2.6. Assays of Myofibroblast Differentiation

The expression of myofibroblast markers in cultured fibroblasts was evaluated by qRT-PCR and immunostaining. To suppress the differentiation of myofibroblasts, fibroblasts were treated with 1 µmol/L SB431542 or 10 µmol/L forskolin for 48 h. Cultured cells were harvested and lysed in Trizol, and the mRNA expression of myofibroblast markers was evaluated by qRT-PCR as described above.

For immunostaining of a myofibroblast marker αSMA and a fibroblast marker Thy1, cultured fibroblasts were fixed with 4% PFA and incubated with primary antibodies of mouse anti-αSMA (1:500, Cat.# MA5–11547, ThermoFisher Sci., Waltham, MA, USA) and APC-conjugated rat anti-Thy1.2 (1:200, Cat.# 13-0903-81, ThermoFisher Scientific) at 4 °C overnight, and then incubated with secondary antibodies of AlexaFluor-488 goat anti-mouse IgG (1:400, Cat#11029, Invitrogen, Waltham, MA, USA). The stained samples were imaged by DMi8 Leica fluorescent microscope (Leica Microsystems, Buffalo Grove, IL, USA) and APC-fluorescence signals were exhibited as pseudo-red color in images.