, Hilden, Germany). RNA from cultured cell lines was isolated using TRIzol (Invitrogen, Carlsbad, CA), as previously described.12 RNA concentration was measured with a Nanodrop ND-100 spectrophotometer (Thermo Scientific, Waltham, MA), and complementary DNA (cDNA) was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), as per the manufacturer’s

instructions. Real-time PCR analysis was performed (FastStart Sybr Green; Roche, Mannheim, Germany) using a Rotor Gene 3000 light cycler (Qiagen Pty Ltd., Sydney, Australia), and the specific target messenger RNA (mRNA) of interest was quantified as a ratio relative to 18S RNA content of the sample. The following mouse primers were used: MMP-2 forward: ACC CAG ATG TGG CCA ACT AC, reverse: TCA TTT TAA GGC CCG AGC AA; TIMP-1 forward: ACG AGA CCA CCT TAT ACC AGC CG, reverse: GCG GTT CTG GGA CTT GTG GGC Small molecule library screening (from Dr. Scott Freidman, Mt. Sinai School of Medicine, New York, NY); 18S forward: GTA ACC CGT TGA ACC CCA TTC, reverse: GCC

TCA CTA AAC CAT CCA Acalabrutinib clinical trial ATC G (from Dr. Eric Morand, Monash University, Melbourne, Victoria, Australia); TGFβ forward: TGC CCT CTA CAA CCA ACA CA, reverse: GTT GGA CAA CTG CTC CAC CT (Primer 3 software); PAR-1 forward: CTC CTC AAG GAG CAG ACC CAC; reverse: AGA CCG TGG AAA CGA TCA AC (Primer 3 software); and PAR-2 and 18S primers from Applied Biosystems TaqMan probe (Mm00433160_m1, Hs03003631_g1) using TaqMan Gene Expression Master Mix (Applied Biosystems). Paraformaldehyde-fixed 4-micron-thick liver tissue sections were stained with primary antibody for alpha smooth muscle actin (αSMA) (monoclonal mouse antimouse α-SMA; Sigma-Aldrich), F4/80 (rat antimouse, 1:200; a gift of Dr. Richard Kitching, Monash University, Clayton, Victoria, Australia) and cluster of differentiation Clomifene (CD)68 (rat antimouse CD68,

FA11, 1:100; a gift of Dr. G. Koch, Cambridge, UK). The following secondary antibodies were used: αSMA biotinylated rabbit antimouse immunoglobulin G (IgG)2a antibody (1:300; Invitrogen, Carlsbad, CA), F4/80 and CD68 polyclonal rabbit antirat IgG (1:150; Dako, Carpinteria, CA). In brief, sections were dewaxed, rehydrated, and then blocked with 0.6% hydrogen peroxide and CAS protein blocking solution (Invitrogen). Primary antibody incubations for 30 minutes at room temperature (αSMA) and overnight at 4°C (F4/80, CD68) were followed by the application of secondary antibody. Staining was amplified using an avidin-biotin complex kit (Vector Laboratories, Burlingame, CA) and was detected with diaminobenzidine (Dako). Slides were counterstained with Harris hematoxylin. For quantitation of immunoreactivity, 15 consecutive nonoverlapping fields at 250× magnification (α-SMA, F4/80, and CD68) were scored using a graticule eyepiece in a blinded fashion.