Fig. 5
REG1CP binds to FANCJ. a Silencing of FANCJ but not DHX36 inhibited REG3A mRNA expression. b FANCJ bound to wild-type REG1CP but not a REG1CP mutant with the G-quadruplex deleted (REG1CP-ΔG4). In vitro-synthesized biotin-labelled REG1CP sense RNA (REG1CP) or antisense RNA (Antisense REG1CP) or REG1CP-ΔG4 was incubated with the immunoprecipitated FANCJ from nuclear extracts of LIM1215 cells followed by EMSA. c Binding of FANCJ to REG1CP was detected using RNA pulldown assays. Proteins co-pulled down with biotin-labelled REG1CP sense probe (REG1CP-S-probe) or REG1CP antisense probe (REG1CP-AS-probe) were analysed using western blotting. d Binding of FANCJ to REG1CP was detected using RNA immunoprecipitation (RIP) assays. RNAs co-precipitated with an antibody against FANJ from nuclear extracts of LIM1215 and HT-29 cells were analysed using qPCR. e FANCJ silencing reduced REG3A mRNA levels. Relative REG3A mRNA abundance was quantitated using qPCR in LIM1215 and HT-29 cells introduced with the control or FANCJ siRNAs. f FANCJ silencing diminished upregulation of REG3A by overexpression of REG1CP. Relative REG3A mRNA abundance was quantitated using qPCR in LIM1215 and HT-29 cells introduced with the control or FANCJ siRNAs with or without overexpressing REC1CP. g An anti-G-quadruplex antibody bound to wild-type REG1CP but not a REG1CP mutant with the G-quadruplex deleted (REG1CP-ΔG4). In vitro-synthesized biotin-labelled REG1CP or REG1CP-ΔG4 were incubated with the isotype control (rabbit IgG) or an anti-FANCJ antibody followed by analysis using EMSA. In vitro-synthesized biotin-labelled MALAT1 was included as a control. h FANCJ bound to wild-type REG1CP but not REG1CP-ΔG4. LIM1215 and HT-29 cells with endogenous REG1CP stably silenced were introduced with exogenous REG1CP or REG1CP-ΔG4. FANCJ-associated REG1CP was detected using RIP assays. n = 3 independent experiments. Data are presented as Mean ± SEM (a, d, e, f, h) or representatives (b, c, g). Statistical significance was calculated using a two-tailed t-test.
