S7903

Microtissues were fixed within the agarose-molded wells of the tissue culture plate with cold PFA solution (4%(w/v) paraformaldehyde, 8% (w/v) sucrose) (F79-500, Sigma-Aldrich) (S7903, Sigma-Aldrich). Microtissues were fixed for 2 h followed by 3 PBS washes. Following several PBS washes, the agarose mold was carefully removed from the bottom of the plate well and the excess gel was cut away while remaining cautious to not cut too close to the tissue itself. The remaining agarose mold containing the microtissue was then placed in warmed PB and heated on a hot plate at 200 °C for 10 min with frequent agitation until the agarose had melted, leaving behind the freely floating fixed microtissue. The microtissue was then transferred to a 35 mm petri dish and washed several times with warmed PBS to remove any residual agarose before transfer to a 48-well plate. Microtissues were left in cold PBS until immunohistochemical labeling was performed.

Cells were fixed in 4% paraformaldehyde (18505, Ted Pella) containing 4% sucrose (S7903, Sigma) in 1×PBS (phosphate-buffered saline, 10010-02, Gibco) for 20 min at RT and subsequently quenched for 20 min with 100 mM NH₄Cl (K298.2, Carl Roth). Next, cells were permeabilized with 0.1% Triton-X 100 (T9284, Sigma) in PBS containing 2.5% BSA (bovine serum albumin, A9085.25G, Sigma) for 15 min at RT. Cells were then incubated for 1 h at RT with rabbit polyclonal anti-ArhGFE37 (1:800, HPA043885, Atlas Antibody) followed by detection with Alexa-Fluor^®488 goat anti-rabbit IgG (1:1000, A1108, Life Technologies).

To establish a diet‐induced myosteatosis model, 7‐week‐old C57BL/6N male mice were fed with a low‐fat diet (LFD; D12450J; Research Diets, New Brunswick, NJ, USA) or a high‐fat diet (HFD; D12492; Research Diets), which contains 60 kcal% fat in the form of lard and soybean oil for 20 weeks. The LFD was made isocaloric to the HFD by adding an ingredient in the form of Lodex 10. After 15 weeks of diet feeding, an adeno‐associated virus (AAV)‐empty vector (EV) or AAV‐RORα (2 × 10¹¹ genome copies/20 μL) was injected to right gastrocnemius (GA) muscles, or after 7 weeks of LFD or HFD feeding, JC1‐40, an RORα agonistic ligand, was suspended in 0.5% carboxymethyl cellulose and administered daily at a dose of 5 mg/kg/day by oral gavage for 5 weeks. JC1‐40 was synthesized as described.
²⁵ (link) GA tissues were dissected from the hind limbs of euthanized mice and immediately placed in 30% sucrose (S7903, Sigma‐Aldrich, St. Louis, MO, USA) solution after dissection. After 20 h, GA tissues were embedded in a formulation of glycols and resins (4583, Sakura Finetek, Torrance, CA, USA) and frozen with liquid nitrogen‐cooled isopentane (M32631, Sigma‐Aldrich).

In-frame deletion mutants were generated using the suicide plasmid pEXG2 (Rietsch et al., 2005 (link)). The primers used in this study are listed in Table S2. First, the flanking regions (consisting of 30 bp at the 3′ end and 30 bp at the 5′ end of the gene of interest plus ~ 800 bp for each flanking region) and a pEXG2 fragment were amplified by PCR and ligated using Gibson assembly (Gibson, 2009 (link)). In general, constructed plasmids were verified by DNA sequencing, transformed into Ec SM10 λ pir and subsequently mobilized by conjugation into PA14. Merodiploids were selected on LB agar plates containing irgasan (25 μg/ml; Sigma Aldrich #72779) and Gm (75 μg/ml). To achieve the second cross-over, counter selection on no-salt lysogeny broth (NSLB) agar containing 15% sucrose was performed (Sigma Aldrich #S7903). Finally, the loss of the plasmid was tested by streaking colonies on LB agar plates containing Gm (75 μg/ml) and in parallel on LB agar plates without antibiotics. In-frame deletion mutants were confirmed by PCR using (i) a primer pair flanking the target gene and (ii) a primer pair where one primer binds to the coding region of the target gene.

Mice were injected with a mix of 100 mg/kg ketamine and 10 mg/kg xylazine and we waited for the mice to be fully asleep. Then, the ribcage was opened, the heart was perfused with 10 ml of cold 1× PBS pH 7.4 (Invitrogen, AM9624) and it was resected just after the perfusion was finished. Then, the mouse trachea was perfused with cold 4% paraformaldehyde (PFA) (Euromedex, 15714-S) until the lungs were fully expanded with no air left inside. The trachea was closed with a thread to avoid PFA leakage. The lungs were resected out of the ribcage and kept in a falcon with cold 4% PFA overnight (o/n) under rotation at 4 °C. After fixation, the 5 lobes were separated and kept individually in cold 1× PBS containing 30% sucrose (Sigma, S7903) during 6 h under rotation at 4 °C. Lobes were rinsed in cold 1× PBS and pre-embedded in cold 50% optimal cutting temperature (OCT) compound (VWR, 411243) diluted in 1× PBS during 30 min under rotation at 4 °C. Finally, each lobe was embedded in square embedding molds (VWR, POLS18646ACODE45) containing OCT, frozen in dry ice during 20 min and stored at −80 °C.

Cells were mechanically homogenised in HIM buffer (200 mM mannitol; M4125; Sigma-Aldrich), 70 mM sucrose (S7903; Sigma-Aldrich), 1 mM EGTA (E4378; Sigma-Aldrich), and 10 mM HEPES-NaOH, pH 7.4 (H3375; Sigma-Aldrich) supplemented with 1 mM DTT (D11000; Melford) to obtain a PNS (post-nuclear supernatant). Briefly, SHSY5Y cells were washed with ice-cold PBS (, 14200067; Gibco) supplemented with 1 mM DTT and then collected by scraping and centrifugation at 1,000g for 2 min. Cell pellets were washed with HIM buffer and then resuspended in HIM buffer supplemented with 1 mM DTT. Cells were mechanically disrupted by shearing through a syringe with a 27G needle, followed by three times through an 8.02 mm diameter “cell cracker” homogenizer using an 8.01 mm diameter ball bearing. The resulting homogenate was cleared from nuclei and unbroken cells by centrifugation at 600g for 10 min to obtain the PNS. Homogenates were incubated with HA-Ub-PA probe (kind gift from Yogesh Kulathu, University of Dundee) at 1:100 (w/w) for 10 min at 37°C. The reaction was stopped by the addition of sample buffer and heating at 95°C. To test drug engagement with USP30, intact cells were treated with CMPD-39 for 2 h at 37°C before homogenization followed by probe incubation.

Mitochondria were isolated from the whole heart according to the method described in [28 (link)]. The heart was purified from blood vessels, crushed and homogenized. The homogenate was suspended in the isolated medium containing 75 mM sucrose (Sigma S7903, Saint-Louis, MO, USA), 10 mM Tris-HCl (pH 7.4), 225 mM mannitol (Sigma M4125, Saint-Louis, MO, USA), 0.5 mM EDTA (Sigma E9884, Saint-Louis, MO, USA), 0.5 mM EGTA (Sigma E3889, Saint-Louis, MO, USA), and 0.1% BSA (Sigma A6003, Saint-Louis, MO, USA). Mitochondria were isolated by differential centrifugation. At first, the homogenate was centrifuged at 1000 × g for 10 min to precipitate blood and destroyed mitochondria. The supernatant was precipitated at 8500 × g for 10 min. The precipitated RHM were washed and in isolation medium without EDTA or BSA (8500× g, 10 min). All procedures were conducted at 4 °C. The concentration of protein RHM was determined using the Bradford assay (30–35 mg/mL).