Ix71 wide field fluorescence microscope

Cells expressing GFP-Atg8 were set up at a density of 1.33 × 10⁶ cells/mL in 2 mL of HL5 media (Formedium, Hunstanton, Norfolk, UK, HLB0103) in six-well plates. Cells were treated with decanoic acid (60 μM), octanoic acid (60 μM), or DMSO control for 22 or 2 h to provide final treatment durations of 24 and 4 h. Autophagy inducer AR-12 (Selleckchem, Houston, TX, USA, OSU-03012), or DMSO control, was added, and shaking was continued for 1 h before addition of protease inhibitors (Roche, Basel, Switzerland, 11873580001) at a final concentration of 2.5-fold or HL5 control. Shaking was continued for 1 h before cells were removed (without additional pipetting) to tubes on ice. For live-cell imaging, cells were centrifuged for 3 min at 500 g at 4 °C and resuspended in KK2 (16.2 mM KH₂PO₄ and 4 mM K₂HPO₄, pH 6.1) and were imaged under a layer of 1% KK2 agar. Time in KK2 and time under agar were kept constant between each condition (20 min in KK2 and 3 min under agar). Cells were imaged on an Olympus IX71 wide-field fluorescence microscope. Images were captured using a Micropix camera, model Elite 2. For measurement of Atg8-positive structure size, cells were analyzed using ImageJ, and cell size was measured across the largest diameter. Thirty cells were analyzed per experiment.

Cells were imaged on an Olympus IX71 wide-field fluorescence microscope. Images were captured using a QICAM FAST 1394 camera. For measurement of Atg8-positive structure size cells were analysed using ImageJ [75] and cell size was measured across the largest diameter. More than 100 GFP-Atg8 positive structures were measured, using around 50 cells per experiment including at least three independent experimental repeats. For live-cell imaging, cells were imaged under a layer of ~1.5-mm thick 1% phosphate buffered agarose [49].

The method for whole teased fibre staining of extensor digitorum longus (EDL), soleus (SOL) and diaphragm was based on a previously published method (Vanhaesebrouck et al., 2019 (link)) and is given in detail in Supplementary material. For nuclei counting, AChRs were stained with 594-α-BuTx as described above and then individual fibres were teased apart. Fibres were mounted in Confocal Matrix containing DAPI onto microscopy slides, which were then coded so that the image acquisition and analysis were carried out blinded. Stained muscles were visualized using an Olympus IX71 wide-field fluorescence microscope. Images were captured using Simple PCI (Digital Pixel) and analysed using ImageJ. Number of mice used for each analysis was as described in the figure legends.