Fig. 4

Proliferation and differentiation are not affected in myoblasts from ΔKlotho mice in vitro. a αklotho mRNA expression during myogenic differentiation of myoblasts d0, growth medium (myoblasts); d1, day1 of differentiation (myocytes); d3 and d5, days 3 and 5 of differentiation (myotubes). (n = 3 mice per time point). All data are presented as means ± SEM. *p < 0.05, **p < 0.01. Expression was normalized to GAPDH. b Immunoblot analysis showing expression of sKlotho in the supernatant from primary myoblasts isolated from wt mice but not in the supernatant from ΔKlotho mice. An image of the Ponceau stained membrane can be found in Additional file 3: Figure S3F. c Primary myoblasts from ΔKlotho and control mice were immunostained for the proliferation marker Ki67 (green) and DAPI (DNA, blue) after 48 h of proliferation time in normal culture medium. Arrow heads mark Ki67 positive cells. Scale bar = 100 μm. d The proportion of Ki67-positive cells was counted in 15 randomly chosen regions of interest per condition (ΔKlotho n = 3 mice, control n = 4 mice). e The mean myotube diameter was measured in six randomly chosen regions of interest per condition (ΔKlotho n = 3 mice, control n = 4 mice). f Representative images of immunostainings from myotubes after 5 days of differentiation. DNA (DAPI, blue), MHC (green), myogenin (red). Scale bar = 100 μm. g The fusion index was calculated as the ratio of nuclei in myotubes in relation to the total number of nuclei in 6 randomly chosen regions of interest per condition (ΔKlotho n = 3 mice, control n = 4 mice). All data are presented as means ± SEM