Hoechst

For fluorescence labelling of 48 h VSM (25 μg/ml) treated cells, the cells were grown in Ibidi dishes (Ibidi GmbH, Martinsried, Germany), fixed in 4% paraformaldehyde (Santa Cruz, Dallas, USA), permeabilized with 0.1% Triton X-100 (Santa Cruz, Dallas, USA) and labelled with F-Actin antibody Phalloidin-Alexa 596 (Invitrogen, USA). Afterwards, a counter-staining with Hoechst (PanReacAppliChem, Darmstadt, Germany) was performed. The labelling procedure was also described previously [21 (link), 30 (link)]. The fluorescence microscopical images were captured on a confocal laser-scanning microscope Leica DMi8 (Leica, Wetzlar, Germany). For subsequent quantification of F-actin fibers (stress fibers induction), the confocal images were mathematical processed with the software FilaQuant (University of Rostock, Institute of Mathematics, Mathematical Optimization, Rostock, Germany) as described previously [22 , 33 (link)].

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).

Our group already described the general confocal microscopy procedure [24 (link)]. For this purpose, continuous and pulsatile electrically stimulated (Group 2a/b either for 3 or 7 days stimulation) as well as non-stimulated ASC, BMSC, and POB cells were fixed in 4% paraformaldehyde (Santa Cruz, Dallas, TX, USA), permeabilized with 0.1% Triton X-100 (Santa Cruz, Dallas, USA) and labeled with Phalloidin-Alexa 488 (Invitrogen) or focal adhesion kinase (FAK) primary antibody (#3285, Cell Signaling, USA) combined Alexa488 secondary antibody (Cell Signaling, Danvers, MA, USA) and counter-stained with Hoechst (PanReacAppliChem, Darmstadt, Germany). Microscopy was performed with an inverted confocal laser-scanning microscope (LSM780, Carl Zeiss, Jena, Germany). All images were taken at identical device settings to guarantee comparable results. The image processing was carried out using ZEN 2011 (Carl Zeiss Jena GmbH, Jena, Germany).