Figure 5.

Absence of AIF1 restrains hematopoietic progenitor differentiation into cDC1 lineage commitment. Bone marrow cells were harvested and silenced for AIF1 by CRISPR RNAi approaches prior to differentiation into DC using Flt3-L as stimuli for 9 days. CRISPR Cas9 plasmids with either gRNA targeting AIF1 (pAIF1) or non-targeting scrambled gRNA (pControl) were used to transfect the cells. Cells were collected and stained with respective antibodies and Live/Dead viability dye to discriminate and gate on the Live⁺ population. (A) To assess common myeloid progenitor (CMP) profiles, dot plots show SCA1 expression in cells gated on Live⁺Lin⁻CD117⁺FcγR⁻CD34⁺ subset. (B) CD11b vs PDCA1 was utilized to assess pDC, where pDC are CD11b-PDCA1⁺. (C) cDC profile shows CD11c vs. MHC class II gated on total Live⁺ cells. CD24 vs CD172a were evaluated on the gated CD11c⁺MHC class II⁺ subsets. Next, cells were assessed for Zbtb46 vs. IRF4 or IRF8. (D) Real-time PCR analysis was performed on control or AIF1 silenced group to assess AIF1, PU.1, Id2, Zbtb46, IRF4, IRF8, BATF3, RelB, and CIITA. Data is representative of six independent experiments. Error bars for all figures indicate standard errors; ^**< 0.01 and NS, not significant.