Brdu assay

Cell proliferation was assessed using chemiluminescent BrdU assay (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Experiments were carried out in triplicate and presented as mean ± SEM.

Hepatic stellate cell proliferation was quantified after 7 days in cell culture by the BrdU assay (Roche, Mannheim, Germany) after labeling for 24 h and antibody incubation for 90 min, according to the manufacturer’s instructions, with the EASY READER SFplus (SLT-Labinstruments, Salzburg, Austria).

Cell proliferation of H1975P and H1975GR was measured using a BrdU assay (Roche Diagnostics Ltd.). Cell viability was measured by the CellTiter-Glo^® assay (Promega), respectively. Cells were seeded at 2 × 10³ cells overnight into adherent 96-well plates in complete cell culture medium. Cells were treated with IBL-301 for 72 h at a range of concentrations (0.0195 μM–1.25 μM) as described in triplicate wells. Absorbance was measured at 450 nm, reference wavelength 690 nm. Following treatment with GDC-0941, AZD1208, or IBL-301 a volume of CellTiter-Glo^® reagent equal to the volume of cell culture medium present in each well (100 μL) was added, contents were mixed for 2 min on an orbital shaker to induce cell lysis and the plate was incubated at room temperature for 10 min to stabilize luminescent signal.

The colorimetric bromodeoxyuridine (BrdU) assay (Roche/Sigma-Aldrich) is based on incorporation of BrdU into newly synthesized DNA in proliferating cells, and quantitative binding of a monoclonal HRP-coupled anti-BrdU-antibody. In brief, IPF-fibroblasts (n = 6) were seeded in 96-well plates (1×10⁴ cells/200 μL/well) and were allowed to grow overnight in normal culture medium. Next day, the medium was replaced, and fibroblasts were incubated for 24h with vehicle, LBH589 or pirfenidone, as described above. Subsequently, fibroblastic cells were labeled with 10 μM BrdU and re-incubated for 12h, followed by fixation (30 min/RT) and incubation with HRP-coupled anti-BrdU-antibody for 90 min at RT. Thereafter, 100 μL substrate solution (tetramethyl-benzidine) per well was added, and the plate was incubated for 5–25 min at RT until color development (blue) was sufficient. The substrate reaction was then stopped by adding 25 μL 1M H₂SO₄ per well and thorough mixing (yellow color). Immediately, absorbance was measured at 450 nm in an ELISA microtiter plate reader. The assay was performed in triplicates.

Harvesting of bone marrow stromal cells from the long bones of 10- to 12-week-old mice was performed by cutting the bones (femur and tibia) at both ends and flushing out the bone marrow using DMEM. Cells were centrifuged, resuspended in DMEM + 10% FCS + 1% P/S and plated in multiwell dishes at a density of 10⁶ cells per cm². After 5 days, the medium was changed. After reaching 70% confluence, the cells were switched to osteogenic medium containing 100 μmol·L⁻¹ ascorbate phosphate and 5 mmol·L⁻¹ β-glycerophosphate. After 5 and 10 days, cell proliferation was assessed using a BrdU assay (Roche) according to the manufacturer’s instructions. After 10 and 21 days, RNA was isolated for gene expression analysis, and the mineralized matrix was stained with alizarin red S (1%, Sigma-Aldrich). alizarin red S was eluted with 100 mmol·L⁻¹ cetylpyridinium chloride and quantified using a spectrophotometer at 540 nm. Cell viability was measured on day 10 and 21 using CellTiter Blue (Promega).

NIH3T3 fibroblasts were transfected with either CTGF-targeting or control siRNA and then stimulated with 5 ng/ml TGF-β₁ for 24 hours. Fibroblast proliferation was determined by BrdU assay (Roche, Mannheim, Germany) according to the manufacture’s protocol. To examine the effect of FG-3019 on fibroblast proliferation, NIH3T3 fibroblasts were pre-treated with 20 μg/ml FG-3019 or control IgG for 30 min prior to the addition of TGF-β₁.