Fig. 3
From: Long noncoding RNA MEG3 blocks telomerase activity in human liver cancer stem cells epigenetically
MEG3 inhibits the expression of telomerase reverse transcriptase gene in human liver cancer stem cells. A DNA pulldown was performed by biotin-labeled TERT promoter probe (Biotin-TERT promoter DNA), and Western blotting analysis was performed with anti-RNAPolII and anti-H3K27me3, respectively. Western blotting with anti-biotin and anti-histone H3 was performed as an internal reference. B Super-DNA-protein complex gel migration assay (Super-EMSA) with biotin-labeled TERT promoter cis-element probe and anti-HK27me3 and anti-biotin. IgG super-EMSA as a negative control. C The CHIP with anti-H3K27me1, anti-H3K27me2, and anti-H3K27me3. IgG chromatin immunoprecipitation was used as a negative control. The promoter of TERT was amplified by using the primers of TERT promoter. D The pEZX-MT-TERT promoter-Luc luciferase activity was tested. E The transcriptional capacity of TERT was detected by RT-PCR. β-actin was used as an internal reference gene. F (a) The expression of TERT was detected by Western blotting. β-actin was used as an internal reference gene. F (b) Semi-quantitative analysis of grayscale scanning of positive bands. G RNA pulldown was performed by biotin-labeled TERC RNA probe (Biotin-TERT), and Western blotting was performed with anti-TERT, anti-HP1α, and anti-P53, respectively. H (a) Super-RNA-protein complex gel migration assay (Super-EMSA) with the biotin-labeled TERC probe (Biotin-TERC) and anti-TERT and anti-biotin. IgG Super-EMSA was used as a negative control. H (b) Quantitative analysis of gray scales of positive bands. I The RNA immunoprecipitation (RIP) using anti-TERT, anti-P53, and anti-HP1α. The TERC was amplified by RT-PCR using primers designed by the TERC sequence. IgG RNA co-immunoprecipitation was used as a negative control. J RNA pulldown was detected by biotin-labeled TERC RNA probe (Biotin-TERT), and Western blotting was performed with anti-TERT, anti-HP1α, and anti-P53, respectively. K RIP using anti-TERT, anti-P53, and anti-HP1α and TERC was detected by RT-PCR. L–M The telomerase activity was detected by quantitative telomerase activity assay (TRAP)