Figure 4.

AIF1 silencing in bone marrow cells restrains macrophage differentiation. Bone marrow was collected from wild type mice prior to immediate transfection with Crispr-Cas9 plasmid carrying gRNA targeting AIF1 gene (pAIF1). Scramble gRNA served as control (pControl). Transfected bone marrow cells were stimulated for 6 days with M-CSF. (A) Western blot analysis was performed on pControl versus pAIF1 transfected bone marrow-derived macrophages to evaluate silencing of AIF1 expression. GAPDH served as internal loading control. Full-length blots/gels are presented in Supplementary Fig. 2. (B) Bar graph denotes percentage knockdown calculated using fluorescence intensity scale (ImageStudio 5.0). (C) For analogous studies, AIF1-silenced and control day 6 bone marrow-derived macrophages were collected and stained for CD11b, F4/80, and Ly6C prior to flow cytometric analysis. (D) Presence of Ly6C + monocytes were analyzed in both CD11b⁺F4/80^neg and CD11b⁺F4/80⁺ subsets. (E) Total RNA was isolated from AIF1-silenced or control bone marrow-derived macrophages differentiated under M-CSF stimulation for 6 days. Real-time PCR was performed to evaluate expression of: AIF1, IRF8, Id2, E2-2, RelB, IRF4, Nr4a1, C/EBPb, KL4 and PU.1. GAPDH was utilized as internal loading control. All data was normalized to the controls. Data shown as mean ± SEM and representative of at least three independent experiments. Statistical significance was determined by unpaired t-test. *p < 0.05 and **p < 0.01.