Human RH30 and murine C2C12 myoblast cell lines were purchased from American Type Culture Collection (ATCC) and cultivated in antibiotic-free standard high-glucose medium without pyruvate (Life Technologies, Darmstadt, Germany). Both cell lines showed irreversible myotube formation by serum-withdrawal (10% fetal bovine serum versus 2% horse serum). For microscopic analysis an inverted microscope (Nikon, Düsseldorf, Germany; TS100) was used and captures were taken with a NIS-Elements Camera (Nikon, Düsseldorf, Germany). For chemical inhibition of ALDH1 enzymatic activity Disulfiram (DSF; Sigma, Munich, Germany) was applied with a concentration of 3 µM of DSF.
Antibodies and reagents
Primary antibodies for Western Blot and immunofluorescent staining were anti-α-Aktin (Santa Cruz Biotechnology, Heidelberg, Germany; monoclonal, J1917, mouse, dilution IF 1:500), anti-ALDH1A1 (Abcam, England, UK; monoclonal, EP1933Y, rabbit, dilution: WB 1:1000, IF 1:500; polyclonal, rabbit, dilution: WB 1:1000, IF 1:500), anti-ALDH1A3 (Abcam, England, UK; polyclonal, rabbit, dilution: WB 1:1000, IF 1:500; Novus Biologicals, Abingdon, UK; polyclonal, rabbit, dilution: WB 1:1000), anti-GFP XP (Cell signaling, Frankfurt, Germany; monoclonal, D5.1, rabbit, dilution: WB 1:1000), anti-Myogenin (Sigma, Munich, Germany; polyclonal, rabbit, dilution: WB 1:500, IF 1:500), anti- MF20/ Myosin 4 (Thermo Fisher, Munich, Germany; monoclonal, 4301341, mouse, dilution: WB 1:1000), anti-Myosin Fast (Sigma, Munich, Germany; monoclonal, MY-32, dilution: WB 1:500) and anti-Vinculin (Abcam, England, UK; polyclonal, dilution WB: 1:20000).
Secondary antibodies for Western Blot were goat anti-rabbit, HRP-linked (Abcam, England, UK; dilution 1:5000), goat anti-mouse, HRP-linked (Cell signaling, Frankfurt, Germany; 1:3000); and for immunofluorescent staining: donkey anti-rabbit (Thermo Fisher, Munich, Germany; AlexaFluor 568, dilution 1:500), donkey anti-mouse (Thermo, Fisher, Munich, Germany; AlexaFluor 546, dilution 1:500).
Cells were cultivated and treated on µ-slides (Ibidi GmbH, Gräfelfing, Germany) for immunofluorescent staining. Slides were washed with PBS and cells were fixed in 4% formalin for 20 minutes at room temperature. Blocking buffer provided with donkey-serum was added and incubated for 30 minutes. After washing, primary antibody, diluted in blocking buffer with additive donkey serum, was applied overnight at 4 °C. Secondary antibody was added in a dilution as indicated with an incubation time of 40 minutes. Nucleic counterstaining with DAPI was conducted for 10 minutes. Samples were mounted with Aqua Poly/Mount (PolySciences, Hirschberg an der Bergstrasse, Germany). Immunofluorescence was examined with a Carl Zeiss Axioplan fluorescence microscope (Zeiss, Munich, Germany; HBO 100 Upright Fluorescence Microscope). Captures were conducted with an AxioCam and AxioVision 4.8 software.
Recombinant overexpression of ALDH1-isoforms
For recombinant ALDH1A1 or ALDH1A3 overexpression plasmids containing GFP-tagged open reading frame of ALDH1A1 and ALDH1A3, respectively, were purchased (OriGENE, Herford, Germany; MG208039, MG222097, respectively). Transfection was performed using 2 µg of DNA and Lipofectamine 3000 reagent (Thermo Fisher Scientific, Munich, Germany) following the manufacturer’s recommendations. Empty GFP-vector transfection served as control. Transfected cells were selected by G418-treatment (Sigma, Munich, Germany), subsequently protein expression was confirmed by Western blotting using antibodies against ALDH1A1-/ALDH1A3 and GFP as indicated before.