Supplementary Material. Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

Transcription

1 Supplementary Material Levels of protein drive the reparative process in acute muscle injury and muscular dystrophy Francesca Riuzzi 1,4 *, Sara Beccafico 1,4 *, Roberta Sagheddu 1,4, Sara Chiappalupi 1,4, Ileana Giambanco 1, Oxana Bereshchenko, Carlo Riccardi, Guglielmo Sorci 1,4 and Rosario Donato 1,3,4 1 Department of Experimental Medicine, Department of Medicine, 3 Centro Universitario per la Ricerca sulla Genomica Funzionale, 4 Istituto Interuniversitario di Miologia, Perugia Medical School, University of Perugia, Piazza Lucio Severi 1, 613 Perugia, Italy 1

2 Supplementary Materials and Methods Supplementary Table 1 Primary antibodies used in immunohistochemistry and immunofluorescence Antibodies Host animal Dilution Source Mouse 1:5 R&D Systems MyoD clone Santa Cruz Mouse 1:5 5.8A Myogenin Mouse 1:5 Santa Cruz RAGE Goat 1:5 Santa Cruz Ki67 Mouse 1: Cell Signaling Technology Collagen IV Rabbit 1:4 Novus Biologicals Rat 1:5 BD Biosciences CD163 Rabbit 1: Bioss NOS Rabbit 1:1 Santa Cruz Mouse 1:1 BD Biosciences FGFR1 Mouse 1:1 Chemicon Secondary antibodies used in immunofluorescence Host Against Fluorophore Dilution Source Donkey Mouse Alexa Fluor 488 1: Invitrogen Donkey Rabbit Alexa Fluor 488 1: Invitrogen Donkey Rabbit Alexa Fluor 594 1: Invitrogen Donkey Rat Alexa Fluor 594 1: Invitrogen

3 Supplementary Table Primary antibodies used in Western blotting Antibodies Host animal Dilution Source Mouse 1:5 R&D Systems MyoD clone 5.8A Mouse 1:1 Santa Cruz Myogenin Mouse 1:1 Santa Cruz RAGE Goat 1:1 Santa Cruz emyhc Mouse 1:5 Monosan Ciclin D1 Rabbit 1:1 Santa Cruz Collagen IV Rabbit 1: Novus Biologicals Rat 1:1 BD Biosciences CD163 Rabbit 1:1 Bioss NOS Rabbit 1:1 Santa Cruz Mouse 1:5 BD Biosciences FGFR1 Mouse 1:1 Chemicon phosphorylated- Mouse 1:1 Santa Cruz Tyr clone PY phosphorylated- (Thr18/Tyr18) p38 MAPK) phosphorylated (Thr/Tyr4) ERK1/ polyclonal ERK1/ phosphorylated ( Se473) Akt phosphorylated (Ser536) NFκB(p65) Rabbit 1:1 Cell Signaling Technology Rabbit 1:1 Cell Signaling Technology Rabbit 1: Sigma Aldrich Rabbit 1:1 Cell Signaling Technology Rabbit 1:1 Cell Signaling Technology NF-κB(p65) Rabbit 1:1 Santa Cruz Mouse 1:5 Santa Cruz α-tubulin Mouse 1:1 Sigma-Aldrich 3

4 Supplementary Table 3 Oligonucleotide primers for real-time PCR Target Gene Accession number Primer Sequence (5-3 ) Il1b NC_68.7 Fwd: TGACGTTCCCATTAGACAACTG Rev: CCGTCTTTCATTACACAGGACA Il4 NC_77.6 Fwd: ATTTTGAACGAGGTCACAGGAGAAG Rev: ACCTTGGAAGCCCTACAGACGAG Il6 M_31168 Fwd: GAACAACGATGATGCACTTGC Rev: CTTCATGTACTCCAGGTAGCTATGGT Il1 NM_1548. Fwd: CAAGGAGCATTTGAATTCCC Rev: GGCCTTGTAGACACCTTGGTC Il1Ra NC_75.6 Fwd: TCATTGCATACGGGACAGAA Rev: TGGATGTCATTCCAGGTTGA Il1a NC_69.6 Fwd: CGCAGCACTTCAGAATCACA Rev: TCTCCCACAGGAGGTTTCTG Tnfa NM_13693 Fwd:TCTTCTGTCTACTGAACTTCGGGGTGA Rev: GTGGTTTGCTACGACGTGGGCTA Nos NM_197 Fwd: AGCCAAGCCCTCACCTACTT Rev: TCTCTGCCTATCCGTCTCGT Arg1 NM_748 Fwd: CAATGAAGAGCTGGCTGGTGT Rev: GTGTGAGCATCCACCCAAATG Mrc1 NC_68.7 Fwd: TCTTTGCCTTTCCCAGTCTCC Rev: TGACACCCAGCGGAATTTC Cd68 NC_77.6 Fwd: CAAAGCTTCTGCTGTGGAAAT Rev: GACTGGTCACGGTTGCAAG Gapdh NM_884. Fwd: GCCTTCCGTGTTCCTACCC Rev: CAGTGGGCCCTCAGATGC Ifng NM_ Fwd: GACAATCAGGCCATCAGCAAC Rev: CGGATGAGCTCATTGAATGCTT Cd86 NM_ Fwd: TTGTGTGTGTTCTGGAAACGGAG Rev: AACTTAGAGGCTGTGTTGCTGGG Cd163a NM_5394. Fwd: GCAAAAACTGGCAGTGGG Rev: GTCAAAATCACAGACGGAGC Tgfb NM_ Fwd: GAGACGGAATACAGGGCTTTC Rev: TCTCGTGGAGCTGAAGCAAT Ccl NC_519.4 Fwd: GCTCAGCCAGATGCAGTTAAC Rev: CTCTCTCTTGAGCTTGGTGAC Ccr NC_75.6 Fwd: CCTGTAAATGCCATGCAAGTTC Rev: GTATGCCGTGGATGAACTGAG Dursp1 NC_715.6 Fwd: CAGATTAGGAGCAGCGAGC Rev: AAAGCGAAGAAGGAGCGAC S1b NC_76.6 Fwd: TGGCTGCGGAAGTTGAGATT Rev: GAAGGGGGTTGGGGTTTCAT 4

5 Supplementary Results Figure S1. Blocking early after acute muscle injury delays regeneration. (a) and S1A1 (5 μg each) were run on SDS polyacrylamide gels (15%) and either stained with blue Coomassie (top panel) or transferred onto nitrocellulose paper for western blotting using a polyclonal antibody (Abcam No. ab41548) (bottom panel). (b) CC1 myoblasts were cultured in differentiation medium in the absence or presence of ng /ml ± increasing doses of a polyclonal antibody (Abcam No. ab41548), as indicated, and left undisturbed for days. Then, cells were lysed and cell lysates were subjected to western blotting for detection of the late myogenic marker, embryonic myosin heavy chain (emyhc) (bottom panel). Immunoblots of α-tubulin are included as loading controls. Results are means ± SEM from three experiments (upper panel). *p<.5, p<.1, p<.1 vs. control. (c) TA muscles were injected with at d, followed by injection with or antibody at d1 p.i. Treated muscle were excised at d3 and d7 p.i. for analyses. (d,e) +, MyoD+, +, Ki67 +, and RAGE + cells were detected at d3 (d) and at d7 (e) p.i. in muscles by immunohistochemistry. The scale bar represents 5 μm in (d and e). Figure S. affects macrophages in acutely injured muscles. (a) TA muscles were treated as described in the legend to Fig a. Muscles were excised at d3 or d7 p.i. (b) Macrophages were detected by immunohistochemistry. (c) Immunofluorescence detection of in macrophages isolated at d3 p.i. from muscles treated with or antibody. (d) Homogenates of muscles excised at d3 or d7 p.i. were subjected to western blotting. Immunoblots of are included as loading controls. (e) Double inos/ and CD163/ immunofluorescence at day 7 p.i. of muscles treated with or antibody at day 1 p.i. Nuclei were counterstained with DAPI. Merged images are shown. Note in -treated muscles the high number of macrophages (red channel) and the co-existence of inos + (M1) and CD163 + (M) macrophages as opposed to the almost complete absence of M1 macrophages and the low number of M macrophages in -treated (control) muscles. Arrows point to double inos + / + (M1) and CD163 + / + (M) macrophages. (f) staining of injured muscles. (g) Peritoneal macrophages were cultured for 4 h in the absence and presence of either, IFN-γ, IL-1 or IL-4 and analyzed by real-time PCR. (h) Macrophages isolated at d3 and d7 p.i. from injured muscles and analyzed for S1b levels by real-time PCR. (i) Detection of in activated ( + ) macrophages and in M1 (inos + ) and M (CD163 + ) macrophages by double immunofluorescence in injured muscles. Nuclei were counterstained with DAPI. The scale bar represents 5 μm in b and f and 1 μm in e and i. Figure S3. regulates muscle regeneration by acting on both myoblasts and macrophages. (a) Mice were intraperitoneally injected with vehicle or clodronate. Injured TA muscles were injected with 5

6 or antibody (day 1 p.i.). Treated muscle were excised at d3 p.i. (b) Histology of muscle tissue (upper panel) and counts of interstitial cells (lower panel). (c) immunohistochemistry (upper panels) and + cell counts (lower panel). (d) Western blots of, CD163 and inos in muscle homogenates. Immunoblots of are included as loading controls. (e) staining. (f) +, MyoD +, + and Ki67 + cell counts. (g) +, MyoD +, + and Ki67 + cells were detected at d3 by immunohistochemistry. Results are means ± SEM from at least six animals. *p<.5, p<.1, p<.1 vs. control. ##p<.1 (clodronate-treated vs. vehicle, b and f). The scale bar in (b, c and e) represents 5 μm. Figure S4. is required during the macrophage M phase for efficient regeneration. (a) Injured TA muscles were injected with or antibody at d4 p.i. Treated muscle were excised at d7 or d14 p.i. (b) +, MyoD+, +, Ki67 +, and + cells were detected at d7 and at d14 p.i. in muscles by immunohistochemistry. (c) staining of injured muscles and collagen IV detection by immunohistochemistry of - and -treated injured muscles at d7. The scale bar represents 5 μm. Figure S5. s ability to promote regeneration of acutely injured skeletal muscles requires RAGE at early, but not mid-late regeneration phase. (a) Macrophages were isolated from wild type and Ager / TA muscles at d7 p.i. and analyzed for expression levels of the indicated genes by real-time PCR. Results are expressed as fold-change of gene expression levels in Ager / vs. wild type macrophages. (b) Injured Ager / TA muscles were injected with or antibody at d1 p.i and excised at d3 or d5 p.i. (c) ) Histology (upper panel) and counts of interstitial cells and centrally nucleated myofibers (lower panel). (d) +, MyoD +, +, + and Ki67 + cell counts. (e) Macrophages isolated at d3 from - and -treated injured Ager / muscles and analyzed by real-time PCR. (f) Peritoneal macrophages from Ager / mice subjected to a migration assay using Boyden chambers in the presence of increasing doses. (g,h) Ager / TA muscles were injected with BaCl at day, followed by injection with or antibody at d4 p.i. and excision at d7 p.i. (g) for histology and collagen IV immunohistochemistry (h). (i) +, MyoD +, +, Ki67 +, and + cells were detected at d7 p.i. by immunohistochemistry. Shown are representative images. The scale bar represents 5 μm. Figure S6. Late blockade of results in altered bfgf/fgfr1 signaling. (a,b) Conditions were as in Fig 5c (a). Ager / muscles were excised at d7 p.i. and analyzed for +, MyoD +, +, Ki67 +, and + cells by immunohistochemistry (b). Shown are representative images. The scale bar represents 5 μm. (c) Same as in a except that macrophages isolated from injured muscles were analyzed for levels of 6

7 proinflammatory and antiinflammatory markers by real-time PCR. (d) Macrophages isolated at d4 p.i. from injured Ager / muscles were incubated for 3 min in the absence or presence of ( ng/ml) and lysed. Cell lysates were subjected to immunoprecipitation with antibody and immunoprecipitates were probed with and FGFR1 antibodies. Figure S7. Full-length blots of cropped blots from the manuscript (relative to Figures 1-3). Figure S8. Full-length blots of cropped blots from the manuscript (relative to Figures 4-7). 7

8 a M 5 µg S1A1 5 µg b.5 CC1, 48 h DM emyhc 1, ng/ml µg/ml tubulin c Treatment Harvest Harvest C57Bl/6 Day 1 p.i. d MyoD Ki67 RAGE MyoD Ki67 RAGE Day 7 post- injury Day 3 post- injury e 96±.5 97±1. Figure S1

9 a C57Bl/6 b Treatment d1 p.i. Harvest Harvest d3 p.i. d7 p.i., day 3 p.i., day 7 p.i. g Relative Quantity, a.u , ng/ml IFN-γ, 1 ng/ml IL-1, 1 ng/ml IL-4, ng/ml Arg MØ, 4h Relative Quantity, a.u. 6 4 Cd163a Relative Quantity, a.u , ng/ml IFN-γ, 1 ng/ml IL-1, 1 ng/ml IL-4, ng/ml Nos Relative Quantity,a.u Cd c -treated muscle MØ -treated muscle MØ h MØ /DAPI d 9 inos CD163 i //DAPI Relative Quantity, a.u S1b 3 7 Day post-injury /inos/dapi Day 5 p.i. /CD163/DAPI 1 α- actinin e Day 7 post-injury inos //dapi CD163//dapi -treated -treated * f staining, day 7 p.i Figure S

10 a C57Bl/6 or clodronate Treatment Harvest b H&E, day 3 p.i. Clodronate -1 d1 p.i. d3 p.i. d 9 CD163 inos e staining, day 3 p.i. Clodronate Interstitial cells/field Clodronate ## MyoD Myogenin Ki67 c, day 3 p.i. Clodronate g + cells/field Clodronate f Mononucletaed cells + /field 15 Ki Clodronate ## ## MyoD * ## ## ## ## ## Clodronate ## Figure S3

11 a Treatment C57Bl/6 Harvest Harvest Day 4 p.i. Day 14 p.i. b MyoD Ki67 c staining Collagen IV Day 7 post- injury MyoD Ki67 Day 14 post- injury Figure S4

12 a f Expression levels in Ager -/- vs. wild type MØ, fold change Nos Cd68 Ifng Il-6 Tnfa MØ, day 7 p.i. Cd86 Il-1β Il-1a Arg1 Cd163a Il-4 Il-1R1 Mrc1 Il-1 tgfb MØ, 4h μg/ml.. 4 Migrated cells/field b g Treatment Harvest Harvest Treatment Harvest Ager -/- Ager -/- Day 1 p.i. Day 5 p.i. Day 4 p.i. c H&E, day 3 p.i. H&E, day 5 p.i. h H&E Collagen IV Interstitial cells/field d e Relative Quantity Nos Cd68 Ifng Tnfa Il6 Arg1 Cd163a Il4 Il1R1 Il1 Tgfb 3 1 Interstitial cells/field Centrally nucleated myofibers/field Day 5 p.i. Day 5 p.i. i MyoD Positive cells/ field Day 3 post-injury Positive cells/ field Day 5 post-injury Ki67 Relative Quantity Figure S5

13 a DMSO or SU54 Ager -/- Treatment Harvest b Day 4 p.i. Day 5 p.i. Day 6 p.i. MyoD Ki67 Day 7 post- injury SU54 + SU54+ DMSO c Expression levels in - vs. - MØ, fold change Nos Cd68 Ifng Il6 Tnfa MØ, day 7 p.i. Cd86 Il1β Il1a Arg1 Cd163a Il4 Il1R1 Mrc1 Il1 Tgfb d MØ Ager -/-, day 4 p.i. IP: Input IP 1 WB:FGFR1 WB: Figure S6

14 GAPDH 18 MyoD cyclin D1 emyhc 18 RAGE 18 d Figure 1 MyoD cyclin D emyhc 18 RAGE GAPDH 18 Figure 1 pho-p65 p65 pho-p38 pho-erk1/ ERK1/ pho-akt tubulin g tubulin MØ, day 3 p.i. Figure 18 Collagen IV 17 1 i l 18 Figure Figure m Figure MØ-conditioned media MØ-conditioned media None IL tubulin None IL MyoD cyclin D1 emyhc 18 d GAPDH 18 Figure 3 #5 #6 GAPDH #5 #6 Figure S7

15 Figure 4 Figure 5 Figure 6 Figure 6 MyoD f pho-tyr a FGFR c MyoD 3X X #5 #6 #7 #8 3X X #5 #6 #7 #8 Collagen IV f 3X #5 3X #5 ogenin emyhc clin D g inos 18 CD163 Figure 7 C57/Bl1 mdx #5 #6 C57/Bl1 mdx #5 #6 C57/Bl1 mdx #5 #6 C57/Bl1 mdx #5 #6 emyhc 18 cyclin D1 GAPDH 3X X #5 #6 #7 #8 3X X #5 #6 #7 #8 3X X #5 #6 #7 #8 3X X #5 #6 #7 # #5 a b 3X Figure 7 Conditioned media C57/Bl1 mdx M #5 C57/Bl1 mdx M #5 18 -actinin APDH 18 C57/Bl1 mdx #5 #6 C57/Bl1 mdx #5 #6 RAGE 18 3X #5 #6 #7 #8 3X #5 X 17 1 GAPDH C57/Bl1 mdx #5 #6 3X #5 GAPDH GAPDH Figure S8