SUPPLEMENTAL INFORMATION (EMM2016177RR)

The maintenance ability and Ca2+ availability of skeletal muscle are enhanced

by sildenafil

Mei Huang1, Keon Jin Lee1, Kyung-Jin Kim2, Mi Kyoung Ahn1, Chung-Hyun Cho2, Do Han Kim3, and Eun

Hui Lee1, *

1Department of Physiology, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu,

Seoul 137-701; 2Department of Pharmacology, College of Medicine, Seoul National University, 103 Daehak-ro,

Jongno-gu, Seoul 110-799, Republic of Korea; 3School of Life Sciences and Systems Biology Research Center,

Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.

* To whom correspondence should be addressed: Eun Hui Lee, Department of Physiology, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Republic of Korea, tel: +82-2-2258- 7279, fax: +82-2-532-9575, [email protected]

1 Supplementary Figure 1. A quantitative analysis of myoblast proliferation with the treatment of various concentrations of sildenafil for various time periods, and the number of places used in the analysis of the sildenafil effect on the proliferation of primary skeletal myoblasts. (a) A quantitative analysis of myoblast proliferation with the treatment of various concentrations of sildenafil for various time periods. The number of myoblasts was normalized by the corresponding number of myoblasts in DMSO control at 0 h, and is presented as histograms (fold increase). The results are presented as the means ± S.E.). The number of myoblasts at each concentration was compared with that of the corresponding DMSO control using t-tests (*, significant, p < 0.05), and Bonferroni correction was conducted. Note that, like in the Fig. 1a, the enhancement in the proliferation of skeletal myoblasts was presented by 'fold-increase'. The proliferation of primary skeletal myoblasts was significantly enhanced by 50 nM sildenafil treatment for 24 h or longer periods. (b) The number of places (0.33 mm2 of the unit area) used in the analysis of the sildenafil effect on the proliferation of primary skeletal myoblasts in A was presented.

Supplementary Figure 2. The effect of 50 nM of sildenafil treatment for 12 h on the proliferation of primary skeletal myoblasts. A quantitative analysis of myoblast proliferation with the treatment of 50 nM of sildenafil for 12 h. The number of myoblasts was normalized by the corresponding number of myoblasts in DMSO control at 0 h (eighteen places per each), and is presented as histograms (fold increase). The results are presented as the means ± S.E. There was no significant change in the proliferation of primary skeletal myoblasts following 50 nM of sildenafil treatment for 12 h.

Supplementary Figure 3. Measurements of the width of primary skeletal myotubes with the sildenafil treatment. To examine the effect of 50 nM of sildenafil on the degree of myotube formations, the width of the myotubes with sildenafil treatment was measured, as described in the Experimental Procedures section. Width values were normalized to the mean value of those from the untreated control, and are presented as histograms (70 myotubes per each). The results are presented as the means ± S.E. There was no significant change in the width of primary skeletal myotubes by treatment with sildenafil.

Supplementary Figure 4. The amount of releasable Ca2+ from the SR to the cytosol. The amount of releasable Ca2+ from the SR to the cytosol in response to TG (2.5 µM) in the myotubes with sildenafil treatment in Figure 5B was analyzed, and histograms are shown for peak amplitude (height), as described in the MATERIALS AND METHODS section. The results are presented as the means ± S.E. for the number of experiments presented in the parentheses of Table 2. There was no significant change in the amount of releasable Ca2+ from the SR to the cytosol by sildenafil (p < 0.05).

Supplementary Figure 5. Expression level of skeletal muscle proteins in primary skeletal myotubes. The immunoblot analysis of proteins mediating Ca2+ movements and handling in skeletal muscle was conducted using the lysate of the myotubes with sildenafil treatment. Fifteen proteins were examined, and α-Actin was used as a loading control. Three-independent experiments per each protein were conducted and presented as bar graphs (a representative image of each is presented in Figure 5A). JP, junctophilin; CSQ, calsequestrin; Mg, mitsugumin; CaM, calmodulin. The expression levels of none were changed by sildenafil (statistically not significant, p < 0.05). Note that there was a significant change in the expression level of MG29 by DMSO (the solvent for sildenafil) or sildenafil compared with that by untreated control, however this was not induced by sildenafil but DMSO itself because there was no significant difference between DMSO and sildenafil.

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