Epoxomicin

NCCIT cells were treated with 0.3 μM epoxomicin (Millipore, catalog #324800) for 24 hours to allow for the stabilization of Np9. NCCIT cells were collected with a cell scraper and lysed in cold RIPA Buffer (Sigma, catalog #R0278) supplemented with cOMPLETE protease inhibitor cocktail (Roche, catalog # 11697498001). Protein lysates (33 μg protein per sample) were separated in Bio-Rad 4–20% Mini-PROTEAN TGX gels (Bio-Rad, catalog #4561094). Proteins were transferred to 0.45 μm pore size polyvinylidene difluoride membranes (PVDF) (Millipore, catalog # IPVH00010), incubated with anti-Np9 rat monoclonal antibody 10B1 or 22E4 (1:25) overnight at 4°C, and then incubated with rabbit anti-rat secondary antibody (1:5000) (Invitrogen, catalog #619520) [47 (link)]. The anti-Np9 rat monoclonal antibodies were kindly provided by Dr. Klaus Römer from the University of Saarland Medical Center. Membranes were incubated with SuperSignal femto chemiluminescent substrate (TFS, catalog #34095). As a loading control, the PVDF membrane was incubated with mouse anti-GAPDH antibody (1:10000) (Santa Cruz, catalog #166545), and then incubated with horse anti-mouse secondary antibody (1:10000) (CST, catalog #7076S). We used ImageJ to analyze the intensity of the Np9 signal to determine the efficiency of the CRISPR/Cas9-mediated gene KD.

Four microliters of each undiluted 2-ml gel filtration fraction was incubated at room temperature with either epoxomicin (Sigma-Aldrich; catalogue no. E3652; 5 μM and 0.5% [vol/vol] DMSO final concentrations) or DMSO (0.5% [vol/vol] final assay concentration) at room temperature for 60 min. Next, 4 μl of the luciferin detection and Proteasome-Glo chymotrypsin-like reagent mixture was added to initiate the biochemical reaction. The reaction was allowed to proceed at room temperature for 20 min, after which the luminescence was read. Data were acquired from a single replicate (n = 1). Gel filtration fractions displaying chymotrypsin-like activity amenable to epoxomicin inhibition were pooled (see Fig. S1 in the supplemental material). Four microliters of the pooled protein material was then added to 4 μl of luciferin detection and Proteasome-Glo chymotrypsin-, trypsin-, or caspase-like reagent mixtures in the presence of either epoxomicin (5 μM and 0.5% [vol/vol] DMSO final assay concentrations) or DMSO (0.5% [vol/vol] final assay concentration). Luminescence was read immediately and then every 90 s for 75 min. Data were acquired from 6 technical replicates (n = 6).

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), epoxomicin and cycloheximide were purchased from Sigma-Aldrich, valinomycin from Axxora. siRNA transfections were performed with 20nM control (all stars negative control) or PINK1-specific siRNA (5′ GACGCTGTTCCTCGTTATGAA-3′) using HiPerfect (all from Qiagen) or Lipofectamine2000 (Invitrogen). siRNA resistant PINK1-V5 constructs have been described before [5 (link), 24 (link)]. The mutations p.F104A, p.L347P and p.I368N were introduced by site-directed mutagenesis and verified by Sanger sequencing. The standard transfection protocol for DNA was as follows: for one well of a 12-well plate 1 μg of DNA and 2.5 μl of Lipofectamine2000 (Invitrogen) were each mixed with 100 μl of Opti-MEM media (Invitrogen), incubated for 5 min at room temperature, mixed together and incubated 20 min before adding to the cells. For low-level expression DNA and lipofectamine amounts were reduced by 50–75% (total DNA per 12 well: 0.5 or 0.25 μg).

HEK293 and HeLa cells at 24 h post-transfection were treated with 0.1 µM or 1 µM Epoxomicin (Sigma E3652). After incubation for 24 h, the cells were washed with PBS and harvested. Cells were lysed in TBS containing 1% Triton X-100, 1% sodium deoxycholate, 50 mM NEM and protease inhibitor cocktail then kept on ice for 30 min followed by centrifugation at 15000 g for 20 min at 4 °C. Supernatants were collected as soluble fraction and pelleted insoluble fraction was lysed in RIPA buffer as insoluble fraction prior to immunoblot analysis. For cytosolic/nuclear fractionation, harvested cells were resuspended in TNE buffer (50 mM Tris–HCl, pH 7.8, 5 mM EDTA, 1% NP-40 and 150 mM NaCl) containing a protease inhibitor cocktail (Roche Diagnostics). Cells were centrifuged at 1000 × g for 10 min at 4 °C and the supernatant was collected as cytosolic fractions. The pellets were resuspended in RIPA buffer, sonicated and centrifuged at 10,000 × g for 10 min to clear the nuclear fraction.

Tumour-associated IMCs were freshly isolated from Braf^{+/LSL−V600E};CreER^+/0 mice and cultured in serum-free Dulbecco's modified Eagle medium (DMEM) (Invitrogen) as previously described (Kamata et al., 2015 (link), 2020 (link)). Atorvastatin (3.3 µM, Generon), lonafarnib (1-5 µM, Tocris Bioscience), GGTI-298 (8 µM, Tocris Bioscience), ML141 (10 µM, Merck) and/or EHT1864 (1-10 µM, Tocris Bioscience) were added to the serum-free IMC culture for 72 h. IMCs cultured for 96 h in serum-free DMEM were treated with the CCR1 inhibitor J113863 (5 µM, Tocris Bioscience) for 1-24 h as indicated. For membrane protein purification and detergent-insoluble protein analysis, primary IMCs were cultured for 48-72 h in DMEM containing 1% foetal bovine serum (Invitrogen) supplemented with Atorvastatin (3.3 µM), epoxomicin (0.05 µM, Sigma-Aldrich) and/or MG132 (3.3 µM, Sigma-Aldrich).