Figure 2

Knockdown of PEX3 compromises peroxisome biogenesis and potentiates Vor-induced cell death. (a) PMP70 immunofluorescence staining (DAPI nuclear stain) of siSCR and siPEX3-1 vehicle (DMSO) and Vor-treated (2 μM, 12 h) U937 cells. Vor was added 72 h post transfection. Graph represents means±S.E.M. (one-way analysis of variance (ANOVA), Tukey) of N=3 with 5 images taken per condition and ~4 cells per image. Scale bar represents 10 μm. *P⩽0.05 and ***P⩽0.001. (b) Immunoblot of PEX3, PEX16, PEX19, and β-actin (loading control) in vehicle, and 6 and 12 h Vor-treated (2 μM) U937 cells post siSCR and siPEX3-1 transfection. (c) Growth curves of siSCR and siPEX3-tranfected U937 cells up to 72 h post transfection. Graph represents means±S.E.M. (two-way ANOVA, Bonferroni) of N=3 (technical triplicate) ***P⩽0.001. (d) Immunoblot of HO-1, CL-CASP3, and β-actin (loading control) in vehicle, and 6 and 12 h Vor-treated (2 μM) U937 cells. (e) DCFDA relative mean fluorescence intensity (MFI) in vehicle and Vor-treated (2 μM, 12 h) U937 cells. Graph represents mean±S.E.M. (one-way ANOVA, Tukey) of N=2 (technical triplicate). **P⩽0.01 and ***P⩽0.001. (f) Percent apoptotic cells as measured by the sum of PI/Annexin V-FITC double-positive and Annexin V-FITC staining. Vor treatment was 2 μM for 18 h. Graph represents means±S.E.M. (one-way ANOVA, Tukey) of N=3 (technical triplicate). *P⩽0.05 and ***P⩽0.001