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Fig. 2 | Stem Cell Research & Therapy

Fig. 2

From: Long noncoding RNA MEG3 blocks telomerase activity in human liver cancer stem cells epigenetically

Fig. 2

MEG3 enhances the P53 expression and promotes methylation modification of histone H3 at lysine 27. A Chromatin immunoprecipitation (CHIP) analysis. hLCSCs were used to extract cross-linked DNA, and CHIP was performed using anti-P300 and anti-RNAPolII. PCR was carried out using a primer designed according to the P53 promoter. IgG CHIP was used as a negative control. B Chromosomal conformation capture (3C)-chromatin immunoprecipitation (CHIP) analysis. The hLCSCs were cross-linked with formaldehyde and then captured by chromosome configuration (3C)-chromatin immunoprecipitation (CHIP) using anti-P300 and anti-RNAPolII, respectively. PCR was carried out using a pair of mixed primers designed according to the P53 promoter and enhancer. IgG CHIP-3C was used as a negative control. C The pEZX-MT-P53 promoter-Luc luciferase activity was tested. D RT-PCR was used to detect the transcriptional capacity of P53. β-actin as an internal reference gene. E (a) The expression of P53 was detected by Western blotting. β-actin was used as an internal reference gene. E (b) Semi-quantitative analysis of gray scale scanning of bands. F The total protein was extracted and subjected to anti-P53 Western blotting analysis. β-actin as an internal reference gene. G The RNA pulldown analysis was performed using biotinylated MEG3 probes and anti-EZH2, anti-SUZ12, anti-EDD, and anti-RbAp46/48. Histone H3 is used as INPUT and biotin is used as an internal reference. H Co-immunoprecipitation with anti-EZH2, anti-SUZ12, anti-EED, anti-RbAp46/48, and anti-Histone H3 was performed. IgG co-immunoprecipitation was used as a negative control. I Co-immunoprecipitation with anti-EZH2, anti-SUZ12, anti-EED, anti-RbAp46/48, and anti-HistoneH3 was performed. IgG co-immunoprecipitation was used as a negative control. J (a) H3K27me1, H3K27me2, and H3K27me3 were detected by Western blotting. Histone H3 was used as an internal reference gene. J (b) Semi-quantitative analysis of grayscale scanning of positive bands

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