Figure 5
MALAT1 combined with HULC promotes the loading of TRF2 onto telomere that prolongs telomere length.
(a) RNA Immunoprecipitation (RIP) with anti-TRF2 followed by RT-PCR with HULC mRNA primers 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 RIP as negative control. HULC mRNA as INPUT. (b) RNA Immunoprecipitation (RIP) with anti-TRA2 followed by RT-PCR with MALAT1 mRNA primers. IgG RIP as negative control. MALAT1 mRNA as INPUT. (c) Super-EMSA (gel-shift) with biotin- telomere DNA probe and anti-TRF2 antibody. The intensity of the band was examined by Western blotting with anti-Bioton. (d) Chromatin Immunoprecipitation (CHIP) with anti-TRF2 followed by PCR with telomere DNA primers. IgG CHIP as negative control 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 CHIP as negative control Telomere DNA as INPUT. anti-Bioton. (e) Chromatin Immunoprecipitation (CHIP) with anti-TRF2 followed by PCR with telomere DNA primers. IgG CHIP as negative control in stable liver cancer stem cell lines transfected with pGFP-V-RS, pGFP-V-RS–HULC, pGFP-V-RS–MALAT1, pGFP-V-RS-HULC plus pGFP-V-RS-MALAT1. IgG CHIP as negative control Telomere DNA as INPUT. (f) Telomere DNA probe Pulldown with anti- TRF2, pTRF2, -POT1, pPOT1, Exo1, SNM1B, CST/AAF, HP1α 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. Histone as internal control. Biotin as INPUT. (g) The PCR detection of telomere repeat sequence 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 (h). The real-time PCR detection of telomere length 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. Each value was presented as mean ± standard error of the mean (SEM).
