Fig. 4.

UBXN3B is crucial for mature B cell survival. ERT2-Cre⁺Ubxn3b^flox/flox mice were treated with tamoxifen (TMX) (Ubxn3b^−/−) or corn oil (Ubxn3^+/+) every two days for 7 days. a) At 14 days after the first treatment, the whole transcriptomes in the bone marrow mature B fraction (N = 2 Ubxn3^+/+, 3 Ubxn3b^−/−) are analysed by bulk RNA-seq. Shown are the top pathways enriched from significantly downregulated genes in Ubxn3b^−/−. The statistical method for p-values is Binomial Test, and for FDR (false discovery rate) is Benjamini-Hochberg. b) The blood B cell ratios of TMX to corn oil-treated ERT2-Cre⁺Ubxn3b^flox/flox mice at various timepoints. Data point: mean ± SD, N = 4 mice/genotype, ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 [repeated measures two-way ANOVA Bonferroni's multiple comparison test with Greenhouse-Geisser correction]. c) The flow cytometry gating strategy and d) percentage of apoptosis (Annexin V positivity) in the splenic B, T, or myeloid populations at 14 days after the first TMX/oil treatment. N = 3 mice/genotype, ∗p < 0.05 [multiple unpaired Welch t-tests with Holm-Šídák correction]. e) Immunofluorescence staining for apoptotic (TUNEL) and B cells (CD19) in the spleens of mice at 14 days after the first TMX/oil treatment. Objective: 20×. Scale bar: 50 μM. DNA is stained with DAPI. f) The numbers of TUNEL-positive cells per microscopic field. Each symbol = one field. N = 11 fields from 3 Ubxn3^+/+, N = 17 fields from 3 Ubxn3b^−/− spleens. ∗∗∗∗p < 0.0001 [unpaired Welch t-test]. g, h) The immunoblots of indicated proteins in g) splenic B and non-B immune cells, h) splenic B cells stimulated with an anti-mouse IgM antibody or isotype control. Spleens are from the mice at 14 days after the first TMX/oil treatment.