Supplementary Figure 3: Tracking resident cardiac macrophages post myocardial infarction.

a-f) 3-week-old Cx3cr1^CreER–YFP:R26^Td mice were fed tamoxifen (TAM)- containing chow for 10 days, and then mice were either left as non-infarcted controls for an additional 6 weeks before a myocardial infarction was performed (MI). a) Ischemic tissue was isolated at Day 2 and Day 28 post-MI and total resident cardiac macrophages (CD45⁺CD64⁺CD11b⁺Td⁺) were analyzed as TIMD4⁺ and TIMD4^– subsets per mg/tissue compared to non-infarcted controls (No MI). N=3; repeated independently 2 times with similar results. b) Total CD45⁺CD64⁺CD11b⁺ cardiac macrophages (MFs) were partitioned into TIMD4⁺, CCR2⁺ and TIMD4^–CCR2^– MF subsets. LYVE1 was detected by flow cytometry. N=3; repeated independently 2 times with similar results. c) Representative images of hearts isolated, fixed and sectioned longitudinally for immunofluorescence from Cx3cr1^CreER–YFP:R26^Td mice post-MI. Td expression was induced as in panel (a) above. Cardiac macrophages (CD68⁺, white) were counted based on their coexpression for the residency marker Td (red) and LYVE1 (green) in the infarct and peri-infarct regions, defined in Fig. 4a, from 3 individual images/region/mouse. N=3; one experiment. d) Hearts were isolated from the ischemic zone of Cx3cr1^CreER–YFP:R26^Td/DTR mice day 4 post-MI after a 2 hr-BrdU pulse. Total Td⁺ cardiac macrophages were subdivided into TIMD4⁺ and TIMD4^– MF populations and %BrdU⁺ macrophages was quantified. N=3,3,4,4; one experiment. e) Heat map of the 20 most differentially expressed genes from clusters from Fig. 5b. Overlapping clusters (#1 to #6a), represent macrophages from control and post-MI samples that clustered together. For the overlapping clusters both the control and MI cells from each cluster are aligned next to each other in the heatmap. f) Means-normalized expression scatter plot produced by comparison of the differentially expressed genes from each unique cluster (7, 8, 9, 10 and 11) in Fig. 5b to the ImmGen data set. For all: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; two-tailed Student’s T test; center value, mean; error bars, SEM.