Dapi 62248

Antibodies were purchased from Cell Signaling Technology (ATG5 12994S, ATG7 8558S, Akt 4685, p-Akt 4060, mTOR 2972, p-mTOR 2971, p70S6K 9202, p-p70S6K 9208, 4E-BP1 9452, p-4E-BP1 9451), Abcam (PARP ab74290, cleaved-PARP ab32064, Ki67 ab66155, Santa Cruz Biotechnology (β-actin sc-1616, horseradish peroxidase-conjugated anti-rabbit secondary antibody sc-2004, horseradish peroxidase-conjugated anti-mouse secondary antibody sc-2005), Thermo Fisher Scientific (goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 488, goat anti-mouse Alexa Fluor 594, goat anti-rabbit Alexa Fluor 594), and Novus (LC3 NB100-2220). DAPI (62248) and Lipofectamine 3000 (L3000015) were purchased from Thermo Fisher Scientific. Unless otherwise indicated, all commercial chemicals were purchased from Med-Chem Express (Monmouth Junction, NJ, USA).

Cyst walls were stained with a 1/300 dilution of biotin-labelled Dolichos biflorus lectin (L-6533, Sigma-Aldrich) for 1 h and revealed using a 1/300 dilution of FITC-conjugated streptavidin (SNN1008, Invitrogen). DNA staining was performed on fixed cells by incubating them for 5 min in 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI 62,248, Thermo Fisher) solution. All images were acquired at the MRI facility on a Zeiss Axio Imager Z2 epifluorescence microscope and analysed using ZEN v3.6 (Zeiss) and FIJI v1.53t (US National Institutes of Health) software. Nile red (72485, Sigma-Aldrich) staining was performed after the fixation and permeabilization steps and prior to antibody or lectin staining: the cells were incubated for 45 min with Nile red at a final concentration of 1 µg/mL. The area of lectin-stained cysts or Nile red-stained lipid droplets was measured using the contour (spline) tool of the ZEN software (Zeiss) after proper scale calibration.

Half of a TA muscle was placed in OCT (Tissue-Tek, Sakura) in plastic molds and frozen in isopentane precooled in liquid nitrogen. Eight-μm-thick sections were cut with a cryostat (Leica CM1950) and stored at −20 °C. Hematoxylin and eosin (Sigma MHS32, HT110232) staining (H&E), nicotinamide adenine dinucleotide (NADH) staining, and Masson’s trichrome (Sigma HT15, HT1079, HT10132) staining were performed on dried sections fixed with paraformaldehyde (PFA) according to the manufacturer’s instructions and mounted with Eukitt mounting medium (O. Kindler). For NADH staining, sections were incubated with NADH (Sigma, N-8129) and nitroblue (Sigma, N-5514) for 30 min at 37 °C and then mounted. Immunostainings were performed on dried fixed sections, blocked with 3 % bovine serum albumin (BSA) in PBS, followed by primary antibody incubation (laminin ab11575 Abcam, DAPI 62248 Thermo Scientific, CD68 MCA1957GA Serotec). After washing with PBS, sections were incubated with secondary antibodies (donkey-anti-rabbit IgG Alexa647 A31573 Life Techologies, goat-anti-rat IgG Alexa488 A11006 Invitrogen) then washed with PBS and mounted with Vectashield (H-1000 Vector).