Softworx software

Coverslips (High-precision, 1.5H, 22 ± 22 mm, 170 ± 5 mm, Marienfeld, 0107052) were immersed in 1 M KOH, sonicated for 15 min, and washed prior to incubation in poly-L-Lysine solution for 30 min. Coverslips were then washed and dried with pressurized air. Fixed cells resuspended in water were dried onto the coverslips with nitrogen and mounted on slides using Slow Fade Diamond mounting medium (Thermo Fisher, S36967). SIM was performed using a v4 DeltaVision OMX 3D-SIM system fitted with a Blaze module (Applied Precision, GE Healthcare, Issaquah, USA) using laser illumination. Images were taken in five phase shifts and three angles for each slice with Z-steps of 0.125 nm. Softworx software (GE Healthcare) was used for reconstruction with OTFs optimisation for 1.516 immersion oil.

THP-1 cells were seeded on glass coverslips and differentiated into macrophages overnight, before fixation with 4% formaldehyde (FA; Sigma-Aldrich, F1635) containing 0.2% Triton X-100 (Sigma-Aldrich, T9284) for 10 minutes and permeabilized with 1% Triton X-100 for 10 minutes. Fixed cells were washed with PBS and non-specific binding was blocked with 0.5% milk in PBS before incubation with primary antibodies in 0.25% milk in PBS for 1hr. Unbound antibody was removed by washing with PBS and secondary antibody in 0.25% milk in PBS was added and incubated for 30 minutes. Unbound secondary antibody was removed and cells were washed with PBS and stained with DAPI (Sigma-Aldrich, D9542) for 1 minute. Coverslips were washed and mounted with Mowiol mountant (Sigma-Aldrich, 324590). Fluorescence was visualized with a DeltaVision Spectris Deconvolution Microscope (GE Healthcare Life Sciences) with a 100x PlanApo 1.35 objective (Olympus). Images were deconvolved with the SoftWoRx software (GE Healthcare Life Sciences). For the experiments studying the effect of PPAR ligands on FAMIN expression, the number of peroxisomes was calculated using a custom ImageJ macro on 10 images per condition and triplicate experiments. Values were normalized according to the controls (untreated cells).

For localisation analysis of endogenously tagged Ctf19-GFP and Ctf19∆C-GFP proteins, cells were grown in synthetic medium without tryptophan at 30°C. For localisation analysis of ectopically expressed Ctf19-Okp1-GFP and Ctf19∆C-Okp1-GFP proteins in the Ctf19-anchor-away (Ctf19-FRB) strain, cells were grown in selective medium (–His/-Trp) until OD₆₀₀ ~0.4, then rapamycin (1 µg/ml) was added and cells were grown for another 3 hr at 30°C. For imaging cells were immobilized on concanavalin-A (Sigma-Aldrich) coated slides (Ibidi). Microscopy was performed using a DeltaVision microscopy system (Applied precision) with a Olympus IX71 microscope controlled by softWoRx software (GE Healthcare). Images were processed using Fiji (Schindelin et al., 2012 (link)).