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
ATCC is a verified supplier 
ATCC manufactures this product 
![( 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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8534/pmc13108534/pmc13108534__sciadv.aec7779-f2.jpg)