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. Author manuscript; available in PMC: 2020 Aug 5.
Published in final edited form as: Cell Rep. 2020 Jun 23;31(12):107815. doi: 10.1016/j.celrep.2020.107815

Figure 7. CD28 Induces Irf4 Expression in BMPCs, and Irf4 Levels Are Associated with BMPC Metabolic Regulation.

Figure 7.

(A) WT SP and BM MNC were stained for B220, CD138, and intracellular IRF4. Comparison of IRF4 expression between SP IRF4−/− B220+ cells and WT B220−CD138+ BMPCs and SPPCs. Representative of three independent experiments. ***p < 0.001.

(B) Purified BMPCs were treated with isotype control versus anti-CD28 mAb × 4 h in serum-free media. Irf4 mRNA expression was determined using qPCR with actin as a loading control; fold change in expression normalized to control-treated cells. Quantitation from pooled results from two independent experiments. *p < .05.

(C) Comparison of IRF4 protein expression between WT, IRF4+/− and IRF4−/− BMPCs. Data are representative of three independent experiments. **p < 0.01.

(D) BM (top panel) and SP (bottom panel) MNCs from age-matched WT, IRF4+/−, and IRF4−/− mice stained for B220 and CD138. Representative of three independent experiments.

(E) WT and IRF4+/− BM and SP MNCs were stained for B220, CD138, and NBDG. Representative of two independent experiments. *p < .05.

(F) Mitochondrial mass between WT and IRF4+/− BMPCs was evaluated by staining MitoTracker Green. Data are representative of three independent experiments. *p < .05.

(G) ROS levels between WT and IRF4+/− BMPCs determined by staining with CM-DCFDA. Data are representative of two independent experiments. **p < 0.01.

(H) BMPCs (2.5 × 103) of the indicated genotype were cocultured with WT BMDCs in a 1:2 ratio in serum-free media and assessed for viability using Live Dead Blue dye after 24 h. (Left) Total live PC quantification, (middle) percentage, and (right) total number of viable WT or IRF4 IRF4+/− BMPCs. ***p < 0.001, **p < 0.01, *p < .05.

(I) MM.1S cells transfected with either siRNA control 1 (siControl) or two different siRNA constructs (siIRF4-1, siIRF4-2). Left: IRF4 knockdown in three separate experiments; densitometry is relative to siControl for each experiment. Right: transfected cells were treated with control IgG or anti-CD28 mAb in full serum (FS) or no serum for 48 h, and survival was compared with FS siControl. *p < .05.

Statistical analysis by Student’s t test. Error bars represent ± SD.