Fig 2: Expression levels of B5R, B5, or CPR in SMCs. Western blots of B5R (A), B5 (B), and CPR (C) obtained from scr-RNA and B5R, B5 and CPR siRNA-treated mouse aortic SMCs. The first three bands correspond to known amounts of purified B5R (A), B5 (B), or CPR (C). Bands 4 and 5 are from scr-RNA and B5R (A), B5 (B), or CPR (C) siRNA-treated cell homogenates, respectively. The bar graphs show the mean value ± SD for n = 3. From the ratio of each measured protein to total cellular protein concentration, the cellular concentrations of B5R, B5, and CPR are estimated at 0.88 µM, 0.38 µM, and 0.15 µM, respectively.

Fig 3: Analysis of NADPH:P450 oxidoreductase (POR) expression and CYP-activity in bladder cancer. (A) Immunoperoxidase labeling of POR was quantified in a group of “normal” non-neoplastic bladders (n = 16) and compared with muscle-invasive bladder cancer (MIBC; n = 59) and organoid models of bladder cancer selected to represent POR suppressed (T24 and SCaBER) and over-expressing (RT4 and RT112) tumors. MIBC showed significant (P < 0.05, Mann-Whitney U-test) suppression of mean POR expression; however, a group of outliers was noted which over-expressed POR (open circles, representing 11.9% of cases). (B) Classification of MIBC as either luminal or basal based on quantification of GATA3 and KRT5/6 immunoperoxidase labeling was performed according to Dadhania et al33, reproducing the logistic regression analysis (LRA) and support vector machine (SVM) cut-off lines derived in that study. All POR over-expressing tumors (red circles) were classified as the more differentiated luminal type of MIBC. (C) Micrographs illustrating the POR immunoperoxidase labeling of normal bladder, MIBC, and cancer cell line (RT4, RT112, T24, and SCaBER) organ culture samples described in panel A. Scale bar represents 50 µm and applies to all images. (D) T24 and SCaBER cells generated peak EROD-activity of 1.6 and 19.5 nM resorufin/min/mg, respectively following ITE exposure. By contrast, 1 µM ITE-induced a peak EROD-activity of 89.3 and 83.1 nM resorufin/min/mg in the POR over-expressing RT4 and RT112 cell lines, respectively. RT4 and RT112 EROD was significantly higher than the mean of 60.0 nM resorufin/min/mg observed for differentiated NHU cells (n = 8 donors; each data point is the mean of six replicate cultures per donor)

Fig 4: The Rabin8–Rab10–KIFC3 complex is implicated in the peroxisome-mediated supply of cholesterol into the ciliary membrane AWhole-cell lysates from HEK293T cells expressing AcGFP1 or AcGFP1-tagged PEX14 and 3×FLAG-tagged KIFC3 were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.BWhole-cell lysates from HEK293T cells expressing AcGFP1-tagged KIFC3 and 3×FLAG-tagged Rab10 or Rab10-Q68L were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.CWhole-cell lysates from HEK293T cells expressing AcGFP1-tagged Pex14 and 3×FLAG-tagged Rab10 or Rab10-Q68L were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.DWestern blot analysis of the ciliary cholesterol trafficking-associated components in Pex1 +/+ and Pex1 -/- hTERT-RPE1 cells. Total cell lysates were separated to crude peroxisomal (Crude Pex), lysosomal and mitochondrial (Lyso/Mito), and peroxisomal (PEX) fractions. CYPOR, a lysosomal and mitochondrial protein, served as a positive control for the Lyso/Mito fractionation. Total cell lysates were gradually injected at 20 µg and 5 µg into a gel for SDS–PAGE, while equal amounts (5 µg) of protein from each fraction were loaded. Rabin8, Rab10, and KIFC3 were concentrated in the PEX fraction in Pex1 +/+ hTERT-RPE1 cells. CYPOR (cytochrome P450 reductase) is a mitochondrial protein.EQuiescent G0-phase hTERT-RPE1 cells transfected with 3×FLAG-tagged KIFC3 were immunostained with anti-FLAG (green), anti-PEX14 (red), and anti-a-tubulin (white) antibodies. Magnified 3D-constituted images of the boxed regions showing peroxisomes are located on the microtubule arrays via KIFC3 (arrows). The scale bars indicate 5 µm.FWestern blot analysis showing depletion of KIFC3 in the KIFC3 -/- hTERT-RPE1 cell clones. GAPDH served as a loading control.G KIFC3 +/+ and KIFC3 -/- hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated-tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Scale bar, 5 µm.HQuantification of (G) indicating that KIFC3 -/- hTERT-RPE1 cells significantly reduced the ciliary accumulation of cholesterol (***P < 0.001: one-way ANOVA with Tukey's HSD, n = 3: 40–50 cells per experiment). In the boxplot, medians, 25th/75th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively.IQuiescent G0-phase KIFC3 +/+ and KIFC3 -/- hTERT-RPE1 cells were immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ninein (green), and anti-PMP70 (white) antibodies. Arrows represent the peroxisomes contacting the ciliary pocket. Scale bar, 5 µm.JQuantification of (I) showing that disruption of the KIFC3 gene significantly interfered with the contact between peroxisomes and primary cilia (mean ± s.d.: ***P < 0.001: one-way ANOVA with Tukey's HSD, n = 3: 45–50 cells per experiment).K KIFC3 -/- hTERT-RPE1 cells were transfected with AcGFP1, AcGFP1-tagged KIFC3, rod domain-deleted KIFC3 mutant (?Rod), or motor domain-deleted KIFC3 mutant (?Motor) and cultured without serum for 24 h before Filipin III (green)-mediated cholesterol staining and immunostaining with anti-GFP (red), anti-pericentrin (white), and anti-acetylated-tubulin (blue) antibodies. Arrows represent ciliary localization of cholesterol. The scale bars indicate 5 µm.LQuantification of the Filipin III intensity at primary cilia from (K). AcGFP1-tagged KIFC3 deletion mutants did not restore the ciliary cholesterol insufficiency in the KIFC3 -/- hTERT-RPE1 cells (mean ± s.d.: ***P < 0.001: one-way ANOVA with Tukey's HSD, n = 3: 40–50 cells per experiment). In the boxplot, medians, 25th/75th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively. Source data are available online for this figure.

Fig 5: Phosphomimetic Pex14 mutant, Pex14-S232D, reduces catalase import into peroxisomes.(A) Cell lysates of CHO-K1, pex14 ZP161, and stable cell lines of ZP161 each expressing wild-type His-Pex14 (WT-6) and its mutants, phosphorylation-deficient Pex14-S232A (SA-13) and phosphomimetic Pex14-S232D (SD-30) (4 × 105 cells each) were analyzed by SDS-PAGE and immunoblotting with antibodies indicated on the left. Only the B-chain of acyl-CoA oxidase (AOx) that is generated by intraperoxisomal proteolytic processing of full-length AOx is shown. (B) Catalase was less imported into peroxisomes in Pex14-S232D-expressing cells. CHO-K1, pex14 ZP161, and stable cell lines each expressing Pex14 variants were immunostained with antibodies to catalase (green) and Pex14 (red). Merged images were also shown. Bar, 10 µm. (C) Stable lines of pex14 ZP161 expressing phosphomimetic Pex14-S232D (SD-30) were likewise immunostained with antibodies to PTS1 (green) and Pex14 (red). Bar, 10 µm. (D) Catalase in cytosolic fraction was increased in Pex14-S232D-expressing cells. Cells indicated at the top (8 × 105 cells each) were separated into cytosolic (S) and organelle (P) fractions by permeabilization with 25 µg/mL digitonin and subsequent ultracentrifugation. Equal aliquots of respective fractions were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. AOx, a typical PTS1 protein; alkyl-dihydroxyacetonephosphate synthase (ADAPS) and 3-ketoacyl-CoA thiolase (thiolase), PTS2 proteins; Cyt. c, cytochrome c. LDH is a marker for cytosolic fraction. (E) Catalase level in the cytosolic and organelle fractions assessed in D was quantified and shown as a ratio of cytosol (S) to total (S plus P). Data represent means ± SEM of three independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett’s post hoc test as compared with the S/(S+P) ratio of catalase in WT-6 cells. *p<0.05 and **p<0.01. (F) Pulse-chase experiment of catalase translocation. Fao cells were labeled with 35S-methionine and 35S-cysteine for 1 hr and were chased for 1 hr in the presence of vehicle or 0.2 mM H2O2. Cells were fractionated into the cytosol (S) and organelle (P) fractions as described in Material and methods. Equal aliquots of respective fractions were solubilized and subjected to immunoprecipitation with antibodies to catalase and DHAPAT. 35S-labeled catalase was analyzed by SDS-PAGE and detected by autoradiography (two upper panels). Equal aliquots of the cytosol and organelle fractions were analyzed by SDS-PAGE and immunoblotting using indicated antibodies. P450r, an ER membrane protein, cytochrome P450 reductase; LDH, a cytosolic protein. *, a putative nonspecific band. (G) 35S-labelled bands in F were quantified and 35S-catalase and 35S-DHAPAT in peroxisomes were shown as the ratio of respective bands in organelle (P) to total (S plus P). The P/(S+P) ratios of 35S-catalase and 35S-DHAPAT were represented as an average of two independent experiments and a single experiment, respectively.Figure 4—source data 1.Data for the level of cytosolic catalase shown in Figure 4E and those of 35S-catalase and 35S-DHAPAT in the organelle fractions shown in Figure 4G.