT-PCR. e 143B cells were infected with lentivirus expressing empty vector control (NC) or ALKBH5. The overexpression efficacy was validated by qRT-PCR. f The qRT-PCR analysis of PVT1 levels in control and ALKBH5-silenced MG63 and U2OS cells. g The qRT-PCR analysis of PVT1 levels in control and ALKBH5-overexpressed 143B cells. h The luciferase reporter containing PVT1 promoter was transfected into MG63 and U2OS cells with ALKBH5 knockdown. The luciferase activity was measured. i The luciferase reporter containing PVT1 promoter was transfected into 143B cells with ALKBH5 overexpression. The luciferase activity was measured. j Control and ALKBH5-silenced MG63 and U2OS cells were treated with α-amanitin(50 mM) to block new RNA synthesis. The stability of PVT1 over time was measured by qRT-PCR relative to time 0. k Control and ALKBH5-overexpressed 143B cells were treated with α-amanitin(50 mM) to block new RNA synthesis. The stability of PVT1 over time was measured by qRT-PCR relative to time 0. *p < 0.05, **p < 0.01, ***p < 0.001