Figure 4
MALAT1 combined with HULC enhances TRF2 expression and sumoylation.
(a) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-P300, anti-RNA polII, anti-CREPT in stable liver cancer stem cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-HULC, pCMV6-A-GFP-MALAT1, pCMV6-A-GFP-HULC plus pCMV6-A-GFP-MALAT1. The chromatin is cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin is immunoprecipitated using an antibody (anti-P300, anti-RNA polII) against the protein of interest. Thereafter, the cross-links are reversed, and the DNA is purified further. The PCR anlysis is applied for detecting TRF2 promoter-enhancer coupling product using TRF2 promoter and enhancer primers. The TRF2 promoter and enhancer as INPUT. (b) The TRF2 promoter luciferase activity assay in stable liver cancer stem cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-HULC, pCMV6-A-GFP-MALAT1, pCMV6-A-GFP-HULC plus pCMV6-A-GFP-MALAT1. (c) RT-PCR analysis of TRF2 mRNA. β-actin as internal control. (d) Western blotting with anti-TRF2, anti-pTRF2, β-actin as internal control. (upper) and anti-TRF2 Co-Immunoprecipitation (IP) followed by Western blotting with anti-SUMO in stable liver cancer stem cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-HULC, pCMV6-A-GFP-MALAT1, pCMV6-A-GFP-HULC plus pCMV6-A-GFP-MALAT1. IgG IP as negative control. INPUT refers to Western blotting with anti-TRF2(lower).
