Pirfenidone

For in vivo studies, Pirfenidone (Esbriet^®, Roche Pharmaceuticals, Switzerland) was solubilized with sterile water followed by warming at 60^°C for 30 min [17 (link)]. Doxorubicin hydrochloride was purchased as already made solution (2 mg/ml, ACTAVIS) and was solubilized in phosphate buffer saline (PBS).

Pirfenidone (Esbriet, Roche
Pharmaceuticals,
Switzerland) was dissolved in deionized water upon heating at 60 °C
for 30 min. The immune checkpoint inhibitors mouse monoclonal anti-PD-1
antibody (CD279, clone RMP1-14) and mouse monoclonal anti-CTLA-4 antibody
(CD152, clone 9D9) were purchased from BioXCell and diluted in InVivoPure
pH 7.0 dilution buffer.

Cell viability was evaluated using a commercial colorimetric assay (Quick Cell Proliferation Assay Kit II. MBL, International Corporation, Woburn, MA, USA) according to the recommended protocol. Briefly, cells (5 × 10⁴/well) were cultured in a 96-well microtiter plate (Nunc Thermo Scientific) and treated with pirfenidone (Hoffmann-La Roche) (1 mg/ml), and rapamycin (Sigma-Aldrich) (1 μg/ml), and the combination of both agents in the presence of TGF−β (5 ng/ml) in a final volume of 100 μl/well of 2% FBS culture medium in triplicates for 72 h. Then, 10 μl/well of WST reagent was added and plates were incubated for 2 h at 37 °C in standard culture conditions. After shaking the plates for 1 min, the absorbance was computed at a wavelength of 450 nm in each well using a microplate reader (Thermo Scientific) with 650 nm of reference wavelength. The amount of the dye generated by activity of dehydrogenase is directly proportional to the number of living cells.