Fig 1: NIPBL reverses the effect of EZH2 on RAD21-mediated PI3K gene transcription through disrupting the association between EZH2 and RAD21 in NSCLC cells.a Representative image of Co-IP using an anti-RAD21 antibody in H1299 and H1650 cells. Rabbit IgG was used as negative control. N = 3. b PI3K mRNA expression detected in scrambled and EZH2 siRNA transfected H1299 and H1650 cells. c Immunoprecipitated chromatin was analyzed by PCR and qPCR for the PI3K gene promoter in H1299 and H1650 cells. d Representative images of Co-IP using an anti-RAD21 antibody in scrambled and NIPBL siRNA transfected H1299 and H1650 cells. Rabbit IgG was used as a negative control. e Immunoprecipitated chromatin was analyzed by PCR and qPCR for the PI3K gene promoter in H1299 and H1650 cells. NC negative control; siEZH2, EZH2 siRNA. Error bars indicate the standard error of the mean (SEM). *P < 0.05.
Fig 2: NIPBL enhances RAD21 gene transcription in NSCLC cells.a NIPBL protein expression and (b) RAD21 mRNA levels in scrambled and NIPBL siRNA transfected H1299 and H1650 cells. c Immunoprecipitated chromatin was analyzed by PCR for the RAD21 gene promoter in scrambled and NIPBL siRNA transfected H1299 and H1650 cells. d Translation activity of RAD21 gene was analyzed by relative luciferase reporter activity assay in scrambled and NIPBL siRNA transfected H1299 and H1650 cells. NC negative control; siNIPBL, NIPBL siRNA. Error bars indicate the standard error of the mean (SEM). N = 3. *P < 0.05, **P < 0.01.
Fig 3: RNA methylation elevates NIPBL mRNA level through improving NIPBL mRNA stability in NSCLC cells.a Comparison of NIPBL mRNA levels in lung cancer cell lines versus control MRC-5 cells. N = 3. b Quantification of methylated NIPBL mRNA by qRT-PCR in MRC-5, H1299 and H1650 cells following MeRIP with m6A antibody. N = 3. c NIPBL mRNA levels in scrambled, METTL3 and METTL14 siRNA transfected H1299 and H1650 cells. N = 3. d qPCR results showing NIPBL RNA stability in scrambled, METTL3 and METTL14 siRNA transfected at 0 h, 1 h, 2 h, 3 h, 4 h and 5 h following treatment with actinomycin D. NC negative control; siMETTL3, METTL3 siRNA; siMETTL14, METTL14 siRNA. Error bars indicate the standard error of the mean (SEM). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: Schematic diagram of molecular mechanisms for the present study.RAD21 mediates its oncogenic role in NSCLC via its activation of the PI3K pathway. Mechanistically, NIPBL promoted RAD21 gene transcription by enhancing H3K27 demethylation by recruitment of KDM6B to the RAD21 gene promoter. Moreover, RAD21 enhanced PI3K gene transcription by serving as a transcription factor. NIPBL reversed the effect of EZH2 on RAD21-mediated PI3K gene transcription by disrupting the association between EZH2 and RAD21 protein. In addition, RNA methylation elevated NIPBL mRNA level by increasing NIPBL mRNA stability.
Fig 5: NIPBL promotes RAD21 gene transcription through enhancing H3K27 demethylation via recruiting KDM6B to RAD21 gene promoter in NSCLC cells.a Representative image of Co-IP using an anti-NIPBL antibody in H1299 and H1650 cells. Rabbit IgG was used as negative control. b Immunoprecipitated chromatin was analyzed by PCR and qPCR for the RAD21 gene promoter in scrambled and NIPBL siRNA transfected H1299 and H1650 cells. c RAD21 mRNA expression detected in scrambled and KDM6B siRNA transfected H1299 and H1650 cells. N = 3. d Immunoprecipitated chromatin was analyzed by PCR and qPCR for the RAD21 gene promoter in scrambled and NIPBL or KDM6B siRNA transfected H1299 and H1650 cells. NC negative control; siKDM6B, KDM6B siRNA. Error bars indicate the standard error of the mean (SEM). *P < 0.05.
Supplier Page from Abcam for Recombinant Human NIPBL protein (GST tag N-Terminus)