Journal: Nature Communications

Article Title: Germinal center output is sustained by HELLS-dependent DNA-methylation-maintenance in B cells

doi: 10.1038/s41467-023-41317-3

Figure Lengend Snippet: a Single-cell transcriptional analysis of day 7 and 14 GC B cells. UMAP displaying 429 day-14 GC B, 466 day-7 GC B Mb1Hells KO , 414 day-14 GC B, and 448 day-7 GC B control cells, colored by shared nearest neighbor clusters collected. Bar charts show the frequencies of GC B cells within the 4 clusters. n = 2 mice for each genotype and timepoint; one of the two control mice for day-7 GC B cells was Mb1 Cre/WT Hells F/WT . b Selected gene expression for the 4 different clusters shown in ( a ), presented in dot plot scaled on normalized UMI counts. c Representative CD98 staining of CD95 + GL7 + CCR6 neg CD38 neg GC B cells and quantification of CD98 geometric MFI (Mean Fluorescent Intensity). d Representative intracellular ATF4 staining within CD95 + GL7 + CCR6 neg GC B cells and quantification of ATF4 geometric MFI (Mean Fluorescent Intensity). e Representative CD98 staining of B220 + day-8 iGB cell cultures and quantification of CD98 geometric MFI (Mean Fluorescent Intensity). f Expression of Asns and Chac1 in sorted B220 + CD138 neg day-8 iGB cells measured by RT-qPCR after normalization to Ubc . g Representative CD98 staining of GFP + and GFP neg day-8 iGB cell cultures after transduction of empty or ATF4 coding retroviral vectors, and quantification of CD98 geometric MFI (Mean Fluorescent Intensity). h GSEA showing enrichment of ASC signature in Mb1Hells KO vs. control day 14 GC B cells. The ASC signature consists in the top 200 genes upregulated in GSE60927. NES normalized enrichment score, FDR false discovery rate. n = 4 for each genotype in ( c ), ( d ), ( e ), ( f ) and ( h ); experiments were done twice and one representative experiment is shown. n = 5 for ( g ); experiment was done twice and data were pooled. Bar charts and error bars represent the mean ± SD. Unpaired two-tailed t -test were performed for ( c ) to ( g ). Source data are provided in Source Data File.

Article Snippet: Murine ATF4 coding sequence without upstream ORFs was PCR-amplified from Mouse ATF4 (CHOP11/cATF)-WT vector, a gift from David Ron (Addgene plasmid # 21845; http://n2t.net/addgene:21845 ; RRID:Addgene_21845), and cloned into Bgl II and Xho I sites of the pMIG retroviral vector, a gift from William Hahn (Addgene plasmid # 9044; http://n2t.net/addgene:9044 ; RRID:Addgene_9044), to generate pMIG-ATF4 expression vector.

Techniques: Control, Gene Expression, Staining, Expressing, Quantitative RT-PCR, Transduction, Retroviral, Two Tailed Test