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. 2022 May 2;13:2370. doi: 10.1038/s41467-022-30120-1

Fig. 3. rFibcd1 preserves myotube size via inducing ERK signaling but does not activate this pathway in some cancer cells.

Fig. 3

a Transfection of C2C12 myotubes for 48 h with Fibcd1 siRNAs reduces ERK signaling, as indicated by lower P-p44/42 MAPK compared to NT siRNAs. A 30-min treatment with rFibcd1 rescues P-p44/42 MAPK. See also Supplementary Fig. 3a. b NT or Fibcd1 siRNAs transfection into mouse C2C12 myotube-enriched cultures for 48 h, followed by treatment for further 24 h with Pyrazolylpyrrole (ERK inhibitor) at 2.5 ng/mL, indicates that ERK is needed for the preservation of myotube size by rFibcd1. Data are mean ± SD, with n indicated in the figure, and ***P < 0.001 (two-way ANOVA with Sidak’s multiple comparisons test). c P-p44/42 MAPK levels do not substantially change upon treatment of LLC, 4T1, and Saos-2 metastatic cancer cells with rFibcd1 (10 and 100 ng/mL for 30 and 120 min), apart for Saos-2 cells treated with the highest rFibcd1 dose. d rFibcd1 induces limited/minimal changes in P-p44/42 MAPK in most osteosarcoma, colorectal adenocarcinoma, melanoma, and breast cancer cell lines. However, rFibcd1 induces P-p44/42 in 67NR and 143B-Luc cells. See also Supplementary Fig. 3b. e rFibcd1 primarily consists of a fibrinogen-related domain (Fig. 2a). Combinations of a and b integrins are known to act as receptors for fibrinogen, including a2b-b3 integrins. f RNA-seq and qRT-PCR indicate that integrin a2b is expressed in C2C12 myotubes, which respond to rFibcd1 by increasing P-p44/42 levels but is not expressed in cancer cells that do not readily respond to rFibcd1 (LLC, 4T1, E0771, Ep5). 67NR cancer cells that respond to rFibcd1 (as indicated by higher P-p44/42; Fig. 3d and Supplementary Fig. 3b) display Itga2b mRNA levels similar to those of C2C12 myotubes, suggesting that Itga2b might be a receptor for rFibcd1; n = 2 (RNA preps from independent cell cultures).   g Itga2b siRNAs impede the rFibcd1-mediated increase in P-ERK, indicating that Itga2b is needed for the optimal response of muscle cells to rFibcd1. The quantitation of P-ERK levels relative to tubulin is shown in a, c, g. Source data are provided in the Source Data file.