Figure 3. Loss of DC subsets upon in vivo silencing of AIF1 in the pancreas.

Six-week-old NOD mice were injected i.p. with siRNA targeting AIF1 (siAIF1) (n = 6) or scrambled control (siScramble) (n = 5) oligonucleotides weekly for a total of 3 weeks. At the end of the 3 weeks, pancreas was harvested from treated NOD mice and prepared for flow cytometric analyses. (A) Cells were stained to assess alterations in the frequency of CD45⁺CD11b^–CD11c⁺F4/80^– DC and CD45⁺CD11b⁺CD11c^–F4/80⁺ macrophages upon in vivo silencing of AIF1. SSC-H and SSC-A evaluation was used to identify singlets. Dot plots show CD11b vs. CD11c gating and subgating to assess F4/80 vs. AIF1 expression in gated singlets. (B) The average percentage of AIF1 expression was determined in the siScramble vs. siAIF1 among a pool of nine NOD mice from each group across 3 independent experiments. Additionally, expression was assessed in key myeloid-associated genes from pancreatic cells sorted for (C) CD45⁺CD11b^–CD11c⁺F4/80^– DC and (D) CD45⁺CD11b⁺CD11c^–F4/80⁺ macrophages. Data set represents a pool of three siScramble and three siAIF1-treated mice from two independent experiments. Data are presented as mean ± SEM. (E) Assessment of MHC class II and CD11c coexpression within pancreas-resident CD45⁺CD11b^– subsets in siScramble vs. siAIF1 cohorts. Data are representative of 4 mice in 2 independent studies. (F) Further interrogation of monocyte infiltration through assessment of CD45⁺CD11c^–CD317^– fraction showing levels of F4/80 in CD11b^hiLy6C^hi and CD11b^hiLy6C^int monocytes. Figure data sets are representative of two independent experiments. (G) Bar graph presenting relative percentage of Ly6C⁺ monocytes within the CD45⁺CD11c^–CD317^– gated subsets in siScramble vs. siAIF1-treated NOD mice. Data are shown as mean ± SEM of 4 groups within 2 independent experiments. Statistical significance was determined by the 2-tailed Student’s unpaired t test. *P < 0.05; **P < 0.01. AIF1, allograft inflammatory factor-1; ns, not significant.