Pa1 650

InSolution Rapamycin [Calbiochem 553,211; 1–100 nM (Foster and Toschi 2009 (link))], bafilomycin A1 [bafilomycin, SML-1661; 10 nM (Redmann et al. 2016 (link))] and Nicotinamide [N0636; 0.1–5 mM Avalos et al. 2005 (link); Hwang and Song 2017 (link))], Yoda1 [REF: SML 1558; 5 µM (Davies et al. 2019 (link))], Filipin complex ready-made solution (REF: SAE0088) were purchased from Sigma-Aldrich (USA). The compounds were dissolved in DMSO or water according to the technical specification of the suppliers. Solvent controls were matched to represent the same solvent concentrations of the treatments which were obtained by diluting stock solutions 1:1000. For the immunofluorescence studies, the following antibodies were used: anti-acetylated Lysine Monoclonal Antibody (1C6) (MA1-2021, Invitrogen), anti-Caveolin-1 (ab192452, Abcam, dil 1:500), anti-Integrin beta 1 (ab30394, Abcam, dil 1:500), anti-PMP70 (PA1-650, Invitrogen dil 1:500), anti-CLUH (PA5-65,101, Invitrogen, dil 1:500), anti-KLF2 (ab203591, Abcam, dil 1:600), anti-TOM20 (sc-17764, Santa Cruz Biotechnology, dil 1:500), Alexa Fluor® 647 donkey anti-mouse IgG (H + L) (A31571, Invitrogen, dil. 1:1000), Alexa Fluor® 647 donkey anti-goat IgG (H + L) (A21447, Invitrogen, dil. 1:1000), Alexa Fluor® 568 donkey anti-rabbit IgG (H + L) (A10042, Invitrogen, dil. 1:1000), Oregon Green® 488 Phalloidin (O7466, Invitrogen, dil. 1:1000).

To perform immunofluorescence microscopy, cells were seeded on coverslips to appropriate density and fixed with 3% formaldehyde/D’PBS for 20 min. After membrane permeabilization with 1% Triton X-100/D’PBS for 5 min a blocking step was performed by incubation in 1% BSA/D’PBS for 1 h. Cells were then incubated with the primary antibody targeting PMP70 (rabbit, 1:500 in D’PBS, Invitrogen PA1-650) for 30 min. Afterwards, the secondary antibody goat-α-rabbit IgG Alexa Fluor 594 (Invitrogen) was added for 10 min under light protection. Cells were mounted on glass slides with Mowiol 4-88 (Calbiochem, USA) supplemented with DAPI. Imaging was performed using the Axio Imager M2 microscope (Zeiss, Germany).
Quantification of peroxisomal protein import was done using the colocalization tool of the Zen blue edition software (Zeiss). Threshold values for the signal intensities of GFP and PMP70 were determined by comparison with stained, non-transfected cells and applied to all analyzed images. The Pearson Colocalization Coefficient was determined for at least 45 individual cells per condition. Statistical analysis was done using Prism (GraphPad) and applying a Kruskal-Wallis test. Correction for multiple comparisons was performed using Dunn’s test.