A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation
Fig 1
Rgs12 expression is high in mouse embryonic skeletal muscle and active satellite cells, upregulated early during muscle repair, and downregulated upon differentiation of the myoblastic C2C12 cell line.
(A) Relative expression (mean ± SEM of Fragments Per Kilobase of transcript per Million mapped reads [FPKM]) of Rgs12, Rgs14, and Pax7 gene transcripts within indicated ages of mouse gastrocnemius muscle samples was obtained via RNA-Seq using an Illumina HiSeq 2000. Based on Gene Expression Omnibus dataset GSE108402 [50]. Inset, detection of RGS12 protein expression via immunoblotting of (a) 100 μg of whole gastrocnemius muscle lysate from a 3-month-old C57BL/6J mouse and (b) 50 μg of lysate from myoblasts isolated from cardiotoxin-injected tibialis anterior (TA) muscle. (B) Normalized expression levels (mean ± SEM) of Rgs12, Rgs14, and Pax7 gene transcripts within indicated ages of flow-sorted, Pax7+ mouse skeletal muscle satellite cells were obtained via an Affymetrix Mouse Gene 1.0 ST microarray, based on Gene Expression Omnibus dataset GSE47401; published in [51]. (C) Relative expression of Rgs12 and eMHC in TA muscle following CTX-induced muscle damage. RNA was extracted from muscle at the indicated time points and quantified using qRT-PCR, with Gapdh abundance as an internal control. *, p < 0.01 Rgs12 level compared to time zero (one-way ANOVA with Dunnett’s test); #, p < 0.0001 eMHC level compared to time zero (one-way ANOVA with Dunnett’s test). Inset, TA muscle was injected with 0.1 ml of 10 μM cardiotoxin (CTX) diluted in PBS; contralateral, PBS-injected TA muscle was used as a control. After four days, the muscles were harvested and used for RGS12 protein expression analysis by immunoblotting (with GAPDH protein levels interrogated in parallel as a loading control). (D) C2C12 cell line cultures (4 x 105 cells/well) were maintained in growth medium (DMEM containing 10% fetal bovine serum [FBS]) for two days. Differentiation was induced by replacing growth medium with differentiation medium (DMEM containing 2% horse serum [HS] instead of FBS). Total RNA and protein lysates were separately isolated from cell cultures at the indicated time points (hours) after the switch to differentiation medium. Rgs12 mRNA and RGS12 protein levels were determined by qRT-PCR and immunoblotting, respectively. GAPDH mRNA and protein levels were used as internal controls for each experiment. **, p < 0.01; ***, p < 0.001 versus level observed at time zero (one-way ANOVA with Dunnett’s test).