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. 2014 Dec 15;211(13):2617–2633. doi: 10.1084/jem.20140317

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© 2014 Di Foggia et al.

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Figure 6. — The satellite cell pool is increased by Bmi1 overexpression in vitro via up-regulation of MT1. (a) Relative expression level of MT1 in Bmi1^−/−, Bmi1^Over, and control satellite cells (mean ± SEM from two independent experiments with n = 3 for each condition; *, P < 0.05; **, P < 0.01). (b) Expression levels of MT1 are also analyzed at the protein level in Bmi1 ^Over satellite cells both in growth and differentiation-inducing conditions (left, three independent experiments with n = 3 for each condition). Efficient knock down of MT1 is also shown in proliferation (P) and differentiation (D) conditions (b, right; representative results from three independent experiments with n = 3 for each condition). WB analysis of the levels of 3-NT modification in Bmi1^Over satellite cells both in proliferation and differentiation-inducing conditions (b, left; three independent experiments with n = 3 for each condition). (c) The amount of ROS production is measured via DCFDA relative fluorescence intensity in Bmi1^Over satellite cells compared with controls and treated with either sh Scramble or knockdown of MT1 (mean ± SEM from two independent experiments with n = 4 for each condition; *, P < 0.05; ***, P < 0.001). (d and e) Quantification of the percentage of strongly 8OHdG^+ve satellite cells in Bmi1^Over cultures with and without knockdown of MT1 (mean ± SEM from two independent experiments with n = 4 for each condition; *, P < 0.05) and representative examples of immunolabeling (e) are shown. (f) Representative examples of immunolabeling for Pax7 and Edu in Bmi1^Over satellite cell cultures upon MT1 knockdown (two independent experiments with n = 4 for each condition). (g) Quantification of the percentage of Edu^+ve or Pax7^+ve or Edu^+ve/Pax7^+ve cells is shown (mean ± SEM from two independent experiments with n = 4 for each condition; *, P < 0.05). (h) Total concentration of nitrosylated proteins (ng/µl) in Bmi1^Over mdx;Bmi1^Over satellite cells compared with A-GFP–infected controls, with and without Mt1 knockdown (mean ± SEM from two independent experiments with n = 4; ***, P < 0.001; *, P < 0.05). (i and j) The level of SNO-Mef2c is shown in mdx;Bmi1^Over satellite cells compared with controls A-GFP–infected satellite cells as assessed by a modified biotin switch assay, followed by immunoblot (i) and quantification (j; mean ± SEM from two independent experiments with n = 4; *, P < 0.05). MT1: Metallothionein1; 8OHdG: 8-HydroxydeoxyGuanosine; MT1 up: MT1 up-regulation; MT1 nc: MT1 not changed. Bars: (e) 125 µm; (f) 62.5 µm.