Targeting the Hedgehog-Gli pathway inhibits bleomycin-induced lung fibrosis in

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1 Targeting the Hedgehog-Gli pathway inhibits bleomycin-induced lung fibrosis in mice Elika Farrokhi Moshai, Lidwine Wémeau-Stervinou, Natacha Cigna, Stephanie Brayer, Joëlle Marchal Sommé, Bruno Crestani, Arnaud A. Mailleux Online data supplement Western blot analysis The ligand SHH, the receptor PTC, and cleaved poly (ADP-ribose) polymerase (cleaved PARP) were quantified by Western Blot in the left mouse lung with standard techniques (E1). Whole mouse lung homogenates were extracted in a RIPA buffer (NaCl 150mM, NP40 1%, desoxycholate 1%, Tris base 20mM, SDS 0,1%, EDTA 1mM). Alpha-actin was used as loading control. The first antibody was a mouse monoclonal anti-shh : Santa- Cruz, sc , Heidelberg, Germany ; rabbit anti-ptc: Abcam ab53715, Cambridge, UK; anti-cleaved PARP : Abcam ab32064 ; anti-beta tubulin: Abcam ab6046)mouse antiα-actin : ss32251, Santa Cruz Biotechnology, Santa Cruz, CA, USA. Then the membranes were incubated with HRP coupled secondary antibodies (anti-rabbit: NA934V, and antimouse: NA931V, GE Healthcare). Immunohistochemistry For immunohistochemistry (IHC), the paraffin-embedded sections were treated as described previously (E2). Primary antibodies were anti-shh (sc-1194, Santa Cruz Biotechnology, Heidelberg, Germany), anti-ptc (ab39266, Abcam Inc, Cambridge, UK), anti-smo (sc-13943, Santa Cruz Biotechnology), anti-gli1 ( , Rockland Immunochemicals Inc., Gilbertsville, PA, USA), anti-gli2 (BAF3526, R&D Systems, Minneapolis, MN, USA), anti-gli3 (ab6050, Abcam), anti-collagen 1 (Ab34710, Abcam,),

2 anti-collagen 3 (Ab7778, Abcam), anti-cleaved-caspase-3 (2305-PC-100, Trevigen, Gaithersburg, MD, USA) and anti-ki-67 (M7249, clone TEC-3, Dako, Les Ulis, France). Staining was followed by revelation with ABC Kit Vectastain (Eurobio Abcys, Courtaboeuf, France) except for GLI2 and Ki-67 that were revealed with N-Histofine Kit (Microm Microtech, Francheville, France). To test for the specificity of immunostaining, antibodies were omitted or replaced by an isotype-matched control antibody. Typical pictures obtained with isotype-matched control antibody are shown as supplemental figure E2. All digital images of light microscopy were acquired with a DM400B microscope (Leica) equipped with a Leica DFC420 CDD camera. The cells positive for cleaved-caspase-3 or anti-ki-67 labeling were counted in at least 20 representative fields per condition and expressed as numbers of positive cells/mm 2. Immunofluorescence Primary antibodies were anti-gli1 ( , Rockland Immunochemicals Inc., Gilbertsville, PA, USA), anti-gli2 (BAF3526, R&D Systems, Minneapolis, MN, USA), anti- GLI3 (ab6050, Abcam), anti-αsma (clone 1A4, Sigma Aldrich) and anti-abca3 (Clone 13- H2-57, Seven Hills bioreagents, Cincinnati, OH, USA). Briefly, the antibody against GLI1, GLI2 and GLI3 were revealed with the tyramide signal amplification systems with FITC (TSA kit, Perkin Elmer, Courtaboeuf, France). The anti-αsma antibody was revealed with an Alexa-568 coupled anti-mouse IgG secondary antibody (Life technologies Invitrogen, St Aubin, France). The anti-abca3 antibody was revealed with a DyLight-594 coupled anti- Mouse IgA secondary antibody (ab97013, Abcam). Nuclei were counterstained with DAPI. Digital images for immunofluorescence were acquired under an IX70 fluorescence microscope (Olympus) equipped with a X-cite Q source and an ORCA-03G CCD camera (Hamamatsu) with Cell-A software (Olympus). E2

3 Page 47 of 51 mrna Analysis Total mrna was extracted from mice lung homogenates with the use of the Trizol Plus RNA Purification Kit (Invitrogen, Saint-Quentin Fallavier, France). The concentrations of mrna were determined by spectrophotometry (Nanodrop, Thermo Fisher Scientific, Wilmington, Delaware, USA). Reverse transcription was performed with Random Hexamer primers, Oligo(d)T and reverse transcriptase MMLV-1 (Moloney Murine Leukemia Virus) (Invitrogen) and with 1µg of mrna. The transcript of beta-2- microglobulin (B2m) was used as a house keeping gene. Specific primers were designed (Table E1) to quantify the expression of the genes of interest by real-time PCR with the use of PCR ABI 7500 (Applied Biosystems, Carlsbad, CA). E3

4 Gene Primer Sequences (5 to 3 ) Forward Reverse Shh TGA TGA CTC AGA GGT GCA AAG GGT CAC TCG CAG CTT CAC T Ihh CCC AAC TAC AAT CCC GAC AT ATG ACA GAG ATG GCC AGT GA Dhh GGG ACC TCG TAC CCA ACT AC CTT TGC AAC GCT CTG TCA TC Ptc TAG TGT GCG CAG TCT TCC TC CAC TCA GCT TGA TCC CAA TG Hhip CAG AAT TGC CAA GTG TGA GC TGA GGA CCA AGA TAG CCC TT Smo CAT GCA CAC TGT CTC CCA TGA T CGG GGA GGT CAA AAG CCA AAC Gli1 CTC CAA TGA GAA GCC ATA CG GGA CCA TGC ACT GTC TTC AC Gli2 Gli3 GAC TCT CAC CTC CAT CAG CA CAC ATT CCA ATG AGA AAC CG TTG TTC TGG TTG GCA TCA TT ACA GTC TTC ACG TGT TTC CG B2m GTG ACC CTG GTC TTT CTG GT GTA TGT TCG GCT TCC CAT TC Acta2 AGTCGCTGTCAGGAACCCTGAGA ATTGTCGCACACCAGGGCTGTG Col1a1 GTG GTG ACA AGG GTG AGA CA GAG AAC CAG GAG AAC CAG GA Col3a1 TACACCTGCTCCTGTGCTTC CATTCCTCCCACTCCAGACT Il1b GCT TCC TTG TGC AAG TGT CT GGT GGC AAT TCA CAG TTG AG Vegfa ATC TTC AAG CCG TCC TGT TGT ATG ATC TGC ATG GTG ATG TTG Tgfb1 ACT GAT ACG CCT GAG TGG CT CCC TGT ATT CCG TCT CCT TG Ctgf GAG TGT GCA CTG CCA AAG AT GGC AAG TGC ATT GGT ATT TG Pai1 TCA CAA GTC TTT CCG ACC AA AGG CTG TGG AGG AAG ACG Supplemental Table E1: primer sequences E4

5 References E1. Lauth M, Bergstrom A, Shimokawa T, Toftgard R. Inhibition of gli-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A 2007;104: E2. Sanchez P, Ruiz i Altaba A. In vivo inhibition of endogenous brain tumors through systemic interference of hedgehog signaling in mice. Mech Dev 2005;122: E5

6 On line supplemental figures Figure E1: Gli2 and Gli1 mrna expression in the lung of naive mice treated with GDC (40 mg/kg every other day for a week) compared with vehicle only (DMSO) in panel A, or with GANT61 (25 mg/kg every other day for a week) compared with vehicle only (DMSO) in panel B. Data are median (25 th -75 th percentile; minimum and maximum) from 5 to 7 mice per condition (*: p<0.05; **: p<0.01).

7 Figure E2 : Immunohistochemistry. Typical pictures obtained for control immunoglobulins (negative controls).