Fig. 3. Peroxisomal constriction can occur independently of DLP1. COS-7 cells stably expressing GFP-PTS1 were transfected with DLP1 siRNA duplexes (siRNA) and processed for immunofluorescence with anti-DLP1 antibodies 48 hours post transfection. (A,B) Co-localization of peroxisomes (green) and DLP1 (red). (A) DLP1 is efficiently silenced in cells that took up the DLP1 siRNA (asterisks). In those cells the remaining DLP1 localizes to a few small spots in the cytoplasm but is not associated with the constriction sites on elongated peroxisomes. Note the untransfected, non-silenced cell on the right, which has a high DLP1 protein level and contains spherical instead of elongated, segmented peroxisomes. (B) Higher magnification view of elongated, segmented peroxisomes (arrows) after efficient silencing of DLP1. Note the absence of co-localization of the remaining DLP1 with constricted peroxisomes. DLP1 is not efficiently silenced in the cell on the left (x). N, nucleus. (C) Immunoblots of peroxisomal fractions isolated from controls treated with buffer (Con) and cells transfected with DLP1 siRNA using anti-PMP70 and anti-DLP1 antibodies. Equal amounts of protein (PMP70, 10 µg/lane; DLP1, 45 µg/lane) were loaded onto the gels. Anti-PMP70 was used as a marker for peroxisomes. (D) Quantitation of peroxisome morphology at different times after transfection with a DLP1-WT construct. Cells were immunostained 24 and 48 hours after transfection by electroporation with antibodies to DLP1, and quantified. The data are from four independent experiments and are expressed as means ± s.d. Con, control cells (untransfected and vector only). For quantitative evaluation, cells were categorized as having elongated (el) (% of total), segmented (seg) (% of cells with elongated peroxisomes) or spherical (sph) (% of total) peroxisomes. Bars, 10 µm.