Figure 3Time series of confocal images showing redistribution of EqtSM - mCherry ( sphingomyelin ( SM ) probe ) and EGFP - TECPR1 in ATG16L1 / TECPR1 - DKO cells with doxycycline - inducible ( dox , 2μg / ml ) expression of EqtSM - mCherry cells following treatment with 400µM LLOMe . Scale bar : 10 µm . LC3B / GABARAP lipidation in ATG16L1 - and TECPR1 - KO HEK293A cells with or without dox - inducible ( 2μg / ml ) expression of nSMase2 - mCherry , treated for 2h as indicated . Cell lysates were immunoblotted against the indicated proteins . γ - Tubulin is loading control . Ratio of LC3B - II to γ - Tubulin quantified from immunoblots in ( B ) and normalized to untreated ATG16L1 - KO cells . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) . Confocal images of ATG16L1 / TECPR1 - DKO HeLa cells expressing EGFP - TECPR1 , with or without dox - inducible ( 2μg / ml ) nSMase2 overexpression , treated for 2h as indicated . Scale bars : 10 µm . Beeswarm - Superplot displaying mean number of EGFP - TECPR1 dots per cell per image of data represented in ( E ) quantified by automated analysis using CellProfiler . Mean ± SEM from each independent experiment ( 3 / group ) presented as large data points , with the black line corresponding to the mean of means . Individual data points ( small ) corresponding to single images are color - coded and superimposed according to the biological replicate they originate from . Total cells per condition / images per condition : 952 / 30 ( LLOMe treated - nSMase2 ) and 1056 / 30 ( LLOMe treated + nSMase2 ) were analyzed . ( P - values determined using Student ' s unpaired t test ) . Liposome co - sedimentation assay of the indicated recombinant proteins and liposomes with or without SM . S : supernatant ; P : pellet . Liposome binding in ( C ) was quantified as percentage of total protein in the pellet . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) .

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Figure 3Time series of confocal images showing redistribution of EqtSM-mCherry (sphingomyelin (SM) probe) and EGFP-TECPR1 in ATG16L1/TECPR1-DKO cells with doxycycline-inducible (dox, 2μg/ml) expression of EqtSM-mCherry cells following treatment with 400µM LLOMe. Scale bar: 10 µm.LC3B/GABARAP lipidation in ATG16L1- and TECPR1-KO HEK293A cells with or without dox-inducible (2μg/ml) expression of nSMase2-mCherry, treated for 2h as indicated. Cell lysates were immunoblotted against the indicated proteins. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (B) and normalized to untreated ATG16L1-KO cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).Confocal images of ATG16L1/TECPR1-DKO HeLa cells expressing EGFP-TECPR1, with or without dox-inducible (2μg/ml) nSMase2 overexpression, treated for 2h as indicated. Scale bars: 10 µm. Beeswarm-Superplot displaying mean number of EGFP-TECPR1 dots per cell per image of data represented in (E) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single images are color-coded and superimposed according to the biological replicate they originate from. Total cells per condition/images per condition: 952/30 (LLOMe treated - nSMase2) and 1056/30 (LLOMe treated + nSMase2) were analyzed. (P-values determined using Student's unpaired t test).Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S: supernatant; P: pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).


Figure 4Immunoblot analysis of LC3B / GABARAP lipidation in ATG16L1 - KO or ATG16L1 / TECPR1 - DKO HEK293A cells with or without rescue with TECPR1 ( WT ) , TECPR1 W77A , TECPR1 W154A , TECPR1 W829A or TECPR1 F908A , and treated with 400 µM LLOMe for 2h . γ - Tubulin is loading control . Ratio of LC3B - II to γ - Tubulin quantified from immunoblots in ( C ) and normalized to ATG16L1 - KO cells . Data information : All data presented as mean + SEM ( in ( D ) n = 3 independent experiments . P - values determined using one - way ANOVA followed by Tukey ' s multiple comparisons test in D and H ; ns = not significant . LC3B / GABARAP lipidation in ATG16L1 / TECPR1 - DKO HEK293A cells rescued with TECPR1 ( WT ) , TECPR1 W77A , TECPR1 W829A or TECPR1 W77A / W829A ( WAWA ) , and treated with 400 µM LLOMe for 2h . γ - Tubulin is loading control . Maximum intensity projection of confocal images of HeLaK cells expressing EGFP - TECPR1 ( WT ) or W77A / W829A ( WAWA ) mutant , with or without 400µM LLOMe for 30min . Nuclei were counterstained with Hoechst 33342 ( in blue ) . Scale bar : 10μm Beeswarm - Superplot displaying mean number of EGFP - TECPR1 dots per cell per image of data represented in ( G ) quantified by automated analysis using CellProfiler . Mean ± SEM from each independent experiment ( 3 / group ) presented as large data points , with the black line corresponding to the mean of means . Individual data points ( small ) corresponding to single images are color - coded and superimposed according to the biological replicate they came from . Total cells per condition / images per condition : 2502 / 59 ( WT untreated ) , 2549 / 48 ( WT LLOMe ) , 2256 / 59 ( WAWA untreated ) and 2816 / 59 ( WAWA LLOMe ) were analyzed .

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Figure 4Immunoblot analysis of LC3B/GABARAP lipidation in ATG16L1-KO or ATG16L1/TECPR1-DKO HEK293A cells with or without rescue with TECPR1 (WT), TECPR1 W77A, TECPR1 W154A, TECPR1 W829A or TECPR1 F908A, and treated with 400 µM LLOMe for 2h. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (C) and normalized to ATG16L1-KO cells. Data information: All data presented as mean + SEM (in (D) n = 3 independent experiments. P-values determined using one-way ANOVA followed by Tukey's multiple comparisons test in D and H; ns= not significant.LC3B/GABARAP lipidation in ATG16L1/TECPR1-DKO HEK293A cells rescued with TECPR1 (WT), TECPR1 W77A, TECPR1 W829A or TECPR1 W77A/W829A (WAWA), and treated with 400 µM LLOMe for 2h. γ-Tubulin is loading control.Maximum intensity projection of confocal images of HeLaK cells expressing EGFP-TECPR1 (WT) or W77A/W829A (WAWA) mutant, with or without 400µM LLOMe for 30min. Nuclei were counterstained with Hoechst 33342 (in blue). Scale bar: 10μm Beeswarm-Superplot displaying mean number of EGFP-TECPR1 dots per cell per image of data represented in (G) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single images are color-coded and superimposed according to the biological replicate they came from. Total cells per condition/images per condition: 2502/59 (WT untreated), 2549/48 (WT LLOMe), 2256/59 (WAWA untreated) and 2816/59 (WAWA LLOMe) were analyzed.


Figure 5Coomassie - stained gel depicting in vitro LC3B lipidation reactions using the indicated combinations of proteins and liposomes ( with or without SM ) , after being subjected to a liposome co - sedimentation assay . LC3B - AMP is denoted with an asterix ( * ) . The extent of LC3B - I to - II conversion in ( A ) was determined and plotted as percentage of total LC3B . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) . Liposome co - sedimentation assay of the indicated recombinant proteins and liposomes with or without SM . S , supernatant ; P , pellet . Liposome binding in ( C ) was quantified as percentage of total protein in the pellet . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) . Liposome co - sedimentation assay of the indicated recombinant proteins and liposomes with or without SM . S , supernatant ; P , pellet . Liposome binding in ( C ) was quantified as percentage of total protein in the pellet . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) .

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Figure 5Coomassie-stained gel depicting in vitro LC3B lipidation reactions using the indicated combinations of proteins and liposomes (with or without SM), after being subjected to a liposome co-sedimentation assay. LC3B-AMP is denoted with an asterix (*). The extent of LC3B-I to -II conversion in (A) was determined and plotted as percentage of total LC3B. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S, supernatant; P, pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S, supernatant; P, pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).


Figure 6LC3B / GABARAP lipidation in WT , ATG16L1 / TECPR1 - DKO , ATG16L1 - KO and TECPR1 - KO HeLa cells with or without dox - inducible ( 2μg / ml ) expression of mCherry - SopF , treated as indicated for 2h , were immunoblotted for the indicated proteins . γ - Tubulin is loading control . MRT68921 and SAR405 are ULK and VPS34 inhibitors , respectively . Ratio of LC3B - II to γ - Tubulin or Vinculin quantified from immunoblots in ( A ) and normalized to WT monensin treated cells . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) . ( Data has skewed distribution . P - values were determined with Kruskal - Wallis followed by Dunn ' s multiple comparisons test ) . LC3B / GABARAP lipidation in WT , ATG16L1 / TECPR1 - DKO , ATG16L1 - KO and TECPR1 - KO HeLa cells with or without dox - inducible ( 2μg / ml ) expression of mCherry - SopF , treated as indicated for 2h , Cell lysates were immunoblotted for the indicated proteins . γ - Tubulin is loading control . MRT68921 and SAR405 are ULK and VPS34 inhibitors , respectively . Ratio of LC3B - II to γ - Tubulin or Vinculin quantified from immunoblots in ( A ) and normalized to WT monensin treated cells . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) . ( Data has skewed distribution . P - values were determined with Kruskal - Wallis followed by Dunn ' s multiple comparisons test ) . Confocal images of WT and TECPR1 - KO cells with or without dox - inducible ( 2μg / ml ) expression of mCherry - SopF , treated as indicated for 2h . Nuclei were counterstained with Hoechst 33342 ( in blue ) . Scale bar : 10μm Beeswarm - Superplot displaying number of LC3 puncta per cell in the images represented in ( E ) quantified by automated analysis using CellProfiler . Mean ± SEM from each independent experiment ( 3 / group ) presented as large data points , with the black line corresponding to the mean of means . Individual data points ( small ) corresponding to single cells are color - coded and superimposed according to the biological replicate they originated from . Total cells per condition / images per condition : 1169 / 30 ( WT LLOMe ) , 1064 / 29 ( WT - mcherrySopF LLOMe ) , 692 / 23 ( TECKO LLOMe ) and 983 / 30 ( TECKO - mcherrySopF untreated ) were analyzed . ( Data has skewed distribution . P - values were determined with Kruskal - Wallis followed by Dunn ' s multiple comparisons test ) .

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Figure 6LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO, ATG16L1-KO and TECPR1-KO HeLa cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h, were immunoblotted for the indicated proteins. γ-Tubulin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Ratio of LC3B-II to γ-Tubulin or Vinculin quantified from immunoblots in (A) and normalized to WT monensin treated cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant). (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO, ATG16L1-KO and TECPR1-KO HeLa cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h, Cell lysates were immunoblotted for the indicated proteins. γ-Tubulin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Ratio of LC3B-II to γ-Tubulin or Vinculin quantified from immunoblots in (A) and normalized to WT monensin treated cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant). (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).Confocal images of WT and TECPR1-KO cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h. Nuclei were counterstained with Hoechst 33342 (in blue). Scale bar: 10μm Beeswarm-Superplot displaying number of LC3 puncta per cell in the images represented in (E) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single cells are color-coded and superimposed according to the biological replicate they originated from. Total cells per condition/images per condition: 1169/30 (WT LLOMe), 1064/29 (WT-mcherrySopF LLOMe), 692/23 (TECKO LLOMe) and 983/30 (TECKO-mcherrySopF untreated) were analyzed. (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).


Figure 1A Analysis of published single - cell RNA sequencing data from lungs of non - infected mice and mice infected with SARS - CoV - 2 MA10 ( GEO accession No . GSE186360 ) . Av Mφ = alveolar macrophages , DC = dendritic cells , infl mo = inflammatory monocytes , non clas mo = non - classical monocytes , trans mo = transitional / intermediate monocytes . D Viral titers in lung homogenates at the indicated days post infection from mice infected as in B ( bars / error bars represent mean ± SD , data points represent distinct biological samples , data points at the x axis represent samples below the limit of detection , day 3 data from two independent experiments , day 2 and 5 data are from single experiments , * * p < 0 . 01 , Mann - Whitney test ) . E IL - 6 levels from lung homogenates and TNFα and IL - 1β levels from serum on day 3 post infection ( bars / error bars represent mean ± SD , data points represent distinct biological samples , data from two independent experiments , * * * p < 0 . 001 , t - test ) . F Viral titers in extrapulmonary organs on day 3 post infection were measured ( bars / error bars represent mean ± SD , data points represent distinct biological samples [ n = 7 for heart , spleen , brain ; n = 5 for kidney and liver ] , data points at the x axis represent samples below the limit of detection , ns , multiple unpaired t - tests ) . G Representative lung sections from WT or IFITM3 KO animals stained for SARS - CoV - 2 N protein . Scale bars represent 2 mm for whole lung images . Colored boxes overlaying the whole lung images correspond to the displayed magnified regions and scale bars in these images represent 0 . 2 mm . Red triangles , infected airways ; solid black arrow , non - infected blood vessels ; open black arrow , infected blood vessels .

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Figure 1A Analysis of published single-cell RNA sequencing data from lungs of non-infected mice and mice infected with SARS-CoV-2 MA10 (GEO accession No. GSE186360). Av Mφ = alveolar macrophages, DC = dendritic cells, infl mo = inflammatory monocytes, non clas mo = non-classical monocytes, trans mo = transitional/intermediate monocytes.D Viral titers in lung homogenates at the indicated days post infection from mice infected as in B (bars/error bars represent mean ± SD, data points represent distinct biological samples, data points at the x axis represent samples below the limit of detection, day 3 data from two independent experiments, day 2 and 5 data are from single experiments, **p < 0.01, Mann-Whitney test).E IL-6 levels from lung homogenates and TNFα and IL-1β levels from serum on day 3 post infection (bars/error bars represent mean ± SD, data points represent distinct biological samples, data from two independent experiments, ***p < 0.001, t-test).F Viral titers in extrapulmonary organs on day 3 post infection were measured (bars/error bars represent mean ± SD, data points represent distinct biological samples [n=7 for heart, spleen, brain; n=5 for kidney and liver], data points at the x axis represent samples below the limit of detection, ns, multiple unpaired t-tests).G Representative lung sections from WT or IFITM3 KO animals stained for SARS-CoV-2 N protein. Scale bars represent 2 mm for whole lung images. Colored boxes overlaying the whole lung images correspond to the displayed magnified regions and scale bars in these images represent 0.2 mm. Red triangles, infected airways; solid black arrow, non-infected blood vessels; open black arrow, infected blood vessels.


Figure 2K18 - hACE2 ( hACE2 ) and K18 - hACE2 / IFITM3 KO mice were infected with 104 TCID SARS - CoV - 2 WA1 . A Weight loss ( data points represent mean and error bars represent SEM , data from two independent experiments [ hACE2 n = 7 , hACE2 / IFITM3 KO n = 8 ] , skull / crossbones indicates that KO mice did not survive beyond this timepoint , * p < 0 . 05 , * * p < 0 . 01 , ANOVA with Bonferoni ' s multiple comparison ' s test ) . K18 - hACE2 ( hACE2 ) and K18 - hACE2 / IFITM3 KO mice were infected with 104 TCID SARS - CoV - 2 WA1 . C Viral titers in extrapulmonary organs on the indicated days post infection were measured ( bars / error bars represent mean ± SD , data points represent distinct biological samples , day 3 data for heart , spleen , and brain are from two independent experiments , day 3 data for liver and kidney and day 5 data are from single experiments , data points at the x axis represent samples below the limit of detection , * * p < 0 . 01 , Mann - Whitney test ) . K18 - hACE2 ( hACE2 ) and K18 - hACE2 / IFITM3 KO mice were infected with 104 TCID SARS - CoV - 2 WA1 . D Representative lung sections from WT mice infected with SARS - CoV - 2 MA10 ( 105 TCID ) or K18 - hACE2 or K18 - hACE2 / IFITM3 KO mice infected with SARS - CoV - 2 WA1 ( 104 TCID ) stained for SARS - CoV - 2 N protein at day 5 post infection . Scale bars represent 2mm .

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Figure 2K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. A Weight loss (data points represent mean and error bars represent SEM, data from two independent experiments [hACE2 n=7, hACE2/IFITM3 KO n=8], skull/crossbones indicates that KO mice did not survive beyond this timepoint, *p < 0.05, ** p < 0.01, ANOVA with Bonferoni's multiple comparison's test).K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. C Viral titers in extrapulmonary organs on the indicated days post infection were measured (bars/error bars represent mean ± SD, data points represent distinct biological samples, day 3 data for heart, spleen, and brain are from two independent experiments, day 3 data for liver and kidney and day 5 data are from single experiments, data points at the x axis represent samples below the limit of detection, **p < 0.01, Mann-Whitney test).K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. D Representative lung sections from WT mice infected with SARS-CoV-2 MA10 (105 TCID) or K18-hACE2 or K18-hACE2/IFITM3 KO mice infected with SARS-CoV-2 WA1 (104 TCID) stained for SARS-CoV-2 N protein at day 5 post infection. Scale bars represent 2mm.


Figure 3WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS - CoV - 2 MA10 or mock infected with PBS ( Non - Infected , NI ) . A Representative lung images from mice at the indicated times post infection were stained with hematoxylin and eosin . Scale bars represent 2mm for whole lung images and 500um for zoomed images . B Staining quantifications from multiple mice are shown ( bars / error bars represent mean ± SD , data points represent distinct biological samples , * p < 0 . 05 , ANOVA with Bonferoni ' s multiple comparison ' s test ) .

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Figure 3WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10 or mock infected with PBS (Non-Infected, NI). A Representative lung images from mice at the indicated times post infection were stained with hematoxylin and eosin. Scale bars represent 2mm for whole lung images and 500um for zoomed images. B Staining quantifications from multiple mice are shown (bars/error bars represent mean ± SD, data points represent distinct biological samples, *p < 0.05, ANOVA with Bonferoni's multiple comparison's test).


Figure 4WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS - CoV - 2 MA10 or mock infected with PBS ( Non - Infected , NI ) . A Representative lung images from mice at the indicated times post infection were stained with anti - CD45 . Scale bars represent 2mm for whole lung images and 500um for zoomed images . B Staining quantifications from multiple mice are shown ( bars / error bars represent mean ± SD , data points represent distinct biological samples , * p < 0 . 05 , ANOVA with Bonferoni ' s multiple comparison ' s test ) .

Figure 4WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10 or mock infected with PBS (Non-Infected, NI). A Representative lung images from mice at the indicated times post infection were stained with anti-CD45. Scale bars represent 2mm for whole lung images and 500um for zoomed images. B Staining quantifications from multiple mice are shown (bars/error bars represent mean ± SD, data points represent distinct biological samples, *p < 0.05, ANOVA with Bonferoni's multiple comparison's test).


Figure 5WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS - CoV - 2 MA10 . RNA sequencing analysis was performed on RNA extracted from infected WT and IFITM3 KO lungs on day 2 post infection . D Relative gene expression heat maps for genes from the top two most significant GO Biological Processes as shown in C .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10. RNA sequencing analysis was performed on RNA extracted from infected WT and IFITM3 KO lungs on day 2 post infection. D Relative gene expression heat maps for genes from the top two most significant GO Biological Processes as shown in C.


Figure 1Primary skin fibroblasts from a normal individual , SLOS patient , ZS patients , X - ALD patient , and NPC patient 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 . The intensity of Filipin III signal at primary cilia from ( a ) was remarkably reduced in SLOS and ZS patient cells ( * * p < 0 . 01 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 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 , representatively . Immunofluorescent staining of primary skin fibroblasts in quiescent G0 phase treated with 50 nM Shh - N for 24 h using anti - Smo ( green ) , anti - acetylated - tubulin ( blue ) , and anti - γ - tubulin ( red ) antibodies . Arrows indicate primary cilia . Scale bar , 2 . 5 μm . Quantification of proportion of Smo - positive ciliated cells upon Shh - N stimulation from ( C ) . The populations of Smo - positive ciliated fibroblasts from the SLOS and ZS patients were significantly reduced ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 100 cells per experiment ) . The Smo intensity at primary cilia in the fibroblasts from ( C ) . The ciliary localization of Smo upon Shh - N stimulation was significantly impaired in fibroblasts from the SLOS and ZS patients ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 90 - 100 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 , representatively . Primary fibroblasts from a normal individual , and SLOS and ZS patients were treated with 1 . 5 % methyl - β - cyclodextrin for 45 min to remove cellular cholesterol , and then incubated with or without cholesterol ( cholesterol / methyl - β - cyclodextrin complex ) for 1 h . After washing out the exogenous cholesterol , the fibroblasts 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 - pericentrin ( 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 . Quantification of the Smo intensity at primary cilia in the fibroblasts from ( F ) . The ciliary accumulation of Smo upon the Shh - N stimulation of fibroblasts from an SLO patient was restored by the complementation of both exogenous cholesterol / methyl - β - cyclodextrin complex and LDL , while that of cells from ZS patients was efficiently rescued by not LDL but cholesterol / methyl - β - cyclodextrin complex ( * 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Primary skin fibroblasts from a normal individual, SLOS patient, ZS patients, X-ALD patient, and NPC patient 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. The intensity of Filipin III signal at primary cilia from (a) was remarkably reduced in SLOS and ZS patient cells (**p<0.01: one-way ANOVA with Tukey's HSD, n=3: 45-50 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, representatively.Immunofluorescent staining of primary skin fibroblasts in quiescent G0 phase treated with 50 nM Shh-N for 24 h using anti-Smo (green), anti-acetylated-tubulin (blue), and anti-γ-tubulin (red) antibodies. Arrows indicate primary cilia. Scale bar, 2.5 μm. Quantification of proportion of Smo-positive ciliated cells upon Shh-N stimulation from (C). The populations of Smo-positive ciliated fibroblasts from the SLOS and ZS patients were significantly reduced (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 100 cells per experiment). The Smo intensity at primary cilia in the fibroblasts from (C). The ciliary localization of Smo upon Shh-N stimulation was significantly impaired in fibroblasts from the SLOS and ZS patients (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 90-100 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, representatively.Primary fibroblasts from a normal individual, and SLOS and ZS patients were treated with 1.5% methyl-β-cyclodextrin for 45 min to remove cellular cholesterol, and then incubated with or without cholesterol (cholesterol/methyl-β-cyclodextrin complex) for 1 h. After washing out the exogenous cholesterol, the fibroblasts 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-pericentrin (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. Quantification of the Smo intensity at primary cilia in the fibroblasts from (F). The ciliary accumulation of Smo upon the Shh-N stimulation of fibroblasts from an SLO patient was restored by the complementation of both exogenous cholesterol/methyl-β-cyclodextrin complex and LDL, while that of cells from ZS patients was efficiently rescued by not LDL but cholesterol/methyl-β-cyclodextrin complex (*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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Figure 2Western 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 . 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 G0 phase . Arrowheads indicate primary cilia . Scale bar , 10 μm . 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 ) . 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 ) . Quiescent G0 - 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively . Quiescent G0 - 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Western 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.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 G0 phase. Arrowheads indicate primary cilia. Scale bar, 10 μm. 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). 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).Quiescent G0-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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.Quiescent G0-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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Figure 3Quiescent G0 - 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 . Quiescent G0 - 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 . 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 ) . Quiescent G0 - 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively . 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 . 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 . 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 . 3D reconstitution of the quiescent G0 - 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Quiescent G0-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.Quiescent G0-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. 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).Quiescent G0-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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.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.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.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.3D reconstitution of the quiescent G0-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.


Figure 4Maximum projection of z - stack sections of hTERT - RPE1 cells expressing MCHR1 - GFP ( blue ) , DsRed2 - PACT ( red ) and ECFP - SKL ( green ) 48 h after serum starvation . The ECFP - SKL - positive cell with primary cilia , indicated by the red box , was analyzed by FIB - SEM . Top , differential interference contrast ( DIC ) ; bottom , merged image . Scale bar , 10 µm . The fluorescent image of primary cilia , enlarged the area boxed in red in panel ( A ) . Arrow indicates the ECFP - SKL positive - peroxisome associated with primary cilium . Scale bar , 5 µm . Single FIB - SEM image ( left ) showing a cross - section the ciliary pocket membrane , and 3D surface model reconstructed from segmented FIB - SEM images ( right ) . Region of interest was coated by a protection layer of platinum before FIB milling . Plasma membrane ( PM ) , yellow ( surface view 75 % transmission ) ; cilia , green ( surface view ) ; ciliary pocket membrane ( CPM ) , red ( surface view 70 % transmission ) ; mitochondria ( Mito ) , orange ( surface view ) ; peroxisome ( PO ) , purple ( surface view ) ; basal body ( BB ) , white ( surface view ) . Scale bar , 1 µm . Enlarged 3D view of peroxisome interacting with ciliary pocket and mitochondria . Left , x - y view ; right , oblique view . Scale bar , 1 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Maximum projection of z-stack sections of hTERT-RPE1 cells expressing MCHR1-GFP (blue), DsRed2-PACT (red) and ECFP-SKL (green) 48 h after serum starvation. The ECFP-SKL-positive cell with primary cilia, indicated by the red box, was analyzed by FIB-SEM. Top, differential interference contrast (DIC); bottom, merged image. Scale bar, 10 µm.The fluorescent image of primary cilia, enlarged the area boxed in red in panel (A). Arrow indicates the ECFP-SKL positive-peroxisome associated with primary cilium. Scale bar, 5 µm.Single FIB-SEM image (left) showing a cross-section the ciliary pocket membrane, and 3D surface model reconstructed from segmented FIB-SEM images (right). Region of interest was coated by a protection layer of platinum before FIB milling. Plasma membrane (PM), yellow (surface view 75% transmission); cilia, green (surface view); ciliary pocket membrane (CPM), red (surface view 70% transmission); mitochondria (Mito), orange (surface view); peroxisome (PO), purple (surface view); basal body (BB), white (surface view). Scale bar, 1 µm. Enlarged 3D view of peroxisome interacting with ciliary pocket and mitochondria. Left, x-y view; right, oblique view. Scale bar, 1 µm.


Figure 5Western blot analysis showing depletion of ORP3 in the ORP3 − / − hTERT - RPE1 cell clones . GAPDH served as a loading control . 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively . Three - dimensional reconstitution of the quiescent G0 - 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 . 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 ) . 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 . 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western blot analysis showing depletion of ORP3 in the ORP3−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.Three-dimensional reconstitution of the quiescent G0-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. 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).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.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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Figure 6Rabin8 + / + 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively . Quiescent G0 - 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 . 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 ) . Quiescent G0 - 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 ( Movie EV10 ) . Scale bar , 5 μm . Quiescent G0 - 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively . 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Rabin8+/+ 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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.Quiescent G0-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. 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).Quiescent G0-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 (Movie EV10). Scale bar, 5 μm.Quiescent G0-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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Figure 7Whole - 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 . 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 . 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 . 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 . Quiescent G0 - 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 . Western blot analysis showing depletion of KIFC3 in the KIFC3 − / − hTERT - RPE1 cell clones . GAPDH served as a loading control . 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively . Quiescent 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 . 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 ) . 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 . 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 , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Whole-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.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.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.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.Quiescent G0-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.Western blot analysis showing depletion of KIFC3 in the KIFC3−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.Quiescent 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. 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).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. 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, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Figure 8Quiescent G0 - phase wild - type , Rab10 − / − , KIFC3 − / − , ORP3 − / − , and EHD3 − / − 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 . Quantification of the Filipin III intensity at primary cilia from ( A ) . Rapamycin - induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10 − / − and KIFC3 − / − hTERT - RPE1 cells , but not in ORP3 − / − and EHD3 − / − hTERT - RPE1 cells ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 35 - 40 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 , representatively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Quiescent G0-phase wild-type, Rab10−/−, KIFC3−/−, ORP3−/−, and EHD3−/− 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.Quantification of the Filipin III intensity at primary cilia from (A). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10−/−and KIFC3−/−hTERT-RPE1 cells, but not in ORP3−/−and EHD3−/− hTERT-RPE1 cells (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 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, representatively.


Figure 1 ( B ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of the sdAbs without heparin . ( C ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of the sdAbs and heparin . ( D ) Bar graph representing residual activated factor X ( FXa ) activity ( ΔOD / min ) in presence of the sdAbs and heparin .

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Figure 1(B) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs without heparin.(C) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs and heparin.(D) Bar graph representing residual activated factor X (FXa) activity (ΔOD/min) in presence of the sdAbs and heparin.


Figure 2 ( B - C ) Graphs reporting the recovery of thrombin - and FXa - mediated substrate conversion in the presence of increasing doses of KB - AT - 23 , in presence of antithrombin and heparin . ( B - C ) Graphs reporting the recovery of thrombin - and FXa - mediated substrate conversion in the presence of increasing doses of KB - AT - 23 , in presence of antithrombin and heparin . ( D - E ) Thrombin generation profiles of FVIII - deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB - AT - 23 . ( D - E ) Thrombin generation profiles of FVIII - deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB - AT - 23 .

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Figure 2(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin.(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin.(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23.(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23.


Figure 3 ( B ) Purified KB - AT - 23 - fus and KB - hFX - 11 - fus were given intravenously to wild - type C57B6 mice ( 10 mg / kg ) . At indicated time - points , blood was collected and residual protein was measured . Plotted is residual protein versus time after injection . Green circles : KB - AT - 23 - fus ; Red circles : KB - hFX - 11 - fus . ( D ) Graph reporting the volume of blood loss ( µL ) in treated mice ( n = 4 - 9 per group ) observed during 30 minutes post TVT . Dosing was 10 mg / kg for KB - AT - 23 and 1 mg / kg for FVIIa . The grey area represents the range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) .

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Figure 3(B) Purified KB-AT-23-fus and KB-hFX-11-fus were given intravenously to wild-type C57B6 mice (10 mg/kg). At indicated time-points, blood was collected and residual protein was measured. Plotted is residual protein versus time after injection. Green circles: KB-AT-23-fus; Red circles: KB-hFX-11-fus.(D) Graph reporting the volume of blood loss (µL) in treated mice (n=4-9 per group) observed during 30 minutes post TVT. Dosing was 10 mg/kg for KB-AT-23 and 1 mg/kg for FVIIa. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016).


Figure 4 ( B ) Western blot analysis of KB - AT - 23 protein secretion in conditioned media 72 hours post - transfection of HuH7 cells . In the lower panel the western blot quantification via densitometry analysis is depicted . Data are presented as mean ± SD , and were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons . * * * * p < 0 . 001 . ( C ) HuH7 - celles were collected 72 hours post - transfection . Presented is the Western blot analysis of lysed cells for the presence of KB - AT - 23 . Cntr - refers to non - transfected cells , while Cntr + refers to purified recombinant KB - AT - 23 .

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Figure 4(B) Western blot analysis of KB-AT-23 protein secretion in conditioned media 72 hours post-transfection of HuH7 cells. In the lower panel the western blot quantification via densitometry analysis is depicted. Data are presented as mean ±SD, and were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons. ****p<0.001.(C) HuH7-celles were collected 72 hours post-transfection. Presented is the Western blot analysis of lysed cells for the presence of KB-AT-23. Cntr- refers to non-transfected cells, while Cntr+ refers to purified recombinant KB-AT-23.


Figure 5 ( B ) Representative western blot analyses of KB - AT - 23 and antithrombin expression on plasma samples ( n = 4 ) collected from animals 27 weeks post - AAV injection . kDa , molecular weight marker . ( C ) Measurement of D - dimers ( D2D ) plasma concentration 27 weeks post vector administration . Data are presented as mean ± SD ( n = 6 - 9 / group ) . ( D ) Measurement of anti - KB - AT - 23 mouse IgG in plasma samples collected from mice ( n = 6 - 9 / group ) 8 weeks post - AAV administration . Data represent mean ± SD . ( E ) TVT test on males and females ( n = 4 - 5 per group ) performed 29 weeks post vector administration . The volume of blood loss ( µL ) during 30 minutes observation time is reported . The grey area represents the range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) . Boxes represent 25 - 75 % quartiles ; the central line indicates the median . Whiskers span min to max . Data were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons . ( F ) Vector genome copy number ( VGCN ) per cell in liver samples collected at sacrifice ( n = 8 / group ) . Data represent mean ± SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Representative western blot analyses of KB-AT-23 and antithrombin expression on plasma samples (n=4) collected from animals 27 weeks post-AAV injection. kDa, molecular weight marker.(C) Measurement of D-dimers (D2D) plasma concentration 27 weeks post vector administration. Data are presented as mean ±SD (n=6-9/group).(D) Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=6-9/group) 8 weeks post-AAV administration. Data represent mean±SD.(E) TVT test on males and females (n=4-5 per group) performed 29 weeks post vector administration. The volume of blood loss (µL) during 30 minutes observation time is reported. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016). Boxes represent 25-75% quartiles; the central line indicates the median. Whiskers span min to max. Data were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons.(F) Vector genome copy number (VGCN) per cell in liver samples collected at sacrifice (n=8/group). Data represent mean±SD.


Figure 6 ( C ) Measurement of anti - hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS ( n = 5 / group ) at different time points . Data represent mean ± SEM . ( D ) Representative western blot analyses on plasma samples ( n = 4 ) collected from animals 8 weeks post - AAV injection . ( E ) Antithrombin activity in plasma samples collected from mice at different time points . Data are presented as mean ± SD ( n = 3 - 4 per datapoint ) . Data were analyzed in a 2 - way ANOVA with Tukey ' s correction for multiple comparisons . * * * p < 0 . 01 * * * * p < 0 . 001 ( 2 - way ANOVA with Tukey ' s multiple comparison test ) ( F ) Graph reporting the volume of blood loss ( µL ) over 30 minutes following the tail clip . WT : wild - type littermates of F9 - / - mice . The grey area represents the range of blood loss of F9 - / - mice that received AAV - vectors expressing human FIX . Data are presented as mean ± SD ( n = 3 - 4 / group ) , and were analyzed in a 1 - way ANOVA with Tukey ' s correction for multiple comparisons . * * p < 0 . 01 Boxes represent 25 % - 75 % quartiles , with the line indicating the median . Whiskers span min to max . G Measurement of anti - KB - AT - 23 mouse IgG in plasma samples collected from mice ( n = 5 / group ) 8 weeks post - AAV administration . Data represent mean ± SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(C) Measurement of anti-hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS (n=5/group) at different time points. Data represent mean±SEM.(D) Representative western blot analyses on plasma samples (n=4) collected from animals 8 weeks post-AAV injection.(E) Antithrombin activity in plasma samples collected from mice at different time points. Data are presented as mean±SD (n=3-4 per datapoint). Data were analyzed in a 2-way ANOVA with Tukey's correction for multiple comparisons. ***p<0.01 ****p<0.001 (2-way ANOVA with Tukey's multiple comparison test)(F) Graph reporting the volume of blood loss (µL) over 30 minutes following the tail clip. WT: wild-type littermates of F9-/- mice. The grey area represents the range of blood loss of F9-/- mice that received AAV-vectors expressing human FIX. Data are presented as mean±SD (n=3-4/group), and were analyzed in a 1-way ANOVA with Tukey's correction for multiple comparisons. **p<0.01 Boxes represent 25%-75% quartiles, with the line indicating the median. Whiskers span min to max.G Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=5/group) 8 weeks post-AAV administration. Data represent mean±SD.


Figure 7 ( C ) Representative western blot analyses on plasma samples ( n = 4 ) collected from animals 4 weeks post - AAV injection . Cntrl + : positive controls represented by the loading of the purified KB - AT - 23 or KB - AT - 113 proteins . ( D - H ) Measurement of anti - sdAbs mouse IgG in plasma samples collected at days 28 and 57 post - AAV injection . The ELISA plate was coated with the purified KB - AT - 01 ( D ) Measurement of anti - sdAbs mouse IgG in plasma samples collected at days 28 and 57 post - AAV injection . The ELISA plate was coated with the purified KB - AT - 01 , - 02 ( E ) , - 03 ( F ) , - 23 ( G ) or - 113 ( H ) sdAbs .

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Figure 7(C) Representative western blot analyses on plasma samples (n=4) collected from animals 4 weeks post-AAV injection. Cntrl +: positive controls represented by the loading of the purified KB-AT-23 or KB-AT-113 proteins.(D-H) Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 (D)Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 , -02 (E), -03 (F), -23 (G) or -113 (H) sdAbs.