Col1 α1

LX2, a human hepatic stellate cell line, and AML-12, a murine hepatocyte cell line, were obtained from Dr. David Moore at BCM. Cells were cultured in DMEM medium containing 5% FBS and 1% penicillin/streptomycin. For experiments, cells were stimulated with: media alone or TGF-β (10 ng/ml) (R&D Systems, 240-B-002) for 24 hours. Cells were harvested at 24 hours post-treatment for RNA analysis using TaqMan Gene Expression Cells-to-CT Kit (Thermo Fisher Scientific, AM1729). Real-time PCR was performed on an Applied Biosystems Step One Plus PCR System using validated TaqMan Gene Expression Assays for CDH11, Col1-α1, α-SMA, Snail, TGF-β, N-Cad, and 18s rRNA (Applied Biosystems). Transcript levels of mRNA in each sample were normalized using the 18s rRNA gene as an endogenous control. Data with each cell type were obtained from 4 individual experiments in triplicates and is presented as mean normalized transcript levels using the comparative Ct method (2ΔΔCt).

The vertebral body of the L3 and L6 (trabecular bone) and the flushed mid-diaphyseal tibias (cortical bone) were stored at −80°C in RNAlater post killing. RNA was extracted using TRIzol reagent (Life Technologies) followed by the RNeasy mini kit (Qiagen). Single-strand cDNA was synthesized using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative real-time PCR (qPCR) analyses were performed by using predesigned Taqman Assays and Taqman Fast Advance Master Mix (Applied Biosystems). The following predesigned real-time PCR assays were used for gene expression analysis: Acp5 (Trap; Mm00475698_m1), Ctsk (Mm00484036_m1), Tnfsf11 (Rankl; Mm00441908_m1), Alpl (Alp; Mm00475834_m1), Tnfrs11a (Rank; Mm00437135_m1), Tnfrsf11b (Opg; Mm0043545_m1), Bglap (Osteocalcin; Mm01741771_g1), Col1α1 (Mm00801666_g1), Runx2 (Mm00501580_m1), Sp7 (Osterix; Mm04209856_m1) (Applied Biosystems). The house-keeping gene 18S was used as the endogenous control in all analyses. Data are displayed as fold change relative to control.