Fluorescence Labeling of Muscle Cell Lines

The fluorescence labeling procedure of HSkM, RH-30 and HA-OH1 cells was performed as described previously [32 (link), 40 (link)]. Briefly, the cells were grown in Ibidi dishes/ slides (Ibidi GmbH, Martinsried, Germany), fixed in 4% paraformaldehyde (Santa Cruz, Dallas, USA), permeabilized with 0.1% Triton X-100 (Santa Cruz, Dallas, USA) and labeled with anti-SGPL1 primary antibody ((H-300) #sc-67368, Santa Cruz, USA) and Alexa Fluor 488 dye secondary antibody (Thermo Fisher Scientific Inc., USA). The co-localization experiments were performed by additional labeling with F-Actin antibody Phalloidin-Alexa 596 (Invitrogen, USA), focal adhesion kinase (FAK) primary antibody (#3285, Cell Signaling, USA) or with cell-permanent ER-Tracker™ Green dye (BODIPY® FL glibenclamide, Molecular Probes, USA). All samples were also counter-stained with Hoechst (PanReacAppliChem, Darmstadt, Germany). Images concerning on SGPL1 expression and localization were captured on a confocal laser-scanning microscope Leica DMi8 (Leica, Germany). Transfection efficiency (GFP signal of SGPL1; TYE-563 positive siRNA control) was controlled with a fluorescence microscope CKX53 (Olympus, Japan).