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. 2023 Jan 5;13:1083119. doi: 10.3389/fimmu.2022.1083119

Figure 5.

Figure 5

Loss of HDAC1 prevents PB differentiation by perturbing the gene regulatory network that specifies PB versus B cell fate. (A) Experimental scheme of co-transduction for activated B cells transduced with combinations of retroviral particles expressing Hdac1-targeting sgRNA (BFP+) and Hdac2 or Bach2-targeting sgRNA (mCherry+). (B) Western blots for HDAC1 and HDAC2 proteins showing gene KO efficiencies of sgRNAs targeting Hdac1 and Hdac2 genes. SgRosa26-1 was used as a negative control. Beta-Tubulin used as a loading control. (C) FACS analysis of one technical repeat experiment at day 6 post transduction showing frequencies of CD138+ PBs in WT (GFP-, top) and Cas9 (GFP+, bottom) cells. (D) Scatter plot showing the ratio of GFP+ (Cas9)/GFP- (WT) PBs corresponding to the experiment shown in (C) plus two independent repeat experiments. Each dot corresponds to one independent biological repeat experiment averaged over two technical replicates. (E) FACS analysis of one representative technical replicate of three independent experiments showing frequencies of GFP+ B cells in Hdac1 KO (sgHdac1-1, blue), Hdac2 KO (sgHdac2-2, black), or Hdac1 and Hdac2 double KO (dKO) (sgHdac1-1 and sgHdac2-2, red) cell populations in a single culture well, at day 2 and 6 post co-transduction. (F) Western blots showing expression levels of HDAC1, BLIMP1, IRF4, BACH2 and PAX5 transcription factors in sorted CD138- activated B cells (aB) and CD138+ PBs (PB) treated with indicated sgRNAs. Beta-Actin was used as a loading control. (G) Pre-gating (left) on Hdac1 KO (sgHdac1-1, blue), Bach2 KO (sgBach2-1, black), or Hdac1 and Bach2 dKO (sgHdac1-1 and sgBach2-1, red) cell populations present in a single culture well for the FACS plot (middle) showing frequencies of CD138+ PBs in WT (GFP-, top) and Cas9 (GFP+, bottom) cells in each pre-gated cell population. Scatter plot (right) showing the ratio of GFP+/GFP- PBs corresponding to the experiment shown on the middle (FACS analysis; one technical replicate of three) plus independent biological repeat experiments with different sgRNA combinations (n=3 independent experiments averaged over three technical replicates each; horizontal line indicates median).