Journal: Open Biology

Article Title: Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

doi: 10.1098/rsob.160274

Figure Lengend Snippet: Ninein depletion in epithelial cells. ( a ) Scramble and ninein siRNA (seq a) depleted TC7 cells methanol fixed and stained for MTs (mAb YL1/2; green; invert) and ninein (mAb N5; red), showing loss of radial MT organization and centrosomal focus in depleted cell. ( b ) Western blot of cell lysates of scramble and ninein (seq a and 3) siRNA showing ninein (mAb Bethyl) and β-actin expression. ( c ) Confocal optical sections and three-dimensional reconstructions of scramble and ninein siRNA (seq a) depleted TC7 cells seeded for apico-basal MT array formation, fixed in methanol and labelled for MTs (mAb YL1/2) and ninein (pAb Pep3). ( d ) Analysis of cell height (scramble n = 284, nin siRNA seq a = 251) and cross-sectional area (scramble n = 190, nin siRNA seq a = 200) in scramble and ninein siRNA-treated TC7 cells show decreased cell height and increased area in depleted cell (Mann–Whitney U -test, *** p < 0.001). Scale bars, 10 µm.

Article Snippet: Ninein depletion in epithelial cells. ( a ) Scramble and ninein siRNA (seq a) depleted TC7 cells methanol fixed and stained for MTs (mAb YL1/2; green; invert) and ninein (mAb N5; red), showing loss of radial MT organization and centrosomal focus in depleted cell. ( b ) Western blot of cell lysates of scramble and ninein (seq a and 3) siRNA showing ninein (mAb Bethyl) and β-actin expression. ( c ) Confocal optical sections and three-dimensional reconstructions of scramble and ninein siRNA (seq a) depleted TC7 cells seeded for apico-basal MT array formation, fixed in methanol and labelled for MTs (mAb YL1/2) and ninein (pAb Pep3). ( d ) Analysis of cell height (scramble n = 284, nin siRNA seq a = 251) and cross-sectional area (scramble n = 190, nin siRNA seq a = 200) in scramble and ninein siRNA-treated TC7 cells show decreased cell height and increased area in depleted cell (Mann–Whitney U -test, *** p < 0.001).

Techniques: Staining, Western Blot, Expressing, MANN-WHITNEY

Journal: Open Biology

Article Title: Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

doi: 10.1098/rsob.160274

Figure Lengend Snippet: CLIP-170 and ninein in confluent and fully differentiated MDCKII cysts. ( a , b ) Cells grown in Matrigel to form three-dimensional cysts. ( a ) Optical sections of cysts fixed in methanol and stained for ninein (pAb Pep3; red) and E-cadherin (mAb, blue) showing apical localization in ( a (i)) and cyst regions showing apico-basal MTs (mAb YL1/2, green in ( a (ii)); pAb α-tubulin, red in ( a (iii))). Optical oblique section though cyst region in ( a (ii)) shows both apical and baso-lateral views with ninein (pAb N5; red) at apical cortex (arrowhead) and centrosomes (arrow) in polarized epithelial cells. Baso-lateral view of cyst epithelial cells in ( a (iii)) shows γ-tubulin (green, ( a (iii))) at centrosomes. ( b ) Optical section of cyst fixed in formaldehyde–methanol and stained for CLIP-170 (pAb, green) and MTs (mAb YL1/2, red) ( b (i)) and cyst regions revealing apico-basal MTs (red; ( b (ii));( b (iii))) and apical concentration of CLIP-170 colocalizing with MTs at apical cortex ( b (iv)). ( c ) Confluent cells fixed in methanol and labelled for ninein (pAb Pep3, green) and CLIP-170 (mAb F-3, red) showing some colocalization (yellow) at cortical regions. ( d ) Western blots of fractionated control and nocodazole-treated cell lysates showing cytosol and membrane fractions, blots probed for CLIP-170 (pAb), E-cadherin (mAb) and α-tubulin (pAb). Note that the double band for CLIP-170 is absent in the nocodazole-treated cell extract and this is most likely due to nocodazole-induced dephosphorylation . ( e ) Nocodazole-treated cells expressing GFP-CLIP-170 (green) fixed in methanol and labelled for β-catenin (pAb, purple) showing cortical rings of GFP-CLIP-170. ( f ) Nocodazole-treated cells fixed in methanol and stained for ninein (pAb Pep3, blue) and E-cadherin (mAb, red). Enlarged inverted junctional region showing cortical ninein remains at the cell cortex following nocodazole treatment. Scale bars, 10 µm except for ( a (iii)) and ( b (ii)), 5 µm.

Article Snippet: Ninein depletion in epithelial cells. ( a ) Scramble and ninein siRNA (seq a) depleted TC7 cells methanol fixed and stained for MTs (mAb YL1/2; green; invert) and ninein (mAb N5; red), showing loss of radial MT organization and centrosomal focus in depleted cell. ( b ) Western blot of cell lysates of scramble and ninein (seq a and 3) siRNA showing ninein (mAb Bethyl) and β-actin expression. ( c ) Confocal optical sections and three-dimensional reconstructions of scramble and ninein siRNA (seq a) depleted TC7 cells seeded for apico-basal MT array formation, fixed in methanol and labelled for MTs (mAb YL1/2) and ninein (pAb Pep3). ( d ) Analysis of cell height (scramble n = 284, nin siRNA seq a = 251) and cross-sectional area (scramble n = 190, nin siRNA seq a = 200) in scramble and ninein siRNA-treated TC7 cells show decreased cell height and increased area in depleted cell (Mann–Whitney U -test, *** p < 0.001).

Techniques: Staining, Concentration Assay, Western Blot, De-Phosphorylation Assay, Expressing

Journal: Open Biology

Article Title: Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

doi: 10.1098/rsob.160274

Figure Lengend Snippet: Ninein and CLIP-170 in mouse small intestinal tissue and organoids. ( a ) Isolated basal region of small intestine crypts fixed in methanol ( a (i–iii)) or formaldehyde–methanol ( a (iv,v)) and stained for ninein (pAB Pep3, red) ( a (i–iii)), γ-tubulin (mAb, green in ( a (i))), β-catenin (mAb, green in ( a (iii))), CLIP-170 (pAb, green; arrow in ( a (iv)) enlarged region in ( a (v))) and MTs (mAb YL1/2, red in ( a (iv))). Apico-basal MTs are evident in cells of the stem cell region ( a (iv)) but ninein is concentrated at the apical centrosome (( a (i–iii))), where it colocalizes with γ-tubulin (inset in ( a (i))). CLIP-170 is present as comets in cells within the stem cell region ( a (iv,v)). ( b ) Confocal images of small intestine villus fixed in methanol ( b (i–iv)) or formaldehyde–methanol ( b (v,vi)) and stained for ninein (pAb Pep3, red) and CLIP-170 (pAb, green) localized at n-MTOCs at cell apices. ( b (i,iii)) Cryostat section of villus with apical ninein localization (invert, arrow enlarged region in ( b (iii))). ( b (ii,iv)) Optical sections through whole mount villus showing apical views of apical surface ( b (ii)) and junctions ( b (iv)) (E-cadherin, mAb, green) with ninein (pAb Pep3, red) puncta at apical surface and AJ-associated n-MTOCs. ( b (v,vi)) Optical sections of whole mount villus stained for CLIP-170 (pAb, green) and MTs (mAb YL1/2, red) showing cross-sectional view ( b (v)) of CLIP-170 at apical junctional n-MTOCs and lateral view ( b (vi)) of villus cells with CLIP-170 concentrated at apical surface n-MTOCs (arrow) and along length of MTs. ( c ) Diagram showing small intestine with crypt and villus regions and organoid generation from isolated mouse small intestinal stem cells initially leading to the formation of cell aggregates that develop into cysts and then into organoids with crypt and villus domains. ( d ) MTs (mAb YL1/2, green in ( d (i,ii)) and red in ( d (iii)), Ninein (pAb Pep3, red) and CLIP-170 (pAb, green) in 7-day cultured gut organoids showing apico-basal MT (mAb YL1/2) arrays in both crypt and villus domains, with ninein concentrated at apical centrosomes (arrow in ( d (i))) in stem cell region of crypt and ninein (arrow in ( d (ii)) and CLIP-170 (arrow in ( d (iii))) at apical surface n-MTOCs in villus-domain cells. Scale bars, 5 µm except for ( b (i)), 10 µm.

Article Snippet: Ninein depletion in epithelial cells. ( a ) Scramble and ninein siRNA (seq a) depleted TC7 cells methanol fixed and stained for MTs (mAb YL1/2; green; invert) and ninein (mAb N5; red), showing loss of radial MT organization and centrosomal focus in depleted cell. ( b ) Western blot of cell lysates of scramble and ninein (seq a and 3) siRNA showing ninein (mAb Bethyl) and β-actin expression. ( c ) Confocal optical sections and three-dimensional reconstructions of scramble and ninein siRNA (seq a) depleted TC7 cells seeded for apico-basal MT array formation, fixed in methanol and labelled for MTs (mAb YL1/2) and ninein (pAb Pep3). ( d ) Analysis of cell height (scramble n = 284, nin siRNA seq a = 251) and cross-sectional area (scramble n = 190, nin siRNA seq a = 200) in scramble and ninein siRNA-treated TC7 cells show decreased cell height and increased area in depleted cell (Mann–Whitney U -test, *** p < 0.001).

Techniques: Isolation, Staining, Cell Culture

Journal: Open Biology

Article Title: Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

doi: 10.1098/rsob.160274

Figure Lengend Snippet: CLIP-170 siRNA knockdown in MDCKII cells leads to reduced cortical ninein and smaller cysts. ( a ) Western blot of lysates of control, scramble and canine CLIP-170 siRNA sequences ( a – d ) showing CLIP-170 and β-actin expression. ( b ) Scramble and CLIP-170 siRNA-treated cells fixed in methanol and stained for ninein (pAb N5, blue and invert) and CLIP-170 (mAb, red). ( c ) Junction fluorescence intensity profile analyses ( n = 128) of ninein in scramble and CLIP-170-depleted cells. ( d ) Relative peak intensities of ninein at junctions in scramble and CLIP-170 siRNA-depleted cells reveal a significant decrease in ninein intensity in depleted cells (Mann–Whitney U -test, *** p < 0.05). ( e ) Western blot of lysates of control, scramble, CLIP-170 siRNA, IQGAP1 siRNA and Rac1 inhibitor NSC23766 (250 µM) treatments showing ninein (pAb Bethyl) and β-actin (pAb) expression. ( f ) Relative centrosomal ninein fluorescence intensity ( n = 50) in control, scramble, CLIP-170 siRNA and Rac1 inhibitor NSC23766 (250 µM) treated cells revealing no significant difference (unpaired t -test). ( g ) Scramble and CLIP-170 siRNA-treated cells grown in Matrigel to induce cyst formation and fixed in formaldehyde–methanol and stained for MTs (mAb YL1/2, red) and CLIP-170 (pAb, green) at day 6 showing apico-basal MTs in both scramble and knockdown cysts. Note the marked decrease in cyst size in CLIP-170 siRNA-treated cysts. Inset shows MTs in depleted cell (arrow). ( h ) Cyst sizes in scramble and CLIP-170-depleted cells based on cross-sectional areas in μm 2 with bars indicating averages showing significantly smaller cyst area in knockdown (Mann–Whitney U -test, *** p < 0.001). Scale bars, 10 µm.

Article Snippet: Ninein depletion in epithelial cells. ( a ) Scramble and ninein siRNA (seq a) depleted TC7 cells methanol fixed and stained for MTs (mAb YL1/2; green; invert) and ninein (mAb N5; red), showing loss of radial MT organization and centrosomal focus in depleted cell. ( b ) Western blot of cell lysates of scramble and ninein (seq a and 3) siRNA showing ninein (mAb Bethyl) and β-actin expression. ( c ) Confocal optical sections and three-dimensional reconstructions of scramble and ninein siRNA (seq a) depleted TC7 cells seeded for apico-basal MT array formation, fixed in methanol and labelled for MTs (mAb YL1/2) and ninein (pAb Pep3). ( d ) Analysis of cell height (scramble n = 284, nin siRNA seq a = 251) and cross-sectional area (scramble n = 190, nin siRNA seq a = 200) in scramble and ninein siRNA-treated TC7 cells show decreased cell height and increased area in depleted cell (Mann–Whitney U -test, *** p < 0.001).

Techniques: Western Blot, Expressing, Staining, Fluorescence, MANN-WHITNEY

Journal: Open Biology

Article Title: Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

doi: 10.1098/rsob.160274

Figure Lengend Snippet: Loss of ninein at n-MTOCs in Clip1/Clip2 double knockout mouse intestine. ( a ) Confocal images of methanol-fixed villus cells stained for ninein (pAb Pep3, red) and β-catenin (mAb, green) showing baso-lateral and apical cross-sectional views and revealing almost total absence of ninein at apical surface n-MTOCs in KO. ( b ) Fluorescence intensity profiles for β-catenin ( n = 112) and ninein ( n = 112) at junctions in WT and KO villus. ( c ) Relative peak fluorescence intensities for β-catenin and ninein at junctional sites in WT and KO villus revealing no significant difference in junctional β-catenin but a significant reduction in ninein (Mann–Whitney U -test, *** p < 0.001). ( d ) Confocal sections showing baso-lateral views of methanol-fixed villus cells stained for γ-tubulin (mAb, green) and β-catenin (pAb, red) revealing γ-tubulin at apical n-MTOCs in both WT and KO. Scale bars, 5 µm.

Article Snippet: Ninein depletion in epithelial cells. ( a ) Scramble and ninein siRNA (seq a) depleted TC7 cells methanol fixed and stained for MTs (mAb YL1/2; green; invert) and ninein (mAb N5; red), showing loss of radial MT organization and centrosomal focus in depleted cell. ( b ) Western blot of cell lysates of scramble and ninein (seq a and 3) siRNA showing ninein (mAb Bethyl) and β-actin expression. ( c ) Confocal optical sections and three-dimensional reconstructions of scramble and ninein siRNA (seq a) depleted TC7 cells seeded for apico-basal MT array formation, fixed in methanol and labelled for MTs (mAb YL1/2) and ninein (pAb Pep3). ( d ) Analysis of cell height (scramble n = 284, nin siRNA seq a = 251) and cross-sectional area (scramble n = 190, nin siRNA seq a = 200) in scramble and ninein siRNA-treated TC7 cells show decreased cell height and increased area in depleted cell (Mann–Whitney U -test, *** p < 0.001).

Techniques: Double Knockout, Staining, Fluorescence, MANN-WHITNEY

Journal: Open Biology

Article Title: Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

doi: 10.1098/rsob.160274

Figure Lengend Snippet: IQGAP1 siRNA depletion leads to loss of MT cortical targeting and reduced ninein at n-MTOCs. ( a ) Western blot of Co-IP experiments using either CLIP-170 or IgG as bait to pull down protein complexes in TC7 cells showing CLIP-170 pulls down endogenous CLIP-170, IQGAP1 and β-catenin but not the IgG control lanes (CLIP-170 pAb was used for probing but mAb used as bait). ( b ) ARPE-19 cells methanol fixed and stained for IQGAP1 (mAb), MTs (YL1/2) and β-catenin (pAb), purple in b (i) indicating colocalization and nocodazole recovery. ( b (ii)) showing CLIP-170-bound MTs targeting cortical IQGAP1 located on the inner face of junctional β-catenin puncta. The arrow indicates region enlarged in the inset to the left. ( c ) Mixed culture of ARPE-19 cells fixed in methanol showing a scramble cell next to a IQGAP1-depleted cell (*) stained for IQGAP1 (mAb, red, invert) and MTs (rab α-tubulin, blue, invert). Enlarged region (arrow) showing lack of cortical MT targeting in IQGAP1-depleted cell. ( d ) Western blots of lysates of control, scramble and IQGAP1 siRNA ARPE-19 and MDCKII cells showing IQGAP1 and β-actin expression. ( e ) Scramble and IQGAP1 siRNA-treated cells methanol fixed and stained for ninein (pAb Pep3, green) and IQGAP1 (mAb, red) showing less cortical ninein in depleted cells. ( f ) Junctional fluorescence intensity profile ( n = 92) for ninein in scramble and IQGAP1 siRNA-treated cells. ( g ) Relative peak fluorescence intensities for β-catenin and ninein at junctions in scramble and IQGAP1 siRNA-treated cells showing no significance in β-catenin intensities (unpaired t -test) but a significant reduction in ninein (non-parametric Mann–Whitney U -test, *** p < 0.001). Scale bars, 10 µm except ( b (ii)), 2 µm.

Article Snippet: Ninein depletion in epithelial cells. ( a ) Scramble and ninein siRNA (seq a) depleted TC7 cells methanol fixed and stained for MTs (mAb YL1/2; green; invert) and ninein (mAb N5; red), showing loss of radial MT organization and centrosomal focus in depleted cell. ( b ) Western blot of cell lysates of scramble and ninein (seq a and 3) siRNA showing ninein (mAb Bethyl) and β-actin expression. ( c ) Confocal optical sections and three-dimensional reconstructions of scramble and ninein siRNA (seq a) depleted TC7 cells seeded for apico-basal MT array formation, fixed in methanol and labelled for MTs (mAb YL1/2) and ninein (pAb Pep3). ( d ) Analysis of cell height (scramble n = 284, nin siRNA seq a = 251) and cross-sectional area (scramble n = 190, nin siRNA seq a = 200) in scramble and ninein siRNA-treated TC7 cells show decreased cell height and increased area in depleted cell (Mann–Whitney U -test, *** p < 0.001).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Staining, Expressing, Fluorescence, MANN-WHITNEY

Journal: Open Biology

Article Title: Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

doi: 10.1098/rsob.160274

Figure Lengend Snippet: Rac1 inhibition leads to reduced cortical ninein and MT junctional targeting. ( a ) Control and Rac1-inhibited (250 µM NSC23766) cells methanol fixed and stained for ninein (pAb N5, green, invert) and β-catenin (mAb, red, invert) showing a marked reduction in cortical ninein in Rac1-inhibited cells. ( b ) Junctional fluorescence intensity profile for ninein ( n = 112) in control and Rac1-inhibited cells. ( c ) Relative peak fluorescence intensity of E-cadherin and ninein at junctions in control and Rac1-inhibited cells showing no significance in E-cadherin but in ninein (non-parametric Mann–Whitney U -test, *** p < 0.001). ( d ) Control and Rac1-inhibited (250 µM NSC23766) ARPE-19 cells methanol fixed and stained for β-catenin (mAb, purple) and MTs (pAb α-tubulin, green), with enlarged regions (arrowed) highlighting cortical MT approaches. Note several MTs aligned parallel to the cortex in Rac1-inhibited cells. ( e ) Graph showing the mean MT orientation to cell junctions ( n = 30), using FibrilTool revealing significant (***) deviation from perpendicular targeting in inhibited cells (non-parametric Mann–Whitney U -test, *** p < 0.001). ( f ) Graph showing the mean ( n = 30) number of MT contacts per 10 µm, junctional β-catenin staining revealing significantly fewer cortical contacts in inhibited cells (unpaired t -test, *** p < 0.001). Scale bars, 10 µm.

Article Snippet: Ninein depletion in epithelial cells. ( a ) Scramble and ninein siRNA (seq a) depleted TC7 cells methanol fixed and stained for MTs (mAb YL1/2; green; invert) and ninein (mAb N5; red), showing loss of radial MT organization and centrosomal focus in depleted cell. ( b ) Western blot of cell lysates of scramble and ninein (seq a and 3) siRNA showing ninein (mAb Bethyl) and β-actin expression. ( c ) Confocal optical sections and three-dimensional reconstructions of scramble and ninein siRNA (seq a) depleted TC7 cells seeded for apico-basal MT array formation, fixed in methanol and labelled for MTs (mAb YL1/2) and ninein (pAb Pep3). ( d ) Analysis of cell height (scramble n = 284, nin siRNA seq a = 251) and cross-sectional area (scramble n = 190, nin siRNA seq a = 200) in scramble and ninein siRNA-treated TC7 cells show decreased cell height and increased area in depleted cell (Mann–Whitney U -test, *** p < 0.001).

Techniques: Inhibition, Staining, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: DGAT1 Inhibitor Suppresses Prostate Tumor Growth and Migration by Regulating Intracellular Lipids and Non-Centrosomal MTOC Protein GM130

doi: 10.1038/s41598-019-39537-z

Figure Lengend Snippet: DGAT1 inhibition decreases ncMTOC-related proteins in prostate cancer cells. ( A ) LNCaP and PC-3 cells were treated with 1 µM DGAT1 inhibitor for 24 h, and, after fixation, the cells were stained with mouse anti-pericentrin antibody (green) and DAPI (blue) to visualize the MTOCs and nucleus, respectively. Size bar: 10 µm. ( B ) The total number of pericentrin-positive MTOCs was counted per single cell (n = 50). ( C ) γ-tubulin (48 kD), ninein (243 kD), GM130 (140 kD), and CETN1 (20 kD) levels were analyzed by western blot in PC-3 control (CTR) and treated with 1 µM DGAT1 inhibitor or 10 nM PEDF. ( D ) γ-tubulin, ( E ) ninein, ( F ) GM130, and ( G ) centrin 1 (CETN1) densities were normalized by GAPDH. For the western blot analysis, each protein has been detected by sequential staining and stripping using the same membrane. Data are presented as mean ± SEM. Student’s unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: PEDF (1:1000 - Cat. No. AB-PEDF4 - BioProducts MD, Middletown, MD), ATGL (1:200 – Cat. No. 10006409 - Cayman Chemical, Ann Arbor, MI), DGAT1 (1:10000 – Cat. No. ab181180 - Abcam, Cambridge, United Kingdom), perilipin 2 (PLIN2) (2 μg/ml – Cat. No. LS-B3121 – Lifespan Biosciences, Seattle, WA), γ-tubulin (1 μg/ml – Cat. No. ab27074 - Abcam, Cambridge, United Kingdom), centrin 1 (CETN1) (1:2000 – Cat. No. ab11257 - Abcam, Cambridge, United Kingdom), ninein (1:5000 – Cat. No. NB100-74631 – Novus Biologicals, Littleton, CO), GM130 (1:5000 – Cat. No. ab52649 - Abcam, Cambridge, United Kingdom), CLASP2 (1:1000 – Cat. No. ab95373 - Abcam, Cambridge, United Kingdom), and α-tubulin (1 μg/ml – Cat. No. ab15246 - Abcam, Cambridge, United Kingdom) antibodies were used.

Techniques: Inhibition, Staining, Western Blot, Control, Stripping Membranes, Membrane

Journal: Scientific Reports

Article Title: DGAT1 Inhibitor Suppresses Prostate Tumor Growth and Migration by Regulating Intracellular Lipids and Non-Centrosomal MTOC Protein GM130

doi: 10.1038/s41598-019-39537-z

Figure Lengend Snippet: Model of DGAT1 inhibition of lipid-MTOC axis in prostate cancer. This is a model of lipid-MTOC crosstalk in prostate cancer. At baseline, PCa cells are lipid-rich with MTOC amplification and a complex and dense MT network. At the site of the Golgi apparatus, GM130 and CLASP2-coated MTs are strongly expressed. PC-3 cells express high levels of DGAT1 and low levels of PEDF protein. After treatment with a DGAT1 inhibitor, the LD density is reduced as well as the number of MTOCs. The expression levels of proteins regulating ncMTOCs (GM130 and CLASP2) and MTOCs in general (γ-tubulin and ninein) were significantly decreased. Structural changes in the MT network post-treatment revealed fragmentation and loss of many linear MTs. Blockade of the lipid-MTOC axis via DGAT1 inhibitor resulted in suppression of prostate cancer growth and invasion both in vitro and in vivo .

Article Snippet: PEDF (1:1000 - Cat. No. AB-PEDF4 - BioProducts MD, Middletown, MD), ATGL (1:200 – Cat. No. 10006409 - Cayman Chemical, Ann Arbor, MI), DGAT1 (1:10000 – Cat. No. ab181180 - Abcam, Cambridge, United Kingdom), perilipin 2 (PLIN2) (2 μg/ml – Cat. No. LS-B3121 – Lifespan Biosciences, Seattle, WA), γ-tubulin (1 μg/ml – Cat. No. ab27074 - Abcam, Cambridge, United Kingdom), centrin 1 (CETN1) (1:2000 – Cat. No. ab11257 - Abcam, Cambridge, United Kingdom), ninein (1:5000 – Cat. No. NB100-74631 – Novus Biologicals, Littleton, CO), GM130 (1:5000 – Cat. No. ab52649 - Abcam, Cambridge, United Kingdom), CLASP2 (1:1000 – Cat. No. ab95373 - Abcam, Cambridge, United Kingdom), and α-tubulin (1 μg/ml – Cat. No. ab15246 - Abcam, Cambridge, United Kingdom) antibodies were used.

Techniques: Inhibition, Amplification, Expressing, In Vitro, In Vivo

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: The primary antibodies used were as follows: mouse anti‐acetylated‐tubulin monoclonal antibody mAb (T7451; Sigma‐Aldrich); mouse anti‐Smo mAb (sc‐166685; Santa Cruz); rabbit anti‐Arl13b polyclonal antibody pAb (17711‐1‐AP; Proteintech); rabbit anti‐PEX1 pAb (13669‐1‐AP; Proteintech); rabbit anti‐PEX14 pAb (10594‐1‐AP; Proteintech); rabbit anti‐pericentrin pAb (A301‐348A; Bethyl Laboratories); mouse anti‐PMP70 mAb (SAB4200181; Sigma‐Aldrich); mouse anti‐ninein mAb (MABT29; Merck Millipore); mouse anti‐GAPDH mAb (sc‐32233; Santa Cruz Biotechnology); rabbit anti‐GFP pAb (598; MBL); mouse anti‐phospho‐S473 Akt mAb (#4051; Cell Signaling Technology); mouse anti‐GFP mAb (11‐814‐460‐001; Roche); rabbit anti‐Rabin8 pAb (12321‐1‐AP; Proteintech); rabbit anti‐Rab10 pAb (27094‐1‐AP; Proteintech); rabbit anti‐KIFC3 pAb (10125‐2‐AP; Proteintech); rat anti‐α‐tubulin mAb (NB600‐506; Novus); mouse anti‐γ‐tubulin mAb (T6557; Sigma‐Aldrich); mouse anti‐CYPOR mAb (sc‐25270; Santa Cruz); rabbit anti‐NPC1 pAb (13926‐1‐AP; Proteintech); rabbit anti‐ABCD1 pAb (18138‐1‐AP; Proteintech); rabbit anti‐EHD1 pAb (24657‐1‐AP; Proteintech); rabbit anti‐EHD3 pAb (25320‐1‐AP; Proteintech); rabbit anti‐Shh pAb (ab53281; Abcam); mouse anti‐ORP3 mAb (sc‐398326; Santa Cruz); rabbit anti‐ACOX1 pAb (10957‐1‐AP; Proteintech); goat anti‐Patched 1 pAb (sc‐6149; Santa Cruz); rabbit anti‐ODF2 pAb (HPA001874; Sigma‐Aldrich); and mouse anti‐DYKDDDDK (FLAG) tag mAb (018‐22381; Fujifilm Wako).

Techniques: Western Blot, Clone Assay, 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: The primary antibodies used were as follows: mouse anti‐acetylated‐tubulin monoclonal antibody mAb (T7451; Sigma‐Aldrich); mouse anti‐Smo mAb (sc‐166685; Santa Cruz); rabbit anti‐Arl13b polyclonal antibody pAb (17711‐1‐AP; Proteintech); rabbit anti‐PEX1 pAb (13669‐1‐AP; Proteintech); rabbit anti‐PEX14 pAb (10594‐1‐AP; Proteintech); rabbit anti‐pericentrin pAb (A301‐348A; Bethyl Laboratories); mouse anti‐PMP70 mAb (SAB4200181; Sigma‐Aldrich); mouse anti‐ninein mAb (MABT29; Merck Millipore); mouse anti‐GAPDH mAb (sc‐32233; Santa Cruz Biotechnology); rabbit anti‐GFP pAb (598; MBL); mouse anti‐phospho‐S473 Akt mAb (#4051; Cell Signaling Technology); mouse anti‐GFP mAb (11‐814‐460‐001; Roche); rabbit anti‐Rabin8 pAb (12321‐1‐AP; Proteintech); rabbit anti‐Rab10 pAb (27094‐1‐AP; Proteintech); rabbit anti‐KIFC3 pAb (10125‐2‐AP; Proteintech); rat anti‐α‐tubulin mAb (NB600‐506; Novus); mouse anti‐γ‐tubulin mAb (T6557; Sigma‐Aldrich); mouse anti‐CYPOR mAb (sc‐25270; Santa Cruz); rabbit anti‐NPC1 pAb (13926‐1‐AP; Proteintech); rabbit anti‐ABCD1 pAb (18138‐1‐AP; Proteintech); rabbit anti‐EHD1 pAb (24657‐1‐AP; Proteintech); rabbit anti‐EHD3 pAb (25320‐1‐AP; Proteintech); rabbit anti‐Shh pAb (ab53281; Abcam); mouse anti‐ORP3 mAb (sc‐398326; Santa Cruz); rabbit anti‐ACOX1 pAb (10957‐1‐AP; Proteintech); goat anti‐Patched 1 pAb (sc‐6149; Santa Cruz); rabbit anti‐ODF2 pAb (HPA001874; Sigma‐Aldrich); and mouse anti‐DYKDDDDK (FLAG) tag mAb (018‐22381; Fujifilm Wako).

Techniques: Staining, 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 Rabin8 +/+ and Rabin8 −/− 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. B Quantification of the Filipin III intensity at primary cilia from (A). Rabin8 −/− 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. C Quiescent G 0 ‐phase Rabin8 +/+ and Rabin8 −/− hTERT‐RPE1 cells were immunostained with anti‐ARL13B (blue), anti‐phospho‐S473‐Akt (red), anti‐ninein (green), and anti‐PMP70 (white) antibodies. Arrow indicates the peroxisomes contacting the ciliary pocket. Scale bar, 5 μm. D Quantification of (C) indicating that disruption of the Rabin8 gene 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). E Schematic representation of the rapamycin‐inducible peroxisome targeting to the ciliary pocket system. F Quiescent G 0 ‐phase hTERT‐RPE1 cells transiently expressing PEX3‐GFP‐FRB (green) and tdTomato‐BicD2‐FKBP (red) treated with rapamycin were imaged live for 10 min by confocal microscopy . Scale bar, 5 μm. G Quiescent G 0 ‐phase Rabin8 +/+ and Rabin8 −/− hTERT‐RPE1 cells transfected with PEX3‐GFP‐FRB (red) and tdTomato‐BicD2‐FKBP (white) were treated with or without 100 μM rapamycin for 30 min, and then immunostained with anti‐acetylated‐tubulin (blue) antibody. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm. H Quantification of the Filipin III intensity at primary cilia from (G). Rapamycin‐induced peroxisome targeting to the ciliary pocket restored the reduction of ciliary cholesterol in the Rabin8 −/− hTERT‐RPE1 cells (*** 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 Rabin8 −/− hTERT‐RPE1 cells were transfected with AcGFP1‐tagged Rabin8, GFP‐tagged Rab8A, GFP‐tagged Rab8A‐Q67L (constitutively active form), GFP‐Rab8B, GFP‐Rab8B‐Q67L (constitutively active form), AcGFP1‐tagged Rab10, or AcGFP1‐tagged Rab10‐Q68L (constitutively active form), and then immunostained with anti‐GFP (red), anti‐acetylated‐tubulin (blue), and anti‐ninein (white) antibodies. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm. J Quantification of (I) showing that AcGFP1‐tagged Rabin8 and AcGFP1‐tagged Rab10‐Q68L effectively restored the defect in ciliary enrichment of cholesterol in Rabin8 −/− hTERT‐RPE1 cells (*** P < 0.001: one‐way ANOVA with Tukey's HSD, n = 3: 35–40 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.

Article Snippet: The primary antibodies used were as follows: mouse anti‐acetylated‐tubulin monoclonal antibody mAb (T7451; Sigma‐Aldrich); mouse anti‐Smo mAb (sc‐166685; Santa Cruz); rabbit anti‐Arl13b polyclonal antibody pAb (17711‐1‐AP; Proteintech); rabbit anti‐PEX1 pAb (13669‐1‐AP; Proteintech); rabbit anti‐PEX14 pAb (10594‐1‐AP; Proteintech); rabbit anti‐pericentrin pAb (A301‐348A; Bethyl Laboratories); mouse anti‐PMP70 mAb (SAB4200181; Sigma‐Aldrich); mouse anti‐ninein mAb (MABT29; Merck Millipore); mouse anti‐GAPDH mAb (sc‐32233; Santa Cruz Biotechnology); rabbit anti‐GFP pAb (598; MBL); mouse anti‐phospho‐S473 Akt mAb (#4051; Cell Signaling Technology); mouse anti‐GFP mAb (11‐814‐460‐001; Roche); rabbit anti‐Rabin8 pAb (12321‐1‐AP; Proteintech); rabbit anti‐Rab10 pAb (27094‐1‐AP; Proteintech); rabbit anti‐KIFC3 pAb (10125‐2‐AP; Proteintech); rat anti‐α‐tubulin mAb (NB600‐506; Novus); mouse anti‐γ‐tubulin mAb (T6557; Sigma‐Aldrich); mouse anti‐CYPOR mAb (sc‐25270; Santa Cruz); rabbit anti‐NPC1 pAb (13926‐1‐AP; Proteintech); rabbit anti‐ABCD1 pAb (18138‐1‐AP; Proteintech); rabbit anti‐EHD1 pAb (24657‐1‐AP; Proteintech); rabbit anti‐EHD3 pAb (25320‐1‐AP; Proteintech); rabbit anti‐Shh pAb (ab53281; Abcam); mouse anti‐ORP3 mAb (sc‐398326; Santa Cruz); rabbit anti‐ACOX1 pAb (10957‐1‐AP; Proteintech); goat anti‐Patched 1 pAb (sc‐6149; Santa Cruz); rabbit anti‐ODF2 pAb (HPA001874; Sigma‐Aldrich); and mouse anti‐DYKDDDDK (FLAG) tag mAb (018‐22381; Fujifilm Wako).

Techniques: Incubation, Staining, Expressing, Confocal Microscopy, Transfection

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: The primary antibodies used were as follows: mouse anti‐acetylated‐tubulin monoclonal antibody mAb (T7451; Sigma‐Aldrich); mouse anti‐Smo mAb (sc‐166685; Santa Cruz); rabbit anti‐Arl13b polyclonal antibody pAb (17711‐1‐AP; Proteintech); rabbit anti‐PEX1 pAb (13669‐1‐AP; Proteintech); rabbit anti‐PEX14 pAb (10594‐1‐AP; Proteintech); rabbit anti‐pericentrin pAb (A301‐348A; Bethyl Laboratories); mouse anti‐PMP70 mAb (SAB4200181; Sigma‐Aldrich); mouse anti‐ninein mAb (MABT29; Merck Millipore); mouse anti‐GAPDH mAb (sc‐32233; Santa Cruz Biotechnology); rabbit anti‐GFP pAb (598; MBL); mouse anti‐phospho‐S473 Akt mAb (#4051; Cell Signaling Technology); mouse anti‐GFP mAb (11‐814‐460‐001; Roche); rabbit anti‐Rabin8 pAb (12321‐1‐AP; Proteintech); rabbit anti‐Rab10 pAb (27094‐1‐AP; Proteintech); rabbit anti‐KIFC3 pAb (10125‐2‐AP; Proteintech); rat anti‐α‐tubulin mAb (NB600‐506; Novus); mouse anti‐γ‐tubulin mAb (T6557; Sigma‐Aldrich); mouse anti‐CYPOR mAb (sc‐25270; Santa Cruz); rabbit anti‐NPC1 pAb (13926‐1‐AP; Proteintech); rabbit anti‐ABCD1 pAb (18138‐1‐AP; Proteintech); rabbit anti‐EHD1 pAb (24657‐1‐AP; Proteintech); rabbit anti‐EHD3 pAb (25320‐1‐AP; Proteintech); rabbit anti‐Shh pAb (ab53281; Abcam); mouse anti‐ORP3 mAb (sc‐398326; Santa Cruz); rabbit anti‐ACOX1 pAb (10957‐1‐AP; Proteintech); goat anti‐Patched 1 pAb (sc‐6149; Santa Cruz); rabbit anti‐ODF2 pAb (HPA001874; Sigma‐Aldrich); and mouse anti‐DYKDDDDK (FLAG) tag mAb (018‐22381; Fujifilm Wako).

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