Figure 3. Bhlhe40 regulates a distinct set of genes related to alternative activation in LPMs.

a,b, Gene expression microarray data were analyzed for genes differentially expressed by ≥2-fold (a) and expression of a macrophage alternative activation gene signature in Bhlhe40^+/+ and Bhlhe40^−/− LPMs (b). c, Flow cytometry of Embigin-1 expression and quantitation of geometric mean fluorescence intensity (gMFI) on LPMs (pooled from 4 experiments, n=8 Bhlhe40^+/+, 7 Bhlhe40^−/−; 1 experiment, n=3 LysM-Cre⁻ Bhlhe40^fl/fl, 4 LysM-Cre⁺ Bhlhe40^fl/fl). d, Flow cytometry of Clec10a expression and frequency of Clec10a⁺ LPMs (pooled from 6 experiments, n=19 Bhlhe40^+/+ and 21 Bhlhe40^−/−; 4 experiments, n=10 LysM-Cre⁻ Bhlhe40^fl/fl and LysM-Cre⁺ Bhlhe40^fl/fl). e Flow cytometry of Lyve-1 expression and frequency of Lyve-1⁺ LPMs (pooled from 7 experiments, n=22 Bhlhe40^+/+, 24 Bhlhe40^−/−; 5 experiments, n=11 LysM-Cre⁻ Bhlhe40^fl/fl and LysM-Cre⁺ Bhlhe40^fl/fl) from Bhlhe40^+/+, Bhlhe40^−/−, LysM-Cre⁻ Bhlhe40^fl/fl, and LysM-Cre⁺ Bhlhe40^fl/fl LPMs. f,g, MSigDB C5 gene set enrichment was analyzed using the lists of genes expressed at ≥1.5-fold in Bhlhe40^+/+ vs. Bhlhe40^−/− (f) or Bhlhe40^−/− vs. Bhlhe40^+/+ LPMs (g). h, qRT-PCR of Maf and Mafb expression relative to Hprt in Bhlhe40^+/+ and Bhlhe40^−/− LPMs (pooled from 2 experiments, n=5 Bhlhe40^+/+, 4 Bhlhe40^−/−). i, Gene expression microarray data were analyzed for shared and unique Bhlhe40-dependent genes in Bhlhe40^+/+ and Bhlhe40^−/− LPMs and AMs (≥2-fold differentially expressed, depicted as a Venn diagram). Heat map depicts all genes differentially expressed by ≥2-fold in Bhlhe40^+/+ and Bhlhe40^−/− AMs. Microarray data from LPMs (n=3/group) and AMs (n=2/group) are from a single experiment. Data in c-e,h are mean ± s.e.m; each symbol represents an individual mouse (c-e,h). *P ≤ 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, (c-e,h) unpaired two-sided Student’s t-test and (f,g) one-sided hypergeometric test.