Fig. 1.

Hsp25 gene expression, protein expression, and subcellular localization in proliferating and differentiating C2C12 cells. a The mRNA expression of gene encoding Hsp25 (hspb1) was examined by semi-quantitative real-time PCR (qPCR) in proliferating myoblasts (MB or day 0) and C2C12 cells differentiated for 1–4 days. Gene transcript levels were normalized to cDNA content and expressed relative to MB levels. b Representative Western blot showing protein expression of Hsp25 in MB and at days 1–4. Densitometry values were normalized to total protein and expressed relative to MB levels. Individual data points and the mean (horizontal line) ± SEM shown; n = 3 replicates/timepoint. βP < 0.05 vs. day 2; γP < 0.05 vs. day 3. c–d Representative immunofluorescence images of C2C12 myoblasts (c) and myotubes (d) stained with DAPI and endogenous Hsp25 in the absence of (control; top row) and after heat-shock treatment at 42 °C for 2 h (bottom row). Arrow heads indicate a subpopulation of cells with low Hsp25 expression. Scale bars as indicated. Boxed regions shown at higher magnification. e–f Western blot analysis showing protein expression of Hsp25 in lysates prepared from control and heat-shock-treated myoblasts (e) and myotubes (f). Densitometry values were normalized to total protein content assessed from the stain-free gel image. Individual data points and the mean (horizontal line) ± SEM are shown; n = 6–9 replicates/timepoint