Figure 4.

LncRNA CASC2 binds to and maintains the stability of RORA protein and promotes the nuclear translocation of RORA. (A) CatRAPID predicts that CASC2 can bind to RORA proteins. (B, C) The RNA immunoprecipitation (RIP) assay was performed in GSC2C or GSC4D after RORA knockdown or overexpression, and negative control was transfected, followed by q-PCR to detect the enrichment of CASC2 and RORA. (D, E) The RNA pull-down assays showed the RORA protein immunoprecipitation with CASC2 as detected by western blotting. (F) qPCR showing the validation of CASC2 knockdown or overexpression. (G) CASC2-silenced GSC2C, and CASC2-overexpressed GSC4D and their control were treated with or without MG132 (50 µM) for 6 h, then, cell lysates were detected by western blotting with indicated antibodies, β-actin was used as the control. (H, J) CASC2-silenced GSC2C, and CASC2-overexpressed GSC4D were treated with cycloheximide (CHX, 100 ng/ml) to indicate periods of time, and cell lysates were detected by western blotting to indicate the half-life of RORA protein. (I) Representative images of subcellular localization of RORA in CASC2-silenced GSC2C and CASC2-overexpressed GSC4D were shown by immunofluorescence. Scale bars=50 µm. (K, L) Nuclear and cytosolic lysates were extracted from CASC2-silenced GSC2C and CASC2-overexpressed GSC4D, followed by western blotting with indicated antibodies. EV, empty vector; OE, overexpression; NC, negative control; KD, knockdown. All data are expressed as the mean ± SD (three independent experiments). ^***P < 0.001.