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. Author manuscript; available in PMC: 2021 Sep 3.
Published in final edited form as: Biochem Biophys Res Commun. 2020 Jul 30;529(4):1011–1017. doi: 10.1016/j.bbrc.2020.07.002

Figure 1. LY83583 stimulated O2•- dependent collagen secretion is independent of COL1a1/2 transcription.

Figure 1.

(a) Incubation of MEF cells with LY83583 induced production of O2•- which could be blocked by O2•-scavenger Tiron. (b) Immunoblotting with Type I collagen specific antibody showed that more Type I collagen was secreted into the culture medium 6 hrs post LY83583 addition. (c) Addition of LY83583 for 6 hrs induced pepsin-resistant Type I collagen in the culture medium. (d) LY83583-induced pepsin-resistant Type I collagen secretion into the culture medium of MEF cells was blocked by O2•- scavenger Tiron. (e) Pepsin-resistant Type I collagen could be detected in the culture medium 3 hrs after addition of LY83583. Accordingly, less Type I collagen was detected from the cell lysates. (f) 0.5 μM of LY83583 induced detectable pepsin-resistant Type I collagen in the culture medium. Accordingly, less Type I collagen was detected in the cell lysates. (g) LY83583 stimulation did not result in significant differences in Col1a1 or Col1a2 mRNA level within 6 hrs of treatment based on RT-qPCR. (h) Immunoblotting with Type I collagen specific antibody showed that blocking mRNA transcription by Act D did not significantly reduce pepsin-resistant Type I collagen in the culture medium induced by LY83583. Laminin and actin were used as loading control for total proteins in the culture medium or cell lysates. Medium or cell lysate collagen to actin was quantified by IMAGE J software. SMC: smooth muscle cell; MEF: mouse embryonic fibroblast; Col 1 α1/2: Type I collagen α1/2; Actinomycin D: Act D. * indicated the position of col 1 α2 or pepsin-resistant col 1 α2. P values were indicated in each panel if available. Other than indicated, all experiments were independently repeated at least three times with similar results.