Extended Data Fig. 3: PAR-activation induces calcium signaling in fibroblasts.
a, Parental or pKO-αV fibroblasts plated on full-length bovine FN-coated WPI dishes were loaded with 1 µM fluorescent calcium indicator (Oregon GreenTM 488 BAPTA-1, 06807, Themo Fisher Scientific) for 2 h and imaged for 10 min before adding PAR1-activating, PAR2-activating or control peptide to a final concentration of 0.1 mM. A representative image frame of spread, calcium-indicator loaded fibroblasts is shown. Five independent samples with at least five fibroblasts were analyzed per condition. Scale bar, 40 µm. b, Fluorescence intensity of fibroblasts monitored over time. The fluorescence intensity traces of four representative fibroblasts (outlined by grey line in a and labeled 1–4) are shown. Representative fluorescence intensity characterized before and after adding PAR1- or PAR2-activating peptide. This indicates a fast calcium increase, common for GPCR signaling. c, Intensity of calcium signaling approximated as the increase of indicator fluorescence intensity after adding control or PAR-activating peptide. In case of adding control peptide, no calcium peak was observed and the calcium level following the peptide addition was taken. Each point represents this calcium ratio for one fibroblast and bars their median. n(cells) gives the number of fibroblasts characterized. Two-tailed Mann-Whitney tests were used to determine P-values between PAR1-activating or PAR2-activating and control peptide. Parental fibroblasts, as well as pKO-αV fibroblasts both show calcium signaling after PAR1- or PAR2-activation.
