Fig. 1: VAT monocyte-derived and resident macrophages have different transcriptomic profiles.

A Flowcytometric analysis of the macrophage subsets in visceral adipose tissue of CX₃CR1^+/GFP mice which express GFP under the CX₃CR1promoter. B tdTomato⁺ (CX₃CR1^high) macrophages were quantified at day 7 after a single i.p injection of tamoxifen in CX₃CR₁^CreER/+ ROSA^tdTomato/+ mice that express YFP under the CX₃CR1 promoter and tdTomato in CX₃CR1-expressing cells upon tamoxifen injection using intravital microscopy (n = 6/group). C Parabiosis between C57BL/6 and CX₃CR1^GFP/+ mice was performed. Flow cytometry was conducted to enumerate chimerism in the macrophage subsets in the C57BL/6 mice six months after parabiosis. (n = 5 for CX₃CR1⁻ and 6 for CX₃CR1⁺). D Heatmap displaying the genes with at least a two-fold difference between the VAT macrophage subsets and with FDR < 0.01 (n = 3/group). E–G Heatmaps displaying the expression of the genes using bulk RNA sequencing comparing CX₃CR1⁺CCR2⁺ and CX₃CR1⁻ CCR2⁻ macrophages (n = 3/group) and CD206⁻ and CD206⁺ macrophage subsets of VAT (n = 3/group). H q-PCR quantification of the genes associated with glycemia and diabetes in CX₃CR1⁺CCR2⁺ and CX₃CR1⁻ CCR2⁻ macrophages sorted from VAT of lean mice (n = 6–12/group). I Bar graph representing the frequency of CX₃CR1⁺CCR2⁺ and CX₃CR1⁻ CCR2⁻ VAT macrophages enriched in the insulin sensitivity and resistance genes shown in (G, H) (n = 3-6 /group). J PCA plot showing the relations among the genes responsible for insulin sensitivity, survival, resident macrophage (ATM) markers, inflammation, insulin resistance, and monocyte-derived macrophages (MDM) markers in the VAT macrophage subsets using bulk RNA sequencing. K, L Frequencies of CCR2⁺ and CCR2⁻ macrophage subsets in human VAT as measured by flow cytometry (K) (n = 5/group) and confocal microscopy (L) (n = 15 for lean and 13 for obese). M–O Quantification of the VAT macrophage subsets in HFD-fed mice by flow cytometry (n = 4/group) (M) and serial intravital microscopy (Scale bar = 10 µm) (N, O) was performed in lean and obese CX₃CR1^CreER/+ ROSA^tdTomato/+ mice (n = 3 for CD, 4 for HFD 2 months, and 5 for HFD 4 months). P–R Apoptosis in VAT resident and monocyte-derived macrophages in lean and obese mice was examined using annexin V by flow cytometry (P) (n = 4/group), and caspase 3 staining by flow cytometry (Q) (n = 5 for CD and 4 for HFD/group) and confocal microscopy (R) (n = 14/group). S Quantification of the VAT macrophage subsets in lean and obese CX₃CR1^creER/+ ROSA^tdTomato mice before and after removal of HFD (n = 7/group). T Evaluation of Ki-67⁺ VAT resident macrophages after HFD withdrawal (n = 10/group). Mean ± s.e.m. *P < 0.05, ** P < 0.01, ***P < 0.001. The Mann–Whitney test (two-tailed) was used to determine the significance between two groups. One-way ANOVA with Bonferoni’s post hoc correction test was performed to determine differences among data obtained from more than two groups (Fig. 1O and S).