*5.5. Confocal Immunofluorescence Analysis*

A monolayer of MRC-5 (2 × 105/well) was grown in DMEM supplemented with 10% SFB for 24 h. Fibroblasts were then treated with CTX (12.5 nM) for 2 h and, washed with PBS and cultured in either DMEM with 1% SFB in the presence of 2 ng/mL of TGF-β1 (Peprotech, cat number 100–21C, Cranbury, NJ, USA) or conditioned media from A549 cells for three days. After this, cells were fixed in 4% paraformaldehyde for 20 min, rehydrated with PBS (3 × 10 min), permeabilized in 0.2% Triton X-100 in PBS for 10 min, and kept overnight in a blocking solution containing PBS with 1% BSA and 0.1% Triton X-100, at 4 ◦C. The slides were incubated for 1 h with anti-α-SMA (1:250) in PBS with 1% BSA, and 0.1% Triton X-100 at 37 ◦C. After three washes with PBS containing 0.05% Tween 20, the slides were incubated with Goat anti-rabbit conjugated with DyLight 549 (1:800) for 1 h at room temperature. The slides were washed twice and then incubated with DAPI (25 mg/mL; Sigma Aldrich, Saint Louis, MO, USA) for 2 min. Mounting medium was applied, and the cells were photographed using a Leica DMi8 confocal microscope (Leica Microsystems, Mannheim, Germany), equipped with a DFC 310 FX digital camera, 63× magnification with oil immersion. Images were captured with the LAS AF and processed with the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
