Fig. 4
SNORA56 upregulates GCLC protein by activating its translation. (A) Differential protein expression in the sgSNORA56 vs. the sgNC group. (B) Volcano plot of differentially expressed proteins. (C) KEGG pathway enrichment analysis of significantly downregulated proteins. (D) GCLC protein levels in HCT116, HCT8, HT29 and HIEC cells were determined using western blotting after indicated transfections. (E–F) GCL enzyme activity in HT29 and HIEC cells with indicated transfection. (G) IHC analysis of GCLC levels in CRC tissue microarray. Mean GCLC density was determined using Image Pro Plus. Scale bar: 100 μm. (H) Analysis of the correlation between the SNORA56 and GCLC protein levels using CRC tissue microarray. (I–J) RT-qPCR analysis of relative GCLC mRNA levels in HT29 and HIEC cells upon indicated transfections. (K) Western blot analysis of GCLC protein levels in paired CRC tissues. (L–M) Western blot analysis of GCLC protein stability in SNORA56-silenced HCT8 and HT29 cells vs. control cells after CHX treatment for indicated durations. Relative band intensities were measured on ImageJ. (N) Western blot analysis of GCLC protein levels in HT29 and HCT8 cells after indicated transfections. GCLC protein levels were normalized to GAPDH.