Journal: Science Advances

Article Title: Conserved noncoding sequence-9 regulates NFATc1-mediated IL-10 expression in B cells to control inflammatory responses

doi: 10.1126/sciadv.aec7779

Figure Lengend Snippet: ( A ) Correlation analysis scheme between Il10 expression and OCR scores in the Il10 locus. ( B ) Il10 expression levels across 11 B cell subsets. ( C ) OCR scores of 33 OCR regions in the Il10 locus across 11 B cell subsets. Circles represent OCRs, with numbers assigned by OCR ID. Red bars mark CNSs, blue bars mark exons, and OCRs overlapping CNSs are highlighted in red. ( D ) Plot of correlations between Il10 expression and OCR scores. The x axis shows Pearson’s r , and the y axis shows −log 10 ( P value). A dotted line indicates a P value of 0.05, and OCRs corresponding to CNSs are highlighted in red. ( E ) Analysis of ATAC-seq (pink), protein ChIP-seq (black), and histone ChIP-seq (blue) data from public datasets ( GSE103057 , GSE82144 , GSE33819 , and GSE92344 ) in the Il10 locus of B cells. ( F ) Reporter assay of Il10 CNSs in A20 cells. Each CNS locus was cloned into the pXPG vector carrying the Il10 minimal promoter. Results are expressed as relative units compared to the luciferase activity of a pXPG vector containing only the minimal promoter. Data are representative of three independent experiments with similar results. Data are presented as means ± SEM. Statistical analysis was performed using a two-tailed unpaired Student’s t test. **** P < 0.0001.

Article Snippet: A20 and Raji B cell lines were obtained from the American Type Culture Collection (MD, USA).

Techniques: Expressing, ChIP-sequencing, Reporter Assay, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Two Tailed Test

Journal: Science Advances

Article Title: Conserved noncoding sequence-9 regulates NFATc1-mediated IL-10 expression in B cells to control inflammatory responses

doi: 10.1126/sciadv.aec7779

Figure Lengend Snippet: ( A ) Correlation analysis of Il10 expression with potential transcription factors using published microarray data; candidates were identified by motif analysis of CNS-9. ( B ) Plot of correlations between the expression levels of Il10 and potential transcription factors. The x axis shows Pearson’s r , and y axis shows −log 10 ( P value). A dotted line indicates P = 0.05, and significant factors ( P < 0.05) are highlighted in red. ( C ) Heatmap of expression profiles of 12 candidate transcription factors across 25 IL-10–producing B cell populations, shown in log scale using RMA values. ( D and E ) qRT-PCR analysis of Il10 mRNA in A20 cells transfected with expression (D) or shRNA (E) vectors for NFATc1, NFATc2, and NFATc3. Results are expressed as fold difference relative to Il10 mRNA levels in unstimulated A20 cells. ( F and G ) Luciferase reporter assay in A20 cells transfected with the pXPG vector containing CNS-9. Results are expressed as relative luciferase activity normalized to the unstimulated condition using a vector with only the minimal promoter. (F) Three NFAT motifs in CNS-9 were mutated individually or additively, and N and X denote NFAT-binding motifs and their mutations, respectively. (G) A pXPG vector containing WT or mutated CNS-9 was cotransfected with an NFATc1 expression vector or a mock vector into A20 cells. ( H ) ChIP assay in mouse B cells with anti-NFATc1 antibody. Immunoprecipitated DNA was quantified by qRT-PCR and normalized to input DNA. Relative enrichment was compared with IgG controls. [(D) and (H)] Data are representative of three independent experiments with similar results. Data are presented as means ± SEM. Statistical analysis was performed using a two-tailed unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. ns, not significant.

Article Snippet: A20 and Raji B cell lines were obtained from the American Type Culture Collection (MD, USA).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Transfection, shRNA, Luciferase, Reporter Assay, Plasmid Preparation, Activity Assay, Binding Assay, Immunoprecipitation, Two Tailed Test