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. 2016 May 31;6:24708. doi: 10.1038/srep24708

Figure 6. Elastase activity is toxic to cultured myoblasts in a substrate-dependent manner.

Figure 6

(a) C2C12 myoblasts were treated with 0.6 U/mL elastase (NT = non-treated) for 2 days prior to fixation and imaging. (b) Quantification of 10–15 technical replicates for 3 biological replicates (N > 30 images per data-point) treated as in (a) with increasing concentrations of elastase as indicated. (c–h) 75 μg of collagen I from rat tail (c,d), collagen IV from human placenta (e,f) and laminin from Engelbreth-Holm-Swarm murine sarcoma (g,h) were incubated with 20 μg of purified elastase and sampled at the indicated time points, boiled with Laemli sample buffer then run on an 8% SDS-PAGE (c) or a two-phase 10–6% SDS-PAGE (e,g). From (c) the bands at ~120 kDa corresponding to Col I α1 and α2 chains and the band at ~200 kDa corresponding to the double chain β resulting from cross-linked single chains were quantified and plotted in (d). From (e) the band at ~160 kDa corresponding to Col IV (any α chain) was quantified and plotted in (f). From (g) the band at ~250 kDa corresponding to laminin was quantified and plotted in (h). Lower molecular weight bands, possibly corresponding to impurities, were ignored. Quantified bands are highlighted by a box in (c,e,g). The arrowhead pointing at a ~26 kDa band indicates elastase. (i,j) TUNEL assay of C2C12 cells treated with elastase (0.75 U/mL) for 24 hours. Representative images from one of three independent experiments are shown in (i) and quantification of 10 image fields per experiment, across three independent experiments (N = 30), in (j). The p-values for elastase-treated samples versus non-treated samples were all significant except for “strong staining” in Col I and Col IV, 0.6 U/mL elastase. (k) DAPI/Annexin-V staining of cells treated with elastase 0.75 U/mL (white bars) or untreated (black bars) 16 hours after treating. The gating scheme is shown in Fig. S7a. p-values could not be accurately calculated due to the relatively small sample size (N = 3 independent experiments). Error bars are S.E.M., **p < 0.01. Scale bars are 100 μm in (a) and 50 μm in (i).