Figure 5.

LINC00526 repressed AXL transcription via interacting with EZH2. A, LINC00526 subcellular location was detected by cytoplasmic and nuclear RNA purification, followed by qRT‐PCR. U6 and β‐actin were used as nuclear and cytoplasmic controls, respectively. B, RIP assays were performed in U87 cells using EZH2 specific antibody or nonspecific IgG. The enriched RNA was measured by qRT‐PCR. HOTAIR was used as positive control, and while GAPDH was used as negative control. C, RNA pull‐down assays were performed in U87 and U251 cells using in vitro transcribed biotin‐labelled LINC00526 or antisense RNA. The enriched proteins were detected by Western blot with EZH2 specific antibody. D, ChIP assays were performed in LINC00526 stably ectopically expressed and control U87 cells using EZH2 specific antibody. The enriched DNA was measured by qRT‐PCR and specific primers corresponding to AXL promoter. E, ChIP assays were performed in LINC00526 stably silenced and control U251 cells using EZH2 specific antibody. The enriched DNA was measured by qRT‐PCR and specific primers corresponding to AXL promoter. F, AXL mRNA expression levels in LINC00526 stably ectopically expressed and control U87 cells were measured by qRT‐PCR. G, AXL mRNA expression levels in LINC00526 stably silenced and control U251 cells were measured by qRT‐PCR. H, AXL protein expression levels in LINC00526 stably ectopically expressed and control U87 cells were measured by Western blot. I, AXL protein expression levels in LINC00526 stably silenced and control U251 cells were measured by Western blot. J, Phosphorylation levels of Akt and IκBα in LINC00526 stably ectopically expressed and control U87 cells were measured by Western blot. K, Phosphorylation levels of Akt and IκBα in LINC00526 stably silenced and control U251 cells were measured by Western blot. **P < 0.01, ***P < 0.001, ns, not significant, Student's t test (B, D, F) or one‐way ANOVA followed by Dunnett's multiple comparison test (E, G)