Figure 2.
Endothelial PFKFB3 Is Crucial for M2-like Polarization of Macrophages in the Muscle
(A–C) Total number of neutrophils (A), monocytes (B), and macrophages (C) in muscle at the indicated times after HLI determined by flow cytometry.
(D) Quantification of EdU+ monocytes (% of total monocytes) at the indicated times after HLI.
(E and F) Representative images of F4/80 immunostainings (red) and hoechst (blue) (E) and quantification of F4/80+ area (F) on muscle at 3 days.
(G) Mean fluorescent intensity (MFI) of Ly-6C, F4/80, and MERTK in muscle macrophages at the indicated times after HLI determined by flow cytometry.
(H) Representative flow cytometric analysis of CD206+ and Relmα+ cells in muscle 3 days after HLI.
(I and J) Quantification of Relmα+CD206+ (I) and Relmα−CD206+ (J) F4/80+CD11b+ macrophages.
(K and L) Representative histograms (K) and quantification of CD206 MFI (L) in the total muscle macrophage population 3 days after HLI.
(M and N) Representative immunostainings for F4/80 (green) and CD206 (red) on CD45+ cells (M) sorted from muscle 3 d after HLI and quantification of CD206 MFI (N).
(O and P) Representative images of CD206 immunostainings (red) and hoechst (blue) (O) and quantification of CD206+ macrophages number (P) 3 days after HLI.
(Q) Quantification of EdU+CD206+ macrophages (% of CD206+ macrophages) at 3 days after HLI determined by flow cytometry
(R and S). Principal-component analysis (R) and Volcano plot from RNA-seq analysis showing differential gene expression (S) of macrophages isolated from pfkfb3WT and pfkfb3ΔEC muscles 3 days after HLI (n = 3).
(T) Expression pattern of pro-inflammatory and anti-inflammatory markers genes in muscle macrophages 3 days after HLI (n = 3).
Arrowheads point at CD206+ cells (O). Scale bar, 50 μm. Student’s t test (two-tailed, unpaired) in (F), (I), (J), (L), (N), (P), and (Q) (∗p < 0.05). Two-way ANOVA with Tukey’s multiple comparisons test in (A), (B), (C), (D), and (G) (∗p < 0.05). Each dot represents a single mouse ([A], [B], [C], [D], [F], [G], [I], [J], [L], [N], [P], and [Q]). Bar graphs represent mean ± SEM. See also Figures S2 and S3.