Journal: Nature Communications

Article Title: Plasma cell differentiation is regulated by the expression of histone variant H3.3

doi: 10.1038/s41467-024-49375-x

Figure Lengend Snippet: a Quantitative RT-PCR of mRNA encoding H3.1/H3.2 ( H3c1 / H3c2 ) and H3.3 ( H3f3a and H3f3b ). B cells in spleen and CD138 hi GFP + plasma cells (PC) in spleen or bone marrow were isolated from naive Prdm1 gfp/+ mice. On the left, the frequency of plasma cells is shown. b Quantitative RT-PCR of mRNA encoding H3.1/H3.2 and H3.3 in naive splenic B cells or CD138 hi B220 low plasma cells cultured with LPS for 4 days. c Western blotting analysis of Hydroxyapatite (HAP)-purified chromatin from naive splenic B cells or LPS-induced plasma cells (PC) with antibodies specific for H3.1/H3.2 and H3.3. d Representative flow cytometry plots to analyze the cell-cycle status of CTV-labeled B cells from Prdm1 gfp/+ mice on day 4 after LPS stimulation. Quantitative RT-PCR of mRNA encoding H3.1 and H3.3 in B cells at each stage of cell division. 0 (D0) and over 6 times division fraction among GFP − CD138 − activated B cells (D6) and GFP + CD138 + plasma cells (PC) among over 6 times division fraction were sorted. Examples of cells were pre-gated on live (PI − ) single lymphocytes ( a , b , d ). e Western blotting analysis of LPS-induced PC and activated B cells with antibodies specific for H3.1/H3.2 and H3.3. Data are pooled from two ( n = 3) ( a ) or three ( n = 4) ( d ) independent experiments or representative of two independent experiments ( b ; n = 4, c , e ; n = 2). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns not significant. The p values were obtained by one-way ANOVA with Tukey’s post hoc test ( a , d ) or a two-tailed unpaired t -test with Welch’s correction ( b ). Source data and exact p values ( a , b , d ) are provided as a Source Data file.

Article Snippet: 5 M NaCl was then added to achieve a concentration of 0.5 M, and the samples were transferred to a spin column Mobicol “Classic” (MoBiTec GmbH), including hydroxyapatite resin (20 mg; Nacalai Tesque Inc.) pre-washed with hydroxyapatite buffer 1 (HAPB1; 5 mM NaPO 4 , 600 mM KCl and 1 mM EDTA).

Techniques: Quantitative RT-PCR, Clinical Proteomics, Isolation, Cell Culture, Western Blot, Purification, Flow Cytometry, Labeling, Two Tailed Test