Supplemental Figures Development of a new macrophage-specific TRAP mouse (MacTRAP) and definition of the renal macrophage translational signature Andreas Hofmeister, Maximilian C. Thomassen, Sabrina Markert, André Marquardt, Mathieu Preußner, Martin Rußwurm, Ralph Schermuly, Ulrich Steinhoff, Hermann-Josef Gröne, Joachim Hoyer, Benjamin D. Humphreys, Ivica Grgic Correspondence: Ivica Grgic MD, Klinikum der Philips-Universität Marburg, Baldingerstrasse 1, 35043 Marburg. Phone: +4964215861736, email: [email protected] Sup. Fig. S1 Δ6.7fmsGFP-L10a plasmid (pGL2 backbone) Sup. Fig. S1: Plasmid map of the engineered c-fms-eGFP-L10a expression vector. Mlu1/Sal1 digestion was used for linearization and extraction of the transgene. Sup. Fig. S2 A Neutrophils Monocytes Lymphocytes B Monocytes Monocytes Neutrophils Lymphocytes H - TRAP Ly6g Count FSC CD115 Mac GFP GFP GFP GFP H - Ly6g Count FSC CD115 Wild Wild type GFP GFP GFP GFP Sup. Fig. S2: FACS analysis detects eGFP-L10a signals in monocytes but not in neutrophils or lymphocytes isolated from peripheral blood of MacTRAP mice. (A) Gating strategy to define monocyte, neutrophil and lymphocyte populations. Only single cells contributed to the analysis. (B) GFP-fluorescence was specifically detected in CD115+ monocytes, but not in Ly6g+ neutrophils or lymphocytes of MacTRAP mice. Blood samples from wild-type mice served as negative controls. Representative plots are shown, n=6. Sup. Fig. S3 A eGFP-L10a Ly6g merge+DAPI kidney 7d UUO B eGFP-L10a Ly6g merge+DAPI incision skin Tail Sup. Fig. S3: Immunostaining for mature neutrophils in fibrotic kidney tissue and tail skin biopsies from MacTRAP mice. (A) Only a very small fraction of Ly6g+ neutrophils is positive for eGFP-L10a in fibrotic kidneys after 7d UUO (0.42% ± 0.29%; 468 cells counted, n=3). (B) Immunostaining of tail skin biopsies shows that only a very small fraction of Ly6g+ neutrophils is double-positive for eGFP-L10a (0.98% ± 0.52%; 517 cells counted, n=3). Scale bar: 20µm. Sup. Fig. S4 Spleen eGFP-L10a F4/80 eGFP-L10a F4/80 DAPI merge DAPI merge Lung eGFP-L10a F4/80 eGFP-L10a F4/80 α-laminin merge + DAPI α-laminin merge + DAPI Liver eGFP-L10a F4/80 eGFP-L10a F4/80 DAPI merge DAPI merge Sup. Fig. S5 Skin eGFP-L10a F4/80 eGFP-L10a F4/80 α-laminin merge + DAPI α-laminin merge + DAPI Heart eGFP-L10a F4/80 eGFP-L10a F4/80 DAPI merge DAPI merge Aorta eGFP-L10a F4/80 eGFP-L10a F4/80 DAPI merge DAPI merge Sup. Fig. S4+S5: Detection of eGFP-L10a+ cells in various other organs and tissues of MacTRAP mice by fluorescence microscopy. EGFP-L10a expressing cells were also identified in other organs including spleen, lung, liver, skin, heart and aorta. Immunostaining confirmed macrophage-specificity (costaining with macrophage surface marker F4/80, red) and interstitial localization (highlighted by anti-laminin staining, white). Scale bar: 20µm. Sup. Fig. S6 28S rRNA peak MacTRAP units WT 18S rRNA peak Marker peak Fluorescence [nt] Sup. Fig. S6: Quality control of TRAP extracted RNA. Representative Agilent 2100 Bioanalyzer run of bound TRAP-RNA from a MacTRAP mouse kidney (red) and WT control (blue). Only TRAP-RNA samples with an RNA integrity number (RIN) >9 indicating excellent quality were used for downstream analyses including RNA-Seq; nt=nucleotides. Sup. Fig. S7 MacTRAP Liu et al. Mass et al. Sup. Fig. S7: Venn diagram between MacTRAP-generated translational profile of adult kidney macrophages and two published kidney macrophage-enriched expression profiles. We cross- referenced the MacTRAP dataset (≥ 2 fold enriched; p < 0.05) with data from Liu et al, 2014 (microarray analysis, ≥ 2 fold enriched, p < 0.05; kidneys from 7 week old Lyz2-L10a mice with Cre-mediated, irreversible recombination) and data published by Mass et al, 2016 (bulk RNA-Seq, top 453 enriched genes; FACS- based approach extracting CD45+, CD11b+ and F4/80+ macrophages from p21 mouse kidneys. 111 genes were co-enriched among all datasets, featuring classical macrophage marker genes such as Csf1r, CD68, CD86, Cd14 and Lyz2 (see also Sup. Table 2). Sup. Fig. S8 A B Cluster 1: Immunity Cluster 2: Actin binding Sup. Fig. S8: DAVID Functional annotation clustering of renal Macrophage translational profile derived from MacTRAP and RNA Seq. Examples of 2D functional annotation clusters. A) Annotation cluster 1: Immunity B) Annotation cluster 2: Actin binding. Full analysis is presented in Supplemental Table 8. Sup. Fig. S9 Sup. Fig. S9: Interaction network of genes with strongest enrichment in MacTRAP kidney macrophages (≥ 8 fold, p < 0.05) generated by StringDB. Red nodes represent genes enriched in immune system processes; blue nodes correspond to actin filament based processes. Sup. Fig. S10 A B MacTRAP + ctrl CLK UUO + - eGFP-L10a -actin GAPDH C Sup. Fig. S10 (A) Full-length Western blots shown in Fig. 1H. Blots were loaded with spleen lysates from MacTRAP mice and lysates from WT controls. Upper blot was probed with an anti-GFP antibody (expected height of the eGFP-L10a band is indicated); lower blot was probed with an anti--actin antibody for loading control. (B) Full-length Western blots shown in Fig. 3D. Blots were loaded with protein lysates of 7d UUO and contralateral (CLK) controls kidneys from MacTRAP mice. Upper blot was probed with an anti-GFP antibody (expected height of the eGFP-L10a band is indicated); lower blot was probed with an anti-GAPDH antibody for loading control. Kidney lysates from PodoTRAP (Grgic et al. 2014) and WT mice served as positive (+) and negative (-) controls for eGFP-L10a. (C) Full-length Coomassie gel depicted in Fig. 2C. Supplementary Methods FACS: Peripheral blood from mice was obtained from the fascial vein and added to 1 ml HBSS prep (0.5% FCS [v/v], 20 mM HEPES in 1x PBS), supplemented with 80 μl heparin (25 I. E./ ml). For the following wash steps, the cells were centrifuged at 300g and 4 °C for 10 min. For erythrocyte lysis, the cell suspension was incubated with 5.0 ml NH4Cl2 at RT for 5 min. After washing with PBS containing 1% FCS, FcγR mouse blocking reagent (Miltenyi) was added according to manufacturers instructions to block unspecific antibody binding to surface FcγRs. For antibody staining, antibodies were diluted in PBS containing 1% FCS and incubated for 20 min at 4°C. Cells were washed with PBS containing 1% FCS for removal of unbound antibodies and subsequently sorted by flow cytometry (Attune NxT cytometer, Thermo Fisher). The data was analyzed using FlowJo software (BD). The following antibodies were used for FACS: rat anti-CD115-BV421 clone AFS98 (BioLegend, 1:300) and rat anti-Ly6g-BV510 clone 1A8 (Biolegend, 1:300). Supplemental Tables Development of a new macrophage-specific TRAP mouse (MacTRAP) and definition of the renal macrophage translational signature Andreas Hofmeister, Maximilian C. Thomassen, Sabrina Markert, André Marquardt, Mathieu Preußner, Martin Rußwurm, Ralph Schermuly, Ulrich Steinhoff, Hermann-Josef Gröne, Joachim Hoyer, Benjamin D. Humphreys, Ivica Grgic Correspondence: Ivica Grgic MD, Klinikum der Philips-Universität Marburg, Baldingerstrasse 1, 35043 Marburg. Phone: +4964215861736, email: [email protected] • Supplemental Table 1: MacTRAP Gene expression list (≥ 2x enriched, p < 0.05, 1448 Genes) • Supplemental Table 2: Core macrophage transcripts (Venn diagramm) • Supplemental Table 3: Genes exclusively identified in MacTRAP dataset • Supplemental Table 4: DAVID functional classification chart • Supplemental Table 5: DAVID functional annotation chart • Supplemental Table 6: DAVID UP_TISSUE • Supplemental Table 7: DAVID UP_TISSUE complete • Supplemental Table 8: DAVID functional annotation clustering • Supplemental Table 9: StringDB GO Biological Processes enrichment • Supplemental Table 10: Primers used for RT-qPCR Supplemental Tables Development of a new macrophage-specific TRAP mouse (MacTRAP) and definition of the renal macrophage translational signature Andreas Hofmeister, Maximilian C. Thomassen, Sabrina Markert, André Marquardt, Mathieu Preußner, Martin Rußwurm, Ralph Schermuly, Ulrich Steinhoff, Hermann-Josef Gröne, Joachim Hoyer, Benjamin D. Humphreys, Ivica Grgic Correspondence: Ivica Grgic MD, Klinikum der Philips-Universität Marburg, Baldingerstrasse 1, 35043 Marburg. Phone: +4964215861736, email: [email protected] • Supplemental Table 1: MacTRAP Gene expression list (≥ 2x enriched, p < 0.05, 1448 Genes) • Supplemental Table 2: Core macrophage transcripts (Venn diagramm) • Supplemental Table 3: Genes exclusively identified in MacTRAP dataset • Supplemental Table 4: DAVID functional classification chart • Supplemental Table 5: DAVID functional annotation chart • Supplemental Table 6: DAVID UP_TISSUE • Supplemental Table 7: DAVID UP_TISSUE complete • Supplemental Table 8: DAVID functional annotation clustering • Supplemental Table 9: StringDB GO Biological Processes enrichment • Supplemental Table 10: Primers used for RT-qPCR Genes ≥ 2 fold enriched p < 0.05 in MacTRAP dataset 1448 Genes external_gene_name baseMean log2FoldChange fold change stat padj Dbn1 5157.23463 6.055316763 66.5015823 19.7637668 1.3821E-82 Myh10 75200.1435 5.980047341 63.1209643 19.4296918 4.8991E-80 Myh9 148852.005 6.290649544 78.2842157 19.0511511 4.8486E-77 Tmod3 22247.4356 5.895186095 59.5151928 18.775043 6.8374E-75 Gsn 27175.7209 5.441014589 43.4418786 18.5999761 1.455E-73 Myh11 4382.93456 5.874074879 58.6506373 16.8272627 5.811E-60 Actn4 52596.2114 5.145304547 35.390851 16.800618 7.8082E-60 Lmo7 8615.91714 4.852990021 28.8998484 16.3444555 1.3477E-56 Myo1e 10004.7452 4.571404327