Figure 3 | Scientific Reports

Figure 3

From: Incorporation of a skeletal muscle-specific enhancer in the regulatory region of Igf1 upregulates IGF1 expression and induces skeletal muscle hypertrophy

Figure 3

Evaluation of the ability of the candidate sites to support IGF1 expression in C2C12 single-cell colonies. (a) Schematic of the strategy used to knock in the MLC enhancer by homologous recombination (HR). The site 2 is chosen as an example; the strategy was the same for the site 1 and site 3. The black and grey blocks represent the 5′ long homology arm (HA-L) and 3′ short homology arms (HA-R), respectively. The white blocks represent the incorporated MLC enhancer. The restriction enzyme used for Southern blot analysis is shown. The 3′ external probe is shown as a short black line. (b) Southern blot analysis of the incorporated MLC enhancer in the site 2. Genomic DNA of the site 2 series of single-cell colonies and the WT control was digested with HindIII and then hybridized with the 3′ external probe. The expected fragment sizes are: WT, 5.9 kb; GM, 3.7 kb. (c) Real-time quantitative PCR analysis of the relative expression levels of Igf1 mRNA in myotubes of the site 1, site 2, site 3 series of single-cell colonies. The results were displayed as fold changes relative to the expression levels in WT C2C12 myotubes using the 2−△△CT method. Values are mean ± SEM of three independent experiments. (d) ELISA analysis of the IGF1 protein levels in myotubes of the site 2 series of single-cell colonies and the WT control. The total IGF1 levels are normalized to the total protein levels. Values are mean ± SEM of three independent experiments. (e) Western blot analysis of the Akt phosphorylation levels in myotubes of the site 2 series of single-cell-colonies and the WT control. Antibodies against phosphorylated Akt (Ser473), Akt (Thr308), and total Akt were used. GAPDH was used as a loading control. The ratio of the phosphorylated Akt (Ser473)/Pan-Akt and the phosphorylated Akt (Thr308)/Pan-Akt were determined by calculating the blot intensities and are shown below each respective blot. All gels/blots were run under the same experimental conditions. Shown are cropped gels/blots (Full-length gels/blots with indicated cropping lines are shown in Supplementary Figure S10).

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