Figure 3

Elevated peroxisome levels promote HDACi resistance. (a) Left: PMP70 immunofluorescence staining (TO-PRO-3 nuclear stain) of vehicle (DMSO) and Vor-treated (2 μM, 12 h) U937 and B8 cells cultured in the presence (2 μM) and absence (DMSO) of Vor. Scale bar represents 10 μm. Right: puncta quantitation. Graph represents means±S.E.M. (one-way analysis of variance, Tukey) of N=3 with 5 images taken per condition with ~4 cells per image. *P⩽0.05 and ***P⩽0.001. (b) Immunoblots of PEX3, PEX16, PEX19, PEX11B, PMP70, ACSL1, and β-actin (loading control) in vehicle, Vor-treated (2 μM) U937 cells at indicated times, and B8 cells cultured in the presence (2 μM, 1 week) and absence (DMSO) of Vor. (c) 16:0, 18:0, and 18:1 PlsEtn levels, relative to vehicle-treated U937 cells. Graphs represent means±S.E.M. (one-way analysis of variance, Tukey) of N=3 (technical triplicate). *P⩽0.05, **P⩽0.01, and ***P⩽0.001. (d) Immunoblots of PEX3, PEX16, PEX19, HO-1, CL-CASP3, and β-actin (loading control), 96 h post transfection with siSCR and siPEX3-1 in B8 cells (2 μM Vor). (e) PMP70 immunofluorescence staining of B8 siSCR versus siPEX3-1-transfected cells, 96 h post transfection with puncta quantitation on right. Graph represents means±S.E.M. (unpaired t-test) of N=3 with 5 images taken per condition and ~4 cells per image. **P⩽0.01. (f) % Apoptotic cells as measured by PI/Annexin V-FITC double-positive and Annexin V-FITC-positive cells corresponding to conditions in dand e. Graph represents means±S.E.M. (unpaired t-test) of N=3 (technical triplicate). ***P⩽0.001