Alexaflour 547

Briefly, 3 × 10⁴ cells were seeded on rounded cover slips in 24-well plates and treated the next day as indicated. Cell monolayers were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Blocking was performed for 2 h at room temperature with 1% BSA in PBS with 0.05% Tween 20 (PBST). Cells were incubated with primary antibodies overnight at 4 °C and washed 3 times for 5 min with 0.1% PBST. Primary antibodies were directed against αSMA (Abcam, Cambridge ab7817, UK), FN1 (Sigma Aldrich, F7387), and pNFκB Ser536 (Cell Signaling, Danvers, MA 3031, USA). Secondary antibodies were conjugated with AlexaFlour-488 and AlexaFlour-547 fluorescent dyes (Thermo Fisher Scientific, Waltham, MA, USA, A-11034 and A-11029, respectively) and incubated with the cells for 1 h at room temperature. Nuclei were counterstained with DAPI (Sigma Aldrich, D-9542). After mounting with DABCO-Mowiol, the samples were examined and photographed using an epifluorescence microscope (Olympus Provis AX70, Tokyo, Japan). Quantification of αSMA-positive cells and the FN1 fluorescence area was performed in ImageJ. The percentage of positive cells and FN1 fluorescence were calculated based on the total number of DAPI-positive nuclei and the total image area, respectively. An average of four sections per patient were quantified.

For immunohistochemistry, tissues were fixed in 10 % buffered formalin and embedded in paraffin. 5 μm paraffin sections were deparaffinized and rehydrated, and antigen retrieval was performed by boiling the samples for 20 min in pH 9 Tris buffer. Sections were blocked for 1 h (PBS with 0.1% Tween 20 and 5% FBS) and incubated overnight at 4 °C with primary antibodies directed against: Lsd1 (Cell signaling, 2184, 1:400), pan-Myosin heavy chain (Myh_fast, Sigma, M4276, 1:1000 and Myh_slow, Sigma, M8421, 1:1000), Dmd (Abcam, ab15277, 1:500), Pax7 (Custom-made, DSHB, 1:80), GFP (Abcam, ab13970, 1:500), Plin1 (Abcam, ab3526, 1:400), or Ucp1(Abcam, ab10983, 1:500). Slides were incubated with appropriate secondary antibodies conjugated with AlexaFlour-488, AlexaFlour-547, or AlexaFluor-633 fluorescent dyes (ThermoFisher Scientific, A-11034, A-11029 and A-21050 respectively, 1:400). Nuclei were visualized by DAPI stain. Slides were mounted in aqueous medium (Fluoromount-G, SouthernBiotech, 0100-01) and observed using Leica confocal microscope.

Immunocytochemistry assay was performed by incubating fixed C2C12 or satellite cells for 15 min in permeabilization buffer (PBS with 0.2% Triton-X 100 and 0.1% Tween 20) followed by 1 h fixation in PBS with 0.1% Tween 20 and 5% FBS (Gibco, 10270-106). Cells were stained over night at 4 °C using the following antibodies: pan-myosin heavy chain (Myh_fast, Sigma, M4276, 1:1000 and Myh_slow, Sigma, M8421, 1:1000), Lsd1 (Custom made, Sigma, 112-4, 1:500), or GFP (Abcam, ab13970, 1:500). Secondary antibodies were conjugated with AlexaFlour-488 and AlexaFlour-547 fluorescent dyes (ThermoFisher Scientific, A-11034 and A-11029 respectively, 1:400) and incubated on the cells for 1 h at room temperature. Nuclei were visualized by DAPI stain (Sigma, D-9542, 1:10000). Stained cells were observed under Leica confocal microscope.