Fig. 4
REG1CP forms an RNA–DNA triplex with the REG3A gene. a Representative microphotographs of ISH staining using probes against REG1CP in HT29 cells. Dihydrodipicolinate reductase (DapB) as a negative control and peptidylprolyl isomerase B (PPIB) as a positive control. Scale bar: 20 µm. b In vitro-synthesized biotin-labelled REG1CP pulled down a DNA fragment containing the TFR of the REG3A gene (-6072/-6248). c In vitro-synthesized biotin-labelled REG1CP at increasing concentrations was incubated with a DNA fragment containing the TFR of the REG3A gene followed by analysis using EMSA. d Deletion of the TFOs of REG1CP or the TFR of the REG3A gene abolished the association between REG1CP RNA and REG3A DNA as shown using EMSA. e In vitro-synthesized biotin-labelled wild-type REG1CP, a REG1CP mutant lacking the TFOs or wild-type REG1CP containing 7-deaza-purine nucleotides was incubated with a DNA fragment containing the TFR of the REG3A gene followed by pulldown with REG1CP sense RNA. f Treatment with RNase A abolished the binding of REG1CP to REG3A as shown using EMSA. g Nuclear extracts from LIM1215 and HT-29 cells were incubated with in vitro-synthesized biotin-labelled REG1CP or antisense RNA in the presence or absence of proteinase K. The baseline amount of REG3A DNA detected in the control assay (pulldown samples without RNA) was arbitrarily designated as one. h Endogenous REG3A DNA containing the TFR (-6072/-6248) was co-pulled down with endogenous REG1CP. LncRNA MALAT1 was included as a control. i UV treatment caused association between the TFR of REG3A and psoralen-modified biotinylated TFOs of REG1CP. Nuclear extracts from cells transfected with psoralen-modified biotinylated TFOs of REG1CP or a biotinylated RNA oligonucleotide without the TFOs with or without UV-crosslinking were subjected to pulldown with streptavidin beads. (j, k) Introduction of a shRNA-resistant REG1CP (REG1CP-R) but not a REG1CP mutant with the TFOs deleted (REG1CP-ΔTFO) into cells with endogenous REG1CP silenced increased REG3A expression (j) and promotes cell proliferation (k). n = 3 independent experiments. Data are presented as Mean ± SEM (b, e, g, i– k) or representatives (a, c, d, f, h). Statistical significance was calculated using a two-tailed t-test.
