Journal: Cell Discovery

Article Title: NF-κB RelB suppresses the inflammatory gene expression programs of dendritic cells by competing with RelA for binding to target gene promoters

doi: 10.1038/s41421-024-00767-9

Figure Lengend Snippet: a Representative image of IFNAR − / − RelB − / − mouse (right) and IFNAR − / − littermate (left) at 4 weeks of age; ruler for scale. b Representative image of thymi from IFNAR − / − RelB − / − mouse (right) and IFNAR − / − littermate (left). c Thymic weights from age-matched WT (dark blue), RelB − / − (dark red), IFNAR − / − (light blue, checkered), IFNAR − / − RelB − / − (light red, checkered) mice. ***P < 0.001, **P < 0.01, *P < 0.05; error bars indicate SD. Statistical analysis was done using an unpaired two-tailed Student’s t -test. d , e Representative images of hematoxylin and eosin (H&E)-stained sections of WT, RelB − / − , IFNAR − / − , and IFNAR − / − RelB − / − mouse lungs ( d ) and livers ( e ). n = 3–4.

Article Snippet: The RelB DB/DB mutant mouse line was generated by Ingenious Targeting Laboratory (iTL, www.genetargeting.com ) using the targeted Neomycin selection cassette for the conventional knockin model. A donor sequence encoding the mutant RelB protein was used to generate, via homologous recombination, a tagged embryonic stem cell line carrying a Neo resistance marker, that was implanted to yield heterozygous mice.

Techniques: Two Tailed Test, Staining

Journal: Cell Discovery

Article Title: NF-κB RelB suppresses the inflammatory gene expression programs of dendritic cells by competing with RelA for binding to target gene promoters

doi: 10.1038/s41421-024-00767-9

Figure Lengend Snippet: a Histogram of log 2 FC ( RelB −/− /WT) in RelA binding (RPKM) for all ChIP peaks with RPKM > 10. Descriptive statistics: mean, log 2 FC = −0.03; 90th percentile, log 2 FC = 0.62; 10th percentile, log 2 FC = −0.74. b Heatmap of z -scored RelA- and RelB-ChIP peaks (RPKM) in all RelB-bound peaks in WT and RelB −/− BMDCs. Peaks selected for all LPS (10 ng/mL)-induced RelB-ChIP binding events (log 2 FC > 1) at 1 h LPS stimulation time point in WT BMDCs, RPKM > 10 in any condition. Heatmaps for RelA- and RelB-ChIP-seq data are ordered by RelA k-means clustering and separately z -scored. c Scatter plot showing the means of log 2 FC ( RelB −/− /WT) in RelA binding for clusters A–C from b . d Top de novo motif analysis results for genomic regions from clusters A–C from b .

Article Snippet: The RelB DB/DB mutant mouse line was generated by Ingenious Targeting Laboratory (iTL, www.genetargeting.com ) using the targeted Neomycin selection cassette for the conventional knockin model. A donor sequence encoding the mutant RelB protein was used to generate, via homologous recombination, a tagged embryonic stem cell line carrying a Neo resistance marker, that was implanted to yield heterozygous mice.

Techniques: Binding Assay, ChIP-sequencing

Journal: Cell Discovery

Article Title: NF-κB RelB suppresses the inflammatory gene expression programs of dendritic cells by competing with RelA for binding to target gene promoters

doi: 10.1038/s41421-024-00767-9

Figure Lengend Snippet: a Histogram of filtered highest FC RelA-ChIP peaks for all genes. Peaks were annotated to the nearest gene promoter region and filtered for the highest log 2 FC ( RelB −/− /WT) in RelA binding (RPKM) for each individual gene; all peaks are RPKM > 10. Descriptive statistics: mean, log 2 FC = 0.24; 90th percentile, log 2 FC = 0.66; 10th percentile, log 2 FC = −0.22. b Scatter plot showing the log 2 FC ( RelB −/− /WT) in RelA binding (RPKM) at 1 h LPS stimulation compared to all CpG-induced genes (Log 2 FC > 1) in WT or RelB −/− BMDCs, 8 h gene expression time point shown. Previously characterized IFN-independent hyper-expressed genes in RelB −/− are highlighted in red, and all other induced genes are colored gray. c Scatter plot comparing the log 2 FC ( RelB −/− /WT) in RelA binding (RPKM) of IFN-independent hyper-expressed genes and genes with unchanged expression (−0.1 < log 2 FC < 0.1) in RelB −/− relative to WT at 8 h CpG stimulation time point. d Representative genome browser tracks for ChIP peaks near promoter regions of TSS for IFN-independent hyper-expressed genes in RelB −/− . IGV tracks from WT BMDCs are represented in blue, and IGV tracks from RelB −/− BMDCs are represented in red. RelB −/− (red) and WT (blue) RelA-ChIP tracks (top row) are overlayed to provide a visual comparison.

Article Snippet: The RelB DB/DB mutant mouse line was generated by Ingenious Targeting Laboratory (iTL, www.genetargeting.com ) using the targeted Neomycin selection cassette for the conventional knockin model. A donor sequence encoding the mutant RelB protein was used to generate, via homologous recombination, a tagged embryonic stem cell line carrying a Neo resistance marker, that was implanted to yield heterozygous mice.

Techniques: Binding Assay, Gene Expression, Expressing, Comparison

Journal: Cell Discovery

Article Title: NF-κB RelB suppresses the inflammatory gene expression programs of dendritic cells by competing with RelA for binding to target gene promoters

doi: 10.1038/s41421-024-00767-9

Figure Lengend Snippet: a Crystal structure of the RelB DNA binding domain that guides the alanine substitutions at R117, Y120, and E123. b EMSA with pBABE puro (EV), RelB WT , RelB Y120A , and RelB DB/DB reconstitution in Relb −/− nfkb2 −/− 3T3 cells stimulated 30 min with TNF (1 ng/mL). c Representative image of RelB DB/DB mouse (left) and WT littermate (right) at 8 weeks of age; ruler for scale. d Spleen weights from age-matched WT (dark blue), RelB DB/DB (dark green), IFNAR −/− (light blue, checkered), IFNAR −/− RelB DB/DB (light green, checkered) mice. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s., not significant; error bars indicate SD. Statistical analysis was done using an unpaired two-tailed Student’s t -test. e , f Representative images from H&E - stained sections of WT, RelB DB/DB , IFNAR −/− , and IFNAR −/− RelB DB/DB lungs ( e ), and livers ( f ). n = 3–4.

Article Snippet: The RelB DB/DB mutant mouse line was generated by Ingenious Targeting Laboratory (iTL, www.genetargeting.com ) using the targeted Neomycin selection cassette for the conventional knockin model. A donor sequence encoding the mutant RelB protein was used to generate, via homologous recombination, a tagged embryonic stem cell line carrying a Neo resistance marker, that was implanted to yield heterozygous mice.

Techniques: Binding Assay, Two Tailed Test, Staining

Journal: Cell Discovery

Article Title: NF-κB RelB suppresses the inflammatory gene expression programs of dendritic cells by competing with RelA for binding to target gene promoters

doi: 10.1038/s41421-024-00767-9

Figure Lengend Snippet: a Heatmap of z -scored CPM of all RelB −/− hyper-expressed genes in WT, RelB −/− , RelB DB/DB , IFNAR −/− , and IFNAR −/− RelB −/− , IFNAR −/− RelB DB/DB BMDCs. Genes selected for CpG (0.1 µM) induced (log 2 FC > 1) in WT or RelB BMDCs at any time point and hyper-expressed (FC > 1.5) in RelB −/− BMDCs relative to WT BMDCs at any time point (271 genes). Each row represents individual genes, and each column is from an individual time point upon CpG stimulation. b Top result of known motif analysis for gene clusters from a . Motif analysis considered –1 kb to +1 kb with respect to the TSS, GO results for gene clusters from a . c Line graphs of gene expression (CPM) for IFN-β and ISGs upon CpG stimulation (0 h, 1 h, 3 h, and 8 h). A dark blue line (circle) represents WT BMDCs, a dark red line (square) represents RelB −/− BMDCs, dark green line (triangle) represents RelB DB/DB BMDCs, light blue line (inverted triangle) represents IFNAR −/− BMDCs, light red line (diamond) represents IFNAR −/− RelB −/− BMDCs, and light green line (circle) represents IFNAR −/− RelB DB/DB BMDCs. d Line graphs of gene expression (CPM) for IFN-independent hyper-expressed genes during CpG stimulation (0 h, 1 h, 3 h, and 8 h). A dark blue line (circle) represents WT BMDCs, a dark red line (square) represents RelB −/− BMDCs, a dark green line (triangle) represents RelB DB/DB BMDCs, the light blue line (inverted triangle) represents IFNAR −/− BMDCs, light red line (diamond) represents IFNAR −/− RelB −/− BMDCs, and light green line (circle) represents IFNAR −/− RelB DB/DB BMDCs.

Article Snippet: The RelB DB/DB mutant mouse line was generated by Ingenious Targeting Laboratory (iTL, www.genetargeting.com ) using the targeted Neomycin selection cassette for the conventional knockin model. A donor sequence encoding the mutant RelB protein was used to generate, via homologous recombination, a tagged embryonic stem cell line carrying a Neo resistance marker, that was implanted to yield heterozygous mice.

Techniques: Gene Expression

Journal: Scientific reports

Article Title: Changes in expression of nuclear factor kappa B subunits in the ovine thymus during early pregnancy.

doi: 10.1038/s41598-022-21632-3

Figure Lengend Snippet: Figure 1. Relative expression values of NF-κB1, NF-κB2 RelA, RelB, and c-Rel mRNA in the thymus from non- pregnant and pregnant ewes (n = 6 for each group). Note: DN16 = day 16 of the estrous cycle; DP13 = day 13 of pregnancy; DP16 = day 16 of pregnancy; DP25 = day 25 of pregnancy. Significance is denoted by letters; if two groups have different letters, this indicates that the difference between these groups was significant with P < 0.05.

Article Snippet: Blot analysis was performed with a mouse anti-NF-κB1 monoclonal antibody (Santa Cruz Biotechnology, Inc., sc-8414, 1:1000), a mouse anti-NF-κB2 monoclonal antibody (Santa Cruz Biotechnology, sc-7386, 1:1000), a mouse anti-RelA monoclonal antibody (Santa Cruz Biotechnology, sc-8008, 1:1000), a mouse anti-RelB monoclonal antibody (Santa Cruz Biotechnology, sc-166416, 1:1000), and a mouse anti-c-Rel monoclonal antibody (Santa Cruz Biotechnology, sc-6955, 1:1000) at 4 °C overnight, respectively.

Techniques: Expressing

Journal: Scientific reports

Article Title: Changes in expression of nuclear factor kappa B subunits in the ovine thymus during early pregnancy.

doi: 10.1038/s41598-022-21632-3

Figure Lengend Snippet: Figure 2. Expression of NF-κB1, NF-κB2 RelA, RelB, and c-Rel proteins in the thymus from non-pregnant and pregnant ewes (n = 6 for each group). Note: DN16 = day 16 of the estrous cycle; DP13 = day 13 of pregnancy; DP16 = day 16 of pregnancy; DP25 = day 25 of pregnancy. Significance is denoted by letters; if two groups have different letters, this indicates that the difference between these groups was significant with P < 0.05.

Article Snippet: Blot analysis was performed with a mouse anti-NF-κB1 monoclonal antibody (Santa Cruz Biotechnology, Inc., sc-8414, 1:1000), a mouse anti-NF-κB2 monoclonal antibody (Santa Cruz Biotechnology, sc-7386, 1:1000), a mouse anti-RelA monoclonal antibody (Santa Cruz Biotechnology, sc-8008, 1:1000), a mouse anti-RelB monoclonal antibody (Santa Cruz Biotechnology, sc-166416, 1:1000), and a mouse anti-c-Rel monoclonal antibody (Santa Cruz Biotechnology, sc-6955, 1:1000) at 4 °C overnight, respectively.

Techniques: Expressing