Journal: bioRxiv

Article Title: Loss of PREPL alters lipid homeostasis due to mitochondrial defects

doi: 10.1101/2025.10.28.685080

Figure Lengend Snippet: (A) Co-staining of PREPL (red) with the peroxisomal marker PEX14 (green). The nuclei were counterstained with DAPI (blue) (B) Peroxisome staining in WT and PREPL KO HEK293T cells (red: PEX14; blue: DAPI). (C) Quantification of peroxisome number per cell and peroxisome length in WT and PREPL KO HEK293T cells. (D) quantitative western blot of peroxisomal proteins in WT and PREPL KO HEK293T cells. Statistical analysis was performed using a T-test * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies used were directed against GAPDH (Cell Signaling Technology, 2118), PEX14 , catalase (Calbiochem, 219010), ACAA1 (Atlas Antibodies, HPA007244), and HSD17B4 (Fisher Scientific, 15116-1-AP).

Techniques: Staining, Marker, Western Blot

Journal: npj Viruses

Article Title: The Wnt/β-catenin pathway is important for replication of SARS-CoV-2 and other pathogenic RNA viruses

doi: 10.1038/s44298-024-00018-4

Figure Lengend Snippet: A A549 cells were treated with DMSO alone or ten different commercially available drugs/compounds (1 μM) that block Wnt/β-catenin signaling. Cells were fixed at 24- and 48-h post-drug treatment and processed for confocal microscopy using an antibody against PEX14 to label peroxisomes and CellMask TM to label the plasma membrane. The numbers of peroxisomes in cells were determined using Volocity software. The peroxisome density (number/μm 3 ) was calculated by dividing the number of peroxisomes by the estimated cell volume. For each sample, peroxisome densities were determined using a minimum of 20 cells. Data from three independent experiments are shown. Error bars represent standard errors of the mean. Two-way ANOVA with Bonferroni post-hoc tests were used to determine significance between samples treated with DMSO and Wnt inhibitors. * P < 0.05; ** P < 0.01. B Calu-3 cells were treated with the indicated Wnt inhibitors (1 μM) or Pyrvinium (100 nM) for 48-h and then total RNA, including small RNAs, were extracted from the samples, and relative levels of miRNAs were determined by RT-qPCR. The average relative levels of miRNAs (normalized to snRNU6) from three independent experiments were determined. Two-way ANOVA with Bonferroni post-hoc tests was used to determine significance between samples treated with DMSO and Wnt inhibitors. Error bars represent standard errors of the mean. *** P < 0.001.

Article Snippet: Primary antibodies were from the following sources: mouse monoclonal against beta-actin (ab8224), rabbit monoclonal against PEX7 (ab133754), rabbit polyclonal antibodies to PEX2 (ab110004), PEX13 (ab190213), PEX11B (ab211508), PEX19 (ab137072), β-catenin (ab32572), and catalase (ab1877) from Abcam (Cambridge, MA); Mouse monoclonal antibody to SARS-CoV/SARS-CoV-2 (COVID-19) spike antibody 1A9 (GTX632604) and rabbit polyclonal antibody to SARS-CoV-2 nucleocapsid (GTX135357) from GeneTex (Irvine, CA); Rabbit polyclonal antibody to PEX14 (NBP2-33455) from Novus Biologicals (Centennial, CO, USA); Mouse monoclonal antibody against the peroxisomal membrane protein PMP70 (SAB4200181) from Sigma (St. Louis, MO).

Techniques: Blocking Assay, Confocal Microscopy, Clinical Proteomics, Membrane, Software, Quantitative RT-PCR

Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 1 – Schematic representation of PEX13 and PEX14 and their interactions with each other and with PEX5. The amino acid positions of the globular domains of Rattus Norvegicus PEX13 and PEX14 proteins are indicated (CC, Coiled coil; NTD, N-terminal domain of PEX14; SH3, Src Homology 3 domain). Putative transmembrane domains (TMDs; light blue cylinders) were predicted with PRALINE™ [83] and PHOBIUS [84] using an alignment of twenty protein sequences representative of eukaryotic evolution. The pentapeptide motifs of PEX5 [43] are represented in orange. Black lines indicate the epitopes recognized by some of the antibodies used in this work. The interactions between PEX13, PEX14 and PEX5 are indicated as red (yeast) or blue (mammals) arrows. The SH3 domain of yeast PEX13 interacts with a PXXP motif (residues 87-90) located between the NTD and TMD of PEX14 [61,85]. In mammals, the SH3 domain of PEX13 interacts with the NTD of PEX14 [41,63]. The region comprising amino acid residues 236-246 of yeast PEX13, which corresponds to a region in the mammalian peroxin located between TMD2 and TMD3, interacts with an undefined region (marked with “?”) of PEX14 [47]. The SH3 of PEX13 also interacts with the N-terminal half of PEX5 in yeast [36,37,85]. A similar interaction involving residues 219-403 of human PEX13 (which includes the SH3 domain) and the N-terminal half of PEX5 has been described [38]. Residues 1-135 of mammalian PEX13 were also reported to interact with pentapeptides motifs 2-4 of PEX5 [45]. The NTD of PEX14 interacts with pentapeptide motifs present in the N-terminal half of PEX5 both in mammals and yeast/fungi [43,45,49,71,86]. A C- terminal domain of yeast PEX14 (residues 235-341) has been shown to interact with PEX5 [49,71], an interaction involving the second pentapeptide motif of PEX5 [49] (Reviewed in [51,57,70]).

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques:

Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 2 – PEX14 is a transmembrane protein with a Nin-Cout topology. A- Expression and purification of H6TEVPEX14 using IMAC. Culture aliquots collected before (lane NI) and after (lane I) protein induction, lysed cell extract (lane T) and the corresponding soluble (lane S) and insoluble protein fractions (lane P), the non-bound protein fraction from the Nickel-NTA beads (NB) and the purified protein () – H6TEVPEX14 (lane P14) were analyzed by SDS-PAGE, blotted onto a nitrocellulose membrane, and stained with Ponceau S. B- Proteoliposomes (Lipos) containing either PEX14 (left panel) or H6TEVPEX14 (H6PEX14; right panel) were treated or not with PK (400 µg/mL) in the presence or absence of TX-100, and analyzed by SDS-PAGE/Western blot. PEX14 was detected with antibodies against the full-length (FL) protein. Equivalent amounts of recombinant proteins (Lanes I; 130 ng) and the corresponding reconstituted proteins were loaded onto the gels. C- As in B, but using PEX14 that was subjected to the reconstitution protocol in the absence of lipids. D- As in B, but using antibodies directed to the full-length protein, the C-terminal residues 327-377 of human PEX14, and the N-terminal histidine-tag. E- PEX14 proteoliposomes were treated with PK (400 µg/mL) or trypsin (Try, 400 µg/mL) and analyzed by SDS-PAGE/Western blot using an antibody against the full-length protein. The ~46-kDa PEX14 fragment obtained after trypsin digestion is recognized by the anti-PEX14(327-377) antibody (data not shown). Identical results were obtained with H6TEVPEX14; in this case, the two fragments with ~20 and ~30 kDa are recognized by the anti-His antibody (data not shown), suggesting that the 30-kDa fragment is a partial proteolysis product. F- Proteoliposomes, PK-treated proteoliposomes, and PK alone were subjected to SDS- PAGE, blotted onto a Sequi-blot PVDF membrane and stained with Coomassie Brilliant Blue. PK- resistant fragments of PEX14 (, 40 kDa and ~20 kDa) and protein bands derived from PK itself (*)

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques: Expressing, Purification, SDS Page, Membrane, Staining, Western Blot, Recombinant, Derivative Assay

Journal: Cell systems

Article Title: Adaptation of human iPSC-derived cardiomyocytes to tyrosine kinase inhibitors reduces acute cardiotoxicity via metabolic reprogramming

doi: 10.1016/j.cels.2019.03.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-PEX14 , GeneTex , Cat# GTX129230.

Techniques: Virus, Recombinant, Cell Viability Assay, Blocking Assay, SYBR Green Assay, Real-time Polymerase Chain Reaction, Software

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division

doi: 10.3390/ijms21218040

Figure Lengend Snippet: Aberrant morphology of peroxisomes in NME3-deficient fibroblasts. ( A – C ) peroxisomes in the fibroblasts from a healthy control (upper panels) and a patient F741 (lower panels) carrying a homozygous mutation in the initiation codon of NME3 were visualized by indirect immunofluorescent staining with antibodies to catalase ( A ), PTS1 ( B ), ADAPS ( C ) and Pex14 ( A – C ). Insets show the images of the boxed areas. Bars, 20 µm and 2 µm (insets). Elongated peroxisomes are frequently observed in the patient-derived fibroblasts. ( D ) proteolytic processing of AOx, thiolase, and ADAPS was accessed by immunoblotting of cell-lysates of fibroblasts from a healthy control and the patient with antibodies to AOx, ADAPS, thiolase, Pex3, and LDHA, respectively. LDHA was used as a loading control. ( E ) histogram of peroxisome length measured in three each fibroblasts from a healthy control (1400 peroxisomes in three cells) and a patient F741 (1038 peroxisomes in three cells).

Article Snippet: Rabbit antibodies to acyl-CoA oxidase (AOx) [ ], 3-ketoacyl-CoA thiolase [ ], ADAPS [ ], PTS1 peptide [ ], Pex3 [ ], Pex14 [ ], Mff [ ], DYNAMO1 [ ], 70-kDa peroxisomal membrane protein (PMP70) [ ], and guinea pig anti-Pex14 antibody [ ] were as described.

Techniques: Control, Mutagenesis, Staining, Derivative Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division

doi: 10.3390/ijms21218040

Figure Lengend Snippet: Intracellular localization of NME3-HA 2 in HeLa cells. ( A ) NME3-HA 2 was expressed in HeLa cells. NME3-HA 2 was stained with rabbit anti-HA antibody (green). Peroxisomes and mitochondria were visualized with guinea pig anti-Pex14 (upper panels) and mouse anti-Tom20 (lower panels) antibodies, respectively. Peroxisomes are shown by a pseudo-color image. Scale bar, 10 µm. Higher magnification images of the boxed regions were shown (Inset). Scale bar, 5 µm. Arrowheads indicate peroxisomal, not mitochondrial, localization of NME3-HA 2 . ( B ) immunoblotting of mock- (-) and NME3-HA 2 - (+) transfected HeLa cells. Approximately ten times more of total proteins were loaded in lanes 1 and 4 than those in lanes 2 and 3. NME3-HA 2 was detected with antibodies to HA (upper panel, lanes 1 and 2) and NME3 (ABclonal) (upper panel, lanes 3 and 4). Two bands (solid and open arrowheads) were detected and termed NME3-1-HA 2 and NME3-2-HA 2 , respectively, by the expression of NME3-HA 2 . Dots indicate non-specific bands. β-actin, a loading control.

Article Snippet: Rabbit antibodies to acyl-CoA oxidase (AOx) [ ], 3-ketoacyl-CoA thiolase [ ], ADAPS [ ], PTS1 peptide [ ], Pex3 [ ], Pex14 [ ], Mff [ ], DYNAMO1 [ ], 70-kDa peroxisomal membrane protein (PMP70) [ ], and guinea pig anti-Pex14 antibody [ ] were as described.

Techniques: Staining, Western Blot, Transfection, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division

doi: 10.3390/ijms21218040

Figure Lengend Snippet: Intracellular localization of non-tagged NME3 in HeLa cells. Non-tagged NME3 was expressed in HeLa cells and stained with the antibody raised to DYNAMO1 (green). Peroxisomes and mitochondria were visualized with guinea pig anti-Pex14 ( upper panels ) and mouse anti-Tom20 ( lower panels ) antibodies, respectively. Peroxisomes are shown by a pseudo-color image. Scale bar, 10 µm. Higher magnification images of the boxed regions were shown (Inset). Scale bar, 5 µm. Arrowheads and arrows indicate peroxisomal and mitochondrial localization of NME3, respectively.

Article Snippet: Rabbit antibodies to acyl-CoA oxidase (AOx) [ ], 3-ketoacyl-CoA thiolase [ ], ADAPS [ ], PTS1 peptide [ ], Pex3 [ ], Pex14 [ ], Mff [ ], DYNAMO1 [ ], 70-kDa peroxisomal membrane protein (PMP70) [ ], and guinea pig anti-Pex14 antibody [ ] were as described.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division

doi: 10.3390/ijms21218040

Figure Lengend Snippet: NME3 is localized to peroxisomes and involved in the fission of peroxisomes. ( A ) intracellular localization of endogenous NME3 in HeLa cells was assessed by indirect immunofluorescent cell staining with antibodies to DYNAMO1 (green), Pex14, and Tom20 as in . Scale bar, 10 µm. Higher magnification images of the boxed regions are shown (Inset). Scale bar, 5 µm. Note that mitochondrial localization of NME3 is distinct (lower panels). ( B ) HeLa cells transfected with dsRNA against NME3 (#28, see ) were verified by indirect immunofluorescent cell staining with anti-bodies to DYNAMO1 and Tom20. Scale bar, 10 µm. ( C ) HeLa cells transfected with dsRNA against ATAD1 were verified by indirect immunofluorescent cell staining as in ( A ). Scale bar, 10 µm. Higher magnification images of the boxed regions are shown (Inset). Scale bar, 5 µm. Note that localization of NME3 in peroxisomes (arrowheads), but not mitochondria, is more readily discernible. ( D ) HeLa cells co-transfected with a set of two dsRNAs against ATAD1 and NME3 were verified by indirect immuno- fluorescent cell staining with antibodies to DYNAMO1 and Pex14. Higher magnification images of the boxed regions are shown (Inset). Note that peroxisomes are frequently elongated. Scale bar, 10 µm. Higher magnification images of the boxed regions were shown (Inset). Scale bar, 5 µm. ( E ) relative fluorescent intensity of NME3 in HeLa cells transfected with mock (-, n = 38) or dsRNA against ATAD1 (+, n = 25) was quantified. * p < 0.05. ( F , G ) transcription level of ATAD1 ( F ) and NME3 ( G ) in HeLa cells treated as in ( C ) was quantified by quantitative real-time PCR (n = 3). ( H ) protein level of Pex14 was verified by immunoblotting (left). β-actin, a loading control. Protein level of Pex14 was represented as values relative to that in mock-treated HeLa cells (right, n = 3). ( I ) the number of peroxisomes in HeLa cells untreated (n = 37), transfected with dsRNA against ATAD1 alone (n = 37), or a set of ATAD1 and NME3 (n = 33) was represented. *** p < 0.001, by Tukey-Kramer test. n.s., not significant. ( J ) NME3 level is elevated by ATAD1 knockdown. siControl and siATAD1 were separately transfected twice with a 24 h interval to HeLa cells that had been transfected for 6 h with a plasmid encoding NME3. After 24-h cell culture, transcription level of ATAD1 (left) was quantified by quantitative real-time PCR (n = 2). Center, NME3 expression levels were assessed by western blotting of respective cell lysates with anti-NME3C antibody. Two bands, NME3-1 and NME3-2, marked by solid and open arrowheads were detected. β-actin, a loading control. Dots indicate non-specific bands; right, NME3-1 and NME3-2 bands were quantified and represented by taking as 1 NME3-1 in siControl -transfected cells in lane 1. ( K ) upper row, NME3 was expressed in PEX11β −/− mouse embryonic fibroblasts (MEF) and its intracellular localization was verified by staining with antibodies to DYNAMO1 ( a ), DLP1 ( b ), and Pex14 ( c ). Scale bar, 10 µm. Higher magnification images of the boxed regions were shown (Inset). Scale bar, 5 µm. Note that NME3 was detected in the limited area of an elongated peroxisome (arrowhead). Panel ( e ), signal intensity of NME3, DLP1, and Pex14 in the elongated peroxisome indicated with its length in the merged view was analyzed by line scanning and represented. Note that NME3 was localized at the DLP1-accumulated potential constriction site (arrowhead in ( a – d )) where signal of Pex14 is weak and the both sides adjacent to this region the constriction site.

Article Snippet: Rabbit antibodies to acyl-CoA oxidase (AOx) [ ], 3-ketoacyl-CoA thiolase [ ], ADAPS [ ], PTS1 peptide [ ], Pex3 [ ], Pex14 [ ], Mff [ ], DYNAMO1 [ ], 70-kDa peroxisomal membrane protein (PMP70) [ ], and guinea pig anti-Pex14 antibody [ ] were as described.

Techniques: Staining, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Control, Knockdown, Plasmid Preparation, Cell Culture, Expressing

Journal: The EMBO Journal

Article Title: Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

doi: 10.15252/embj.2019103499

Figure Lengend Snippet: A Western blot analysis showing the depletion of PEX1 and PEX14 and the normal processing of Shh protein in the PEX1 −/− and PEX14 −/− hTERT‐RPE1 cell clones. GAPDH served as a loading control. B Immunostaining with anti‐PEX1 (red) or PEX14 (red), anti‐PMP70 (green), anti‐ninein (blue), and anti‐acetylated‐tubulin (white) in wild‐type, PEX1 −/− , and PEX14 −/− hTERT‐RPE1 cells in quiescent G 0 phase. Arrowheads indicate primary cilia. Scale bar, 10 μm. C Quantification of the number of PMP70‐positive peroxisomes per cell from (B). Peroxisome formation in PEX1 −/− and PEX14 −/− hTERT‐RPE1 cells was significantly impaired compared with that of the parental cells (mean ± s.d.: *** P < 0.001: one‐way ANOVA with Tukey's HSD, n = 3: 20–25 cells per experiment). D Quantification of proportion of ciliated cells from (B). Ciliogenesis in PEX1 −/− and PEX14 −/− hTERT‐RPE1 cells was not significantly altered compared with that of the parental cells (mean ± s.d.: one‐way ANOVA with Tukey's HSD, n = 3: 190–200 cells per experiment). E Quiescent G 0 ‐phase wild‐type, PEX1 −/− , and PEX14 −/− hTERT‐RPE1 cells transfected with AcGFP1‐tagged D4 as a cholesterol probe were immunostained with anti‐pericentrin (white) and anti‐acetylated‐tubulin (blue) antibodies. Cholesterol was stained with Filipin III (red). Arrows and arrowheads indicate primary cilia and cytosolic accumulations of AcGFP1‐tagged D4, respectively. Scale bar, 5 μm. F Quantification of the Filipin III intensity at primary cilia from (E). PEX1 −/− and PEX14 −/− hTERT‐RPE1 cells had significant reductions in the ciliary signal of Filipin III (*** P < 0.001: one‐way ANOVA with Tukey's HSD, n = 3: 40–50 cells per experiment). In the boxplot, medians, 25 th /75 th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively. G Quantification of the AcGFP1‐tagged D4 intensity at primary cilia from (E). The ciliary signal of AcGFP1‐tagged D4 in PEX1 −/− and PEX14 −/− hTERT‐RPE1 cells was significantly diminished (*** P < 0.001: one‐way ANOVA with Tukey's HSD, n = 3: 25–30 cells per experiment). In the boxplot, medians, 25 th /75 th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively. H Quiescent G 0 ‐phase wild‐type, PEX1 −/− , and PEX14 −/− hTERT‐RPE1 cells were treated with or without 1.5% methyl‐β‐cyclodextrin for 45 min and then incubated with or without cholesterol (cholesterol/methyl‐β‐cyclodextrin complex) for 1 h. After removing exogenous cholesterol, they were stimulated with 50 nM Shh‐N for 24 h in the presence of pravastatin, and then immunostained with anti‐Smo (green), anti‐acetylated‐tubulin (blue), and anti‐γ‐tubulin (red) antibodies. For the alternative cholesterol complementation, LDL (0.06 mg/ml) was co‐incubated with Shh‐N and pravastatin for 24 h after methyl‐β‐cyclodextrin‐mediated cholesterol depletion. Scale bar, 2.5 μm. I Quantification of the Smo intensity at primary cilia in wild‐type, PEX1 −/− , and PEX14 −/− hTERT‐RPE1 cells from (H). PEX1 −/− and PEX14 −/− hTERT‐RPE1 cells exhibited the dampened Shh‐N ligand‐induced ciliary accumulation of Smo. The complementation of exogenous cholesterol (cholesterol/methyl‐β‐cyclodextrin complex) restored the ciliary accumulation of Smo in both PEX ‐knockout cells, while the LDL complementation did not rescue the ciliary phenotypes efficiently (* P < 0.05, ** P < 0.01, *** P < 0.001: one‐way ANOVA with Tukey's HSD, n = 3: 90–100 cells per experiment). In the boxplot, medians, 25 th /75 th 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.

Article Snippet: GST (0.2 μg, Abcam: ab81793) or GST fused to PEX14 proteins (1 μg, Abnova: H00005195‐P01) was loaded to glutathione‐Sepharose beads (GE Healthcare) and then incubated with 6×His‐EHD3 (1 μg, ORIGENE: TP761227), 6×His‐Rabin8 (1 μg, ORIGENE: TP760069), or 6×His‐Rab10 (1 μg, NOVUS: NBP2‐23392).

Techniques: Western Blot, Clone Assay, Control, Immunostaining, Transfection, Staining, Incubation, Knock-Out

Journal: The EMBO Journal

Article Title: Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

doi: 10.15252/embj.2019103499

Figure Lengend Snippet: A Quiescent G 0 ‐phase hTERT‐RPE1 cells were immunostained with anti‐PEX14 (red), anti‐ninein (white), and anti‐acetylated‐tubulin (blue) antibodies. Cholesterol was stained with Filipin III (Green). Magnified images of the boxed regions showing peroxisome accompanied by cholesterol (arrows). Three‐dimensional reconstitution of the same cell indicates that Filipin III stains the membrane regions of ciliary axonemes and peroxisomes. The scale bars indicate 2.5 μm and 1.25 μm in lower‐ and higher‐magnified images, respectively. B Quiescent G 0 ‐phase wild‐type hTERT‐RPE1 cells treated with Cytochalasin‐D (200 nM), colcemid (50 nM), or Ciliobrevin‐D (10 μM) for 6 h were immunostained with anti‐ARL13B (blue), anti‐phospho‐S473‐Akt (red), anti‐ninein (green), and anti‐PMP70 (white) antibodies. Arrows indicate the peroxisomes interacting with the ciliary pocket. Scale bar, 5 μm. C Quantification of proportion of primary cilia interacting with peroxisomes from (B). Colcemid (50 nM) and Ciliobrevin‐D (10 μM) significantly inhibited the spatial interaction 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). D Quiescent G 0 ‐phase wild‐type hTERT‐RPE1 cells were treated with Cytochalasin‐D (200 nM), Colcemid (50 nM), or Ciliobrevin‐D (10 μM) for 6 h and then immunostained with anti‐acetylated‐tubulin (blue) and anti‐pericentrin (green) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 2.5 μm. E Quantification of the Filipin III intensity at primary cilia from (D). Colcemid and Ciliobrevin‐D interfered with the distribution of cholesterol in the ciliary membrane (*** P < 0.001: one‐way ANOVA with Tukey's HSD, n = 3: 40–50 cells per experiment). In the boxplot, medians, 25 th /75 th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively. F hTERT‐RPE1 cells were transfected with AcGFP1‐tagged EHD3 and cultured without serum for 24 h before immunostaining with anti‐GFP (green), anti‐ARL13B (blue), anti‐PMP70 (white), and anti‐phospho‐S473‐Akt (red) antibodies. The scale bars represent 2.5 μm. G 3×FLAG‐tagged EHD1 or EHD3 and AcGFP1‐tagged PEX14 were coexpressed in HEK293T cells and then immunoprecipitated from whole‐cell lysates using the anti‐FLAG antibody. AcGFP1‐tagged PEX14 and 3×FLAG‐tagged EHD1 or EHD3 fragments in the IP fractions and inputs were detected by Western blotting. H Recombinant GST (0.2 μg) or GST fused to PEX14 proteins (1 μg) and 6×His‐EHD3 protein (1 μg) were pulled down using glutathione‐Sepharose beads. GST‐tagged PEX14 and 6×His‐tagged EHD3 proteins in the pull‐down fractions and inputs were detected by western blotting. I 3D reconstitution of the quiescent G 0 ‐phase hTERT‐RPE1 cells transfected with AcGFP1‐tagged EHD3 (white) immunostained with anti‐acetylated‐tubulin (blue) and anti‐PEX14 (red) antibodies. Cholesterol was stained with Filipin III (green). Cholesterol‐containing peroxisome interacted with the ciliary pocket (arrow). The scale bars indicate 2 μm. Source data are available online for this figure.

Article Snippet: GST (0.2 μg, Abcam: ab81793) or GST fused to PEX14 proteins (1 μg, Abnova: H00005195‐P01) was loaded to glutathione‐Sepharose beads (GE Healthcare) and then incubated with 6×His‐EHD3 (1 μg, ORIGENE: TP761227), 6×His‐Rabin8 (1 μg, ORIGENE: TP760069), or 6×His‐Rab10 (1 μg, NOVUS: NBP2‐23392).

Techniques: Staining, Membrane, Transfection, Cell Culture, Immunostaining, Immunoprecipitation, Western Blot, Recombinant

Journal: The EMBO Journal

Article Title: Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

doi: 10.15252/embj.2019103499

Figure Lengend Snippet: A Schematic of ORP3 structure. ORP3 contains an N‐terminal pleckstrin homology (PH) domain that interacts with phospholipids, an FFAT motif that specifically interacts with VAP‐A/B, and a highly conserved C‐terminal OSBP‐homology domain (OHD). B Western blot analysis showing depletion of ORP3 in the ORP3 −/− hTERT‐RPE1 cell clones. GAPDH served as a loading control. C ORP3 +/+ and ORP3 −/− 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). Arrows indicate primary cilia. Scale bar, 5 μm. D Quantification of the Filipin III intensity at primary cilia from (C). ORP3 −/− hTERT‐RPE1 cells exhibited a significant reduction of ciliary cholesterol (*** P < 0.001: one‐way ANOVA with Tukey's HSD, n = 3: 40–50 cells per experiment). In the boxplot, medians, 25 th /75 th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively. E Three‐dimensional reconstitution of the quiescent G 0 ‐phase ORP3 +/+ and ORP3 −/− hTERT‐RPE1 cells immunostained with anti‐ARL13B (blue), anti‐phospho‐S473‐Akt (red), anti‐ORP3 (green), and anti‐PMP70 (white) antibodies indicates that ORP3 at the ciliary pocket (arrow and arrowhead) mediates the membrane regions of ciliary pocket and peroxisomes (arrowhead). The scale bars indicate 5 μm. F Quantification of proportion of primary cilia interacting with peroxisomes from (E). Depletion of ORP3 significantly interfered with the spatial interaction 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). G 3×FLAG‐tagged ORP3 and AcGFP1‐tagged PEX14, EHD1, or EHD3 were coexpressed in HEK293T cells and then immunoprecipitated from whole‐cell lysates using the anti‐FLAG antibody. 3×FLAG‐tagged ORP3 and AcGFP1‐tagged PEX14, EHD1 or EHD3 fragments in the IP fractions and inputs were detected by Western blotting. H ORP3 −/− hTERT‐RPE1 cells were transfected with AcGFP1‐tagged ORP3, PH domain‐deleted‐ORP3 mutant (ΔPH), FFAT motif‐deleted‐ORP3 mutant (ΔFFAT), or OHD domain‐deleted‐ORP3 mutant (ΔOHD) 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 AcGFP1‐tagged ORP3 or the mutants localized to the ciliary pocket. AcGFP1‐tagged ORP3ΔPH mutant mis‐localized to the ciliary pocket. The scale bars indicate 5 μm. I Quantification of the Filipin III intensity at primary cilia from (H). AcGFP1‐tagged ORP3 deletion mutants did not restore the ciliary cholesterol insufficiency in the ORP3 −/− hTERT‐RPE1 cells (* P < 0.05, *** P < 0.001: one‐way ANOVA with Tukey's HSD, n = 3: 40–50 cells per experiment). In the boxplot, medians, 25 th /75 th 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.

Article Snippet: GST (0.2 μg, Abcam: ab81793) or GST fused to PEX14 proteins (1 μg, Abnova: H00005195‐P01) was loaded to glutathione‐Sepharose beads (GE Healthcare) and then incubated with 6×His‐EHD3 (1 μg, ORIGENE: TP761227), 6×His‐Rabin8 (1 μg, ORIGENE: TP760069), or 6×His‐Rab10 (1 μg, NOVUS: NBP2‐23392).

Techniques: Western Blot, Clone Assay, Control, Incubation, Staining, Membrane, Immunoprecipitation, Transfection, Mutagenesis, Cell Culture, Immunostaining

Journal: The EMBO Journal

Article Title: Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

doi: 10.15252/embj.2019103499

Figure Lengend Snippet: A Whole‐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. B Whole‐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. C Whole‐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. D Western 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. E Quiescent G 0 ‐phase hTERT‐RPE1 cells transfected with 3×FLAG‐tagged KIFC3 were immunostained with anti‐FLAG (green), anti‐PEX14 (red), and anti‐α‐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. F Western 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. H Quantification 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, 25 th /75 th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively. I Quiescent G 0 ‐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. J Quantification 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 (ΔRod), or motor domain‐deleted KIFC3 mutant (ΔMotor) 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. L Quantification 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, 25 th /75 th 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.

Article Snippet: GST (0.2 μg, Abcam: ab81793) or GST fused to PEX14 proteins (1 μg, Abnova: H00005195‐P01) was loaded to glutathione‐Sepharose beads (GE Healthcare) and then incubated with 6×His‐EHD3 (1 μg, ORIGENE: TP761227), 6×His‐Rabin8 (1 μg, ORIGENE: TP760069), or 6×His‐Rab10 (1 μg, NOVUS: NBP2‐23392).

Techniques: Expressing, Immunoprecipitation, Western Blot, Positive Control, Fractionation, Injection, SDS Page, Transfection, Clone Assay, Control, Incubation, Staining, Disruption, Mutagenesis, Cell Culture, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Peroxisome protein import recapitulated in Xenopus egg extracts

doi: 10.1083/jcb.201901152

Figure Lengend Snippet: Peroxisome targeting of SKL-containing fluorescent proteins in Xenopus egg extracts. (A) Cleared egg extract was incubated with 0.9 µM purified mCherry-SKL for 1 h at 18°C. The formation of bright puncta was visualized with a spinning-disk confocal microscope. (B) As in A, but with mCherry lacking the SKL targeting sequence. (C) As in A, but in the presence of a synthetic peptide (300 µM) with a C-terminal SKL sequence. (D) As in C, but with a peptide containing a scrambled import signal at its C terminus (KLS). (E) Cleared egg extract was incubated with 0.6 µM purified mScarlet-SKL for 5 h at 18°C. The sample was subjected to flotation in a discontinuous sucrose gradient. Fractions were collected and analyzed with a fluorescence microscope. Shown is the bottom fraction containing nonimported substrate. (F) As in E, but for the top fraction, containing peroxisome-associated substrate. (G) Quantification of the number of fluorescent peroxisome foci in the different fractions of the sucrose gradient. Shown are the mean and standard deviation from >40 images of two different experiments. (H) All fractions of the sucrose gradient were analyzed by SDS-PAGE, followed by immunoblotting with antibodies against the peroxisome membrane protein Pex14. Bars, 5 µm.

Article Snippet: The open reading frame of Xenopus Pex14 was synthesized (Genscript) based on a Pex14 protein sequence obtained from mass spectrometry data ( ).

Techniques: Incubation, Purification, Microscopy, Sequencing, Fluorescence, Standard Deviation, SDS Page, Western Blot, Membrane

Journal: The Journal of Cell Biology

Article Title: Peroxisome protein import recapitulated in Xenopus egg extracts

doi: 10.1083/jcb.201901152

Figure Lengend Snippet: Protein targeting to peroxisomes depends on Pex5 and Pex14. (A) Cleared Xenopus egg extract was incubated for 1 h at 18°C with 0.5 µM GFP-SKL in the presence of 6 µM of a cytosolic fragment of the peroxisome docking protein Pex14 (cytPex14). The sample was imaged with a spinning-disk confocal microscope. (B) As in A, but without adding cytPex14. (C) Egg extract was incubated with beads containing immobilized cytPex14 and subjected to SDS-PAGE, followed by immunoblotting with Pex5 antibodies (lane 2). A control was done with beads lacking cytPex14 (lane 1; mock depletion). Purified, recombinant Pex5 was analyzed either without added extract (lane 3) or after addition of different amounts to depleted extract (lanes 4–6). MW, molecular weight. (D) Pex5-depleted extract was incubated for 1 h at 18°C with 0.5 µM GFP-SKL and imaged with a spinning-disk confocal microscope. (E) As in D, but with mock-depleted extract. (F) As in D, but in the presence of 1 µM purified Pex5. (G) As in F, but with 1 µM purified Pex5 A510W , a Pex5 mutant defective in SKL binding. All experiments were performed at least three times. Bars, 10 µm.

Article Snippet: The open reading frame of Xenopus Pex14 was synthesized (Genscript) based on a Pex14 protein sequence obtained from mass spectrometry data ( ).

Techniques: Incubation, Microscopy, SDS Page, Western Blot, Control, Purification, Recombinant, Molecular Weight, Mutagenesis, Binding Assay