Western blot showing depletion of PEX1 PEX14 normal processing of Shh in PEX1 − / − PEX14 − / − hTERT - RPE1 clones . GAPDH served as a loading control .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Western blot analysis showing the depletion of PEX1 and PEX14 and the normal processing of Shh protein in the PEX1−/− and PEX14−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.


Immunostaining with anti - PEX1 ( red ) or PEX14 ( red ) , anti - PMP70 ( green ) , anti - ninein ( blue ) , anti - acetylated tubulin ( ) in wild - type , PEX1 − / − , PEX14 − / − hTERT - RPE1 in quiescent G0 phase . Arrowheads indicate cilia . Scale bar , 10 μm . of number of PMP70 - positive peroxisomes per from ( B ) . Peroxisome formation in PEX1 − / − PEX14 − / − hTERT - RPE1 was significantly impaired compared with that of parental ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 20 - 25 per experiment ) . of proportion of ciliated from ( B ) . Ciliogenesis in PEX1 − / − PEX14 − / − hTERT - RPE1 was not significantly altered compared with that of parental ( mean ± s . d . : one - way ANOVA with Tukey ' s HSD , n = 3 : 190 - 200 per experiment ) .

O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 − / − , PEX14 − / − hTERT - RPE1 transfected with AcGFP1 - tagged D4 as a cholesterol probe were immunostained with anti - pericentrin ( ) anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( red ) . Arrows arrowheads indicate cilia accumulations of AcGFP1 - tagged D4 , respectively . Scale bar , 5 μm . of Filipin III at cilia from ( E ) . PEX1 − / − PEX14 − / − hTERT - RPE1 had significant reductions in ciliary signal of Filipin III ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively . of AcGFP1 - tagged D4 at cilia from ( E ) . ciliary signal of AcGFP1 - tagged D4 in PEX1 − / − PEX14 − / − hTERT - RPE1 was significantly diminished ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 25 - 30 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 − / − , PEX14 − / − hTERT - RPE1 were treated with or without 1 . 5 % methyl - β - cyclodextrin for 45 min , then incubated with or without cholesterol ( cholesterol / methyl - β - cyclodextrin ) for 1 h . After removing exogenous cholesterol , they were stimulated with 50 nM Shh - N for 24 h in presence of pravastatin , then immunostained with anti - Smo ( green ) , anti - acetylated - tubulin ( blue ) , anti - γ - tubulin ( red ) antibodies . For alternative cholesterol complementation , LDL ( 0 . 06 mg / ml ) was co - incubated with Shh - N pravastatin for 24 h after methyl - β - cyclodextrin - mediated cholesterol depletion . Scale bar , 2 . 5 μm . of Smo at cilia in wild - type , PEX1 − / − , PEX14 − / − hTERT - RPE1 from ( H ) . PEX1 − / − PEX14 − / − hTERT - RPE1 exhibited dampened Shh - N ligand - induced ciliary accumulation of Smo . complementation of exogenous cholesterol ( cholesterol / methyl - β - cyclodextrin ) restored ciliary accumulation of Smo in both PEX - knockout , while LDL complementation did not rescue ciliary efficiently ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 90 - 100 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Quiescent G0 - phase hTERT - RPE1 were immunostained with anti - PEX14 ( red ) , anti - ninein ( ) , anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( Green ) . Magnified of boxed regions showing peroxisome accompanied by cholesterol ( arrows ) . Three - dimensional reconstitution of same indicates that Filipin III stains regions of ciliary axonemes peroxisomes . scale bars indicate 2 . 5 μm 1 . 25 μm in lower - higher - magnified , respectively .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quiescent 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 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 ) , anti - PMP70 ( ) antibodies . Arrows indicate peroxisomes interacting with ciliary pocket . Scale bar , 5 μm . of proportion of cilia interacting with peroxisomes from ( B ) . Colcemid ( 50 nM ) Ciliobrevin - D ( 10 μM ) significantly inhibited spatial interaction between peroxisomes cilia ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 per experiment ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 were treated with Cytochalasin - D ( 200 nM ) , Colcemid ( 50 nM ) , or Ciliobrevin - D ( 10 μM ) for 6 h then immunostained with anti - acetylated tubulin ( blue ) anti - pericentrin ( green ) antibodies . Cholesterol was stained with Filipin III ( green ) . Arrows indicate cilia . Scale bar , 2 . 5 μm . of Filipin III at cilia from ( D ) . Colcemid Ciliobrevin - D interfered with distribution of cholesterol in ciliary ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 were transfected with AcGFP1 - tagged EHD3 cultured without serum for 24 h before immunostaining with anti - GFP ( green ) , anti - ARL13B ( blue ) , anti - PMP70 ( ) , anti - phospho - S473 - Akt ( red ) antibodies . scale bars represent 2 . 5 μm .

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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 AcGFP1 - tagged PEX14 were coexpressed in HEK293T then immunoprecipitated from whole - lysates using anti - FLAG antibody . AcGFP1 - tagged PEX14 3 × FLAG - tagged EHD1 or EHD3 fragments in IP fractions inputs were detected by western blotting .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O

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 ( 1 μg ) 6 × His - EHD3 ( 1 μg ) were pulled down using glutathione - Sepharose beads . GST - tagged PEX14 6 × His - tagged EHD3 in pull - down fractions inputs were detected by western blotting .

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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 quiescent G0 - phase hTERT - RPE1 transfected with AcGFP1 - tagged EHD3 ( ) immunostained with anti - acetylated - tubulin ( blue ) anti - PEX14 ( red ) antibodies . Cholesterol was stained with Filipin III ( green ) . Cholesterol - containing peroxisome interacted with ciliary pocket ( arrow ) . scale bars indicate 2 μm .

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-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

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.


Maximum projection of z - stack sections of hTERT - RPE1 expressing MCHR1 - GFP ( blue ) , DsRed2 - PACT ( red ) ECFP - SKL ( green ) 48 h after serum starvation . ECFP - SKL - positive with cilia , indicated by red box , was analyzed by FIB - . Top , differential interference contrast ( DIC ) ; bottom , merged image . Scale bar , 10 µm .

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Maximum 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.


image of cilia , enlarged boxed in red in panel ( A ) . Arrow indicates ECFP - SKL positive - peroxisome associated with cilium . Scale bar , 5 µm .

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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 - image ( left ) showing a cross - section ciliary pocket , 3D surface model reconstructed from segmented FIB - ( right ) . Region of interest was coated by a protection layer of platinum before FIB milling . Plasma ( PM ) , yellow ( surface view 75 % transmission ) ; cilia , green ( surface view ) ; ciliary pocket ( CPM ) , red ( surface view 70 % transmission ) ; mitochondria ( Mito ) , orange ( surface view ) ; peroxisome ( PO ) , purple ( surface view ) ; basal ( BB ) , ( surface view ) . Scale bar , 1 µm . Enlarged 3D view of peroxisome interacting with ciliary pocket mitochondria . Left , x - y view ; right , oblique view . Scale bar , 1 µm .

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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.


Western blot showing depletion of ORP3 in ORP3 − / − hTERT - RPE1 clones . GAPDH served as a loading control .

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Western blot analysis showing depletion of ORP3 in the ORP3−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.


ORP3 + / + ORP3 − / − hTERT - RPE1 incubated for 24 h without serum were immunostained with anti - pericentrin ( red ) anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( green ) . Arrows indicate cilia . Scale bar , 5 μm . of Filipin III at cilia from ( C ) . ORP3 − / − hTERT - RPE1 exhibited a significant reduction of ciliary cholesterol ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 quiescent G0 - phase ORP3 + / + ORP3 − / − hTERT - RPE1 immunostained with anti - ARL13B ( blue ) , anti - phospho - S473 - Akt ( red ) , anti - ORP3 ( green ) , anti - PMP70 ( ) antibodies indicates that ORP3 at ciliary pocket ( arrow arrowhead ) mediates regions of ciliary pocket peroxisomes ( arrowhead ) . scale bars indicate 5 μm . of proportion of cilia interacting with peroxisomes from ( E ) . Depletion of ORP3 significantly interfered with spatial interaction between peroxisomes cilia ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 per experiment ) .

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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 AcGFP1 - tagged PEX14 , EHD1 or EHD3 were coexpressed in HEK293T then immunoprecipitated from whole - lysates using anti - FLAG antibody . 3 × FLAG - tagged ORP3 AcGFP1 - tagged PEX14 , EHD1 or EHD3 fragments in IP fractions inputs were detected by western blotting .

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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 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 ) cultured without serum for 24 h before Filipin - III ( green ) - mediated cholesterol immunostaining with anti - GFP ( red ) , anti - pericentrin ( ) , anti - acetylated - tubulin ( blue ) antibodies . Arrows represent AcGFP1 - tagged ORP3 or mutants localized to ciliary pocket . AcGFP1 - tagged ORP3ΔPH mutant mis - localized to ciliary pocket . scale bars indicate 5 μm . of Filipin III at cilia from ( H ) . AcGFP1 - tagged ORP3 deletion mutants did not restore ciliary cholesterol insufficiency in ORP3 − / − hTERT - RPE1 ( * p < 0 . 05 , * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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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.


Rabin8 + / + Rabin8 − / − hTERT - RPE1 incubated for 24 h without serum were immunostained with anti - pericentrin ( red ) anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( green ) . Scale bar , 5 μm . of Filipin III at cilia from ( A ) . Rabin8 − / − hTERT - RPE1 exhibited a significant reduction of ciliary cholesterol ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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Rabin8+/+ and Rabin8−/− hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Scale bar, 5 μm. 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 + / + Rabin8 − / − hTERT - RPE1 were immunostained with anti - ARL13B ( blue ) , anti - phospho - S473 - Akt ( red ) , anti - ninein ( green ) , anti - PMP70 ( ) antibodies . Arrow indicates peroxisomes contacting ciliary pocket . Scale bar , 5 μm . of ( C ) indicating that disruption of Rabin8 gene significantly inhibited spatial interaction between peroxisomes cilia ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 per experiment ) .

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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 transiently expressing PEX3 - GFP - FRB ( green ) tdTomato - BicD2 - FKBP ( red ) treated with rapamycin were imaged live for 10 min by confocal microscopy ( Movie EV10 ) . Scale bar , 5 μm .

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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 + / + Rabin8 − / − hTERT - RPE1 transfected with PEX3 - GFP - FRB ( red ) tdTomato - BicD2 - FKBP ( ) were treated with or without 100 μM rapamycin for 30 min , then immunostained with anti - acetylated - tubulin ( blue ) antibody . Cholesterol was stained with Filipin III ( Green ) . Arrows indicate enrichment of ciliary cholesterol . scale bars represent 5 μm . of Filipin III at cilia from ( G ) . Rapamycin - induced peroxisome targeting to ciliary pocket restored reduction of ciliary cholesterol in Rabin8 − / − hTERT - RPE1 ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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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 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 ) , then immunostained with anti - GFP ( red ) , anti - acetylated - tubulin ( blue ) , anti - ninein ( ) antibodies . Cholesterol was stained with Filipin III ( Green ) . Arrows indicate enrichment of ciliary cholesterol . scale bars represent 5 μm . of ( I ) showing that AcGFP1 - tagged Rabin8 AcGFP1 - tagged Rab10 - Q68L effectively restored defect in ciliary enrichment of cholesterol in Rabin8 − / − hTERT - RPE1 ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 35 - 40 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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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.


Whole - lysates from HEK293T expressing AcGFP1 or AcGFP1 - tagged PEX14 3 × FLAG - tagged KIFC3 were immunoprecipitated with anti - FLAG antibody immunoblotted with anti - GFP or anti - FLAG antibody .

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Whole-cell lysates from HEK293T cells expressing AcGFP1 or AcGFP1-tagged PEX14 and 3×FLAG-tagged KIFC3 were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.


Whole - lysates from HEK293T expressing AcGFP1 - tagged KIFC3 3 × FLAG - tagged Rab10 or Rab10 - Q68L were immunoprecipitated with anti - FLAG antibody immunoblotted with anti - GFP or anti - FLAG antibody .

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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 - lysates from HEK293T expressing AcGFP1 - tagged Pex14 3 × FLAG - tagged Rab10 or Rab10 - Q68L were immunoprecipitated with anti - FLAG antibody 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 of ciliary cholesterol trafficking - associated components in Pex1 + / + Pex1 − / − hTERT - RPE1 . Total lysates were separated to crude peroxisomal ( Crude Pex ) , lysosomal mitochondrial ( Lyso / Mito ) , peroxisomal ( PEX ) fractions . CYPOR , a lysosomal mitochondrial , served as a positive control for Lyso / Mito fractionation . Total lysates were gradually injected at 20 µg 5 µg into a gel for SDS - PAGE , while equal amounts ( 5 µg ) of from each fraction were loaded . Rabin8 , Rab10 , KIFC3 were concentrated in PEX fraction in Pex1 + / + hTERT - RPE1 . CYPOR ( cytochrome P450 reductase ) is a mitochondrial .

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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 transfected with 3 × FLAG - tagged KIFC3 were immunostained with anti - FLAG ( green ) , anti - PEX14 ( red ) , anti - α - tubulin ( ) antibodies . Magnified 3D - constituted of boxed regions showing peroxisomes are located on microtubule arrays via KIFC3 ( arrows ) . scale bars indicate 5 μm .

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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 showing depletion of KIFC3 in KIFC3 − / − hTERT - RPE1 clones . GAPDH served as a loading control .

Western blot analysis showing depletion of KIFC3 in the KIFC3−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.


KIFC3 + / + KIFC3 − / − hTERT - RPE1 incubated for 24 h without serum were immunostained with anti - pericentrin ( red ) anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( green ) . Scale bar , 5 μm . of ( G ) indicating that KIFC3 − / − hTERT - RPE1 significantly reduced ciliary accumulation of cholesterol ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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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 + / + KIFC3 − / − hTERT - RPE1 were immunostained with anti - ARL13B ( blue ) , anti - phospho - S473 - Akt ( red ) , anti - ninein ( green ) , anti - PMP70 ( ) antibodies . Arrows represent peroxisomes contacting ciliary pocket . Scale bar , 5 μm . of ( I ) showing that disruption of KIFC3 gene significantly interfered with contact between peroxisomes cilia ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 per experiment ) .

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 were transfected with AcGFP1 , AcGFP1 - tagged KIFC3 , rod domain - deleted KIFC3 mutant ( ΔRod ) , or motor domain - deleted KIFC3 mutant ( ΔMotor ) cultured without serum for 24 h before Filipin - III ( green ) - mediated cholesterol immunostaining with anti - GFP ( red ) , anti - pericentrin ( ) , anti - acetylated - tubulin ( blue ) antibodies . Arrows represent ciliary localization of cholesterol . scale bars indicate 5 μm . of Filipin III at cilia from ( K ) . AcGFP1 - tagged KIFC3 deletion mutants did not restore ciliary cholesterol insufficiency in KIFC3 − / − hTERT - RPE1 ( mean + / - s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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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.


Quiescent G0 - phase wild - type , Rab10 − / − , KIFC3 − / − , ORP3 − / − , EHD3 − / − hTERT - RPE1 transfected with PEX3 - GFP - FRB ( red ) tdTomato - BicD2 - FKBP ( ) were treated with or without 100 μM rapamycin for 30 min , then immunostained with anti - acetylated - tubulin ( blue ) antibody . Cholesterol was stained with Filipin III ( Green ) . Arrows indicate enrichment of ciliary cholesterol . scale bars represent 5 μm .

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Quiescent 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.


of Filipin III at cilia from ( A ) . Rapamycin - induced peroxisome targeting to ciliary pocket restored insufficiency of ciliary cholesterol in Rab10 − / − KIFC3 − / − hTERT - RPE1 , but not in ORP3 − / − EHD3 − / − hTERT - RPE1 ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 35 - 40 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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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.


( B ) Example of bead survival distributions for 1G4 TCR binding NYE 9V . survival probability at 1 s is shown as a dotted vertical is used to calculate off - rate under force : − ln ( survival at 1 s ) / 1s . ( C ) Off - rates under force obtained from ( B ) are fitted with Bell ' s model ( solid ) . Data information : number of independent experiments performed on different days that were combined to produce estimated off - rates are : 7 ( 1G4 / 9V )

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(B) Example of bead survival distributions for the 1G4 TCR binding NYE 9V. The survival probability at 1 s is shown as a dotted vertical line and is used to calculate the off-rate under force: −ln(survival at 1 s)/1s. (C) Off-rates under force obtained from (B) are fitted with Bell's model (solid line). Data information: The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 7 (1G4/9V)


( D ) Off - rates under force for additional pMHCs interacting with 1G4 TCR fitted to Bell ' s model ( solid ) . ( E ) Correlation of dissociation constant ( KD ) measured previously by SPR extrapolated zero - force flow chamber off - rate ( k0off ) from fitting Bell ' s model for 1G4 TCR . ( F ) Correlation of SPR dissociation constant ( KD ) with fitted force sensitivity parameter ( xβ ) for 1G4 TCR . Data information : Error bars ( SE ) in E , F , are obtained from fit . number of independent experiments performed on different days that were combined to produce estimated off - rates are : 7 ( 1G4 / 9V ) , 8 ( 1G4 / 6V ) , 8 ( 1G4 / 4E5E8E ) , 10 ( 1G4 / 3Y ) , 6 ( 1G4 / 4D )

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(D) Off-rates under force for additional pMHCs interacting with the 1G4 TCR fitted to Bell's model (solid line). (E) Correlation of the dissociation constant (KD) measured previously by SPR and the extrapolated zero-force flow chamber off-rate (k0off) from fitting Bell's model for the 1G4 TCR. (F) Correlation of the SPR dissociation constant (KD) with the fitted force sensitivity parameter (xβ) for the 1G4 TCR. Data information: Error bars (SE) in E,F, are obtained from the fit. The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 7 (1G4/9V), 8 (1G4/6V), 8 (1G4/4E5E8E), 10 (1G4/3Y), 6 (1G4/4D)


( G ) Off - rates under force for pMHCs interacting with A6 TCR fitted to Bell ' s model ( solid ) . ( H ) Correlation of dissociation constant ( KD ) measured previously by SPR extrapolated zero - force flow chamber off - rate ( k0off ) from fitting Bell ' s model for A6 TCR . ( I ) Correlation of SPR dissociation constant ( KD ) with fitted force sensitivity parameter ( xβ ) for A6 TCR . Data information : Error bars ( SE ) in H , I are obtained from fit . number of independent experiments performed on different days that were combined to produce estimated off - rates are : 9 ( A6 / Tax WT ) , 9 ( A6 / 7Q ) , 8 ( A6 / 7R ) , 8 ( A6 / 5H ) .

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(G) Off-rates under force for pMHCs interacting with the A6 TCR fitted to Bell's model (solid line). (H) Correlation of the dissociation constant (KD) measured previously by SPR and the extrapolated zero-force flow chamber off-rate (k0off) from fitting Bell's model for the A6 TCR. (I) Correlation of the SPR dissociation constant (KD) with the fitted force sensitivity parameter (xβ) for the A6 TCR. Data information: Error bars (SE) in H,I are obtained from the fit. The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 9 (A6/Tax WT), 9 (A6/7Q), 8 (A6/7R), 8 (A6/5H).


( A ) response force ( F ) on TCR / pMHC interaction when pulled at acting on spring attached to C - terminus of MHC which is being pulled with constant velocity ( ν ) . A second spring is attached to C - terminus of TCRβ fixed in space . Insets : TCR is shown in grey , MHC in blue , peptide in red . sites where springs are attached are marked in orange . direction of pulling is marked by a dashed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The response force (F) on the TCR/pMHC interaction when pulled at acting on the spring attached to the C-terminus of the MHC which is being pulled with constant velocity (ν). A second spring is attached to the C-terminus of the TCRβ and fixed in space. Insets: the TCR is shown in grey, the MHC in blue, and the peptide in red. The sites where the springs are attached are marked in orange. The direction of pulling is marked by a dashed line.


( B ) of productively signalling TCRs ( CN ) over antigen for antigens with indicated zero - force off - rates ( koff ) . Shown is effect of forces at 100 pN ( left ) 10 pN ( right ) . horizontal dashed is threshold value of CN required for activation in model ( λ ) . vertical dashed shows antigen ( P ) required to elicit activation by antigen with largest value of koff .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The concentration of productively signalling TCRs (CN) over the antigen concentration for antigens with the indicated zero-force off-rates (koff). Shown is the effect of forces at 100 pN (left) and 10 pN (right). The horizontal dashed line is the threshold value of CN required for activation in the model (λ). The vertical dashed line shows the antigen concentration (P) required to elicit activation by the antigen with the largest value of koff.


( C ) antigen required to elicit activation ( P ) over koff for indicated molecular forces on TCR / pMHC interaction . ( D ) Antigen sensitivity quantified as value of P from ( C ) over applied force for a higher - affinity antigen with koff = 0 . 5 s - 1 . ( E ) Antigen discrimination quantified by discrimination power ( α ) over applied force . Discrimination power is defined as of logarithm of fold - change in P over fold - change in koff for a higher - affinity ligand ( koff = 0 . 5 s - 1 ) a lower - affinity ligand ( koff = 101 . 5 s - 1 ) from panel ( C ) .

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(C) The antigen concentration required to elicit activation (P) over koff for the indicated molecular forces on the TCR/pMHC interaction. (D) Antigen sensitivity quantified as the value of P from (C) over the applied force for a higher-affinity antigen with koff = 0.5 s-1. (E) Antigen discrimination quantified by the discrimination power (α) over the applied force. Discrimination power is defined as the ratio of the logarithm of the fold-change in P over the fold-change in koff for a higher-affinity ligand (koff = 0.5 s-1) and a lower-affinity ligand (koff = 101.5 s-1) from panel (C).


( F ) Experimental value of P ( symbols ) over antigen affinity fitted by kinetic proofreading model with force ( dashed solid lines ) . value of P is defined as of pMHC required for 15 % upregulation of CD69 above baseline is shown as mean with from 4 independent experiments . ( G ) fitted value of applied force with . ( H ) fitted value of λ with . Data information : An F - is used to produce a p - value for null hypothesis that applied force or λ is same across three conditions . data fitting ( F - H ) used N = 2 . 46 kp = 2 . 15 s − 1 ( average of CD58 ICAM - 1 conditions )

O O O O O O O O O O O B-CONTROLLED_VAR O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O

(F) Experimental value of P (symbols) over antigen affinity fitted by the kinetic proofreading model with force (dashed and solid lines). The value of P is defined as the concentration of pMHC required for 15% upregulation of CD69 above baseline and is shown as the mean with SEM from 4 independent experiments. (G) The fitted value of the applied force with SEM. (H) The fitted value of λ with SEM. Data information: An F-test is used to produce a p-value for the null hypothesis that the applied force or λ is the same across the three conditions. All data fitting (F-H) used N = 2.46 and kp = 2.15 s−1 (average of CD58 and ICAM-1 conditions) taken


F . Influence of mutating σ4 on promoter recognition .

F. Influence of mutating σ4 on promoter recognition.


B , C . Effects of deleting β ′ ZBD on transcriptional activities of RNAP on different promoters . fliCp ( B ) was used as template for σ28 - dependent transcription , lacUV5p ( C ) was used in σ70 - dependent transcription . products indicated by solid triangles were quantified signal obtained from 100 nM wild - type RNAP was normalized to 1 in each respectively .

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B, C. Effects of deleting β′ ZBD on transcriptional activities of RNAP on different promoters. The fliCp (B) was used as template for σ28-dependent transcription, and the lacUV5p (C) was used in σ70-dependent transcription. RNA products indicated by solid triangles were quantified and the signal obtained from 100 nM wild-type RNAP was normalized to 1 in each test respectively.


D . Transcriptional activities of ZBD mutated σ28 - RNAP on fliCp . products indicated by solid triangles were quantified .

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D. Transcriptional activities of ZBD mutated σ28-RNAP on fliCp. RNA products indicated by solid triangles were quantified.


E . Binding of σ28 - RNAP with fliCp as detected by EMSA ( left ) . heparin - resistant RNAP - promoter open is indicated as " RPo " , heparin - sensitive RNAP - promoter closed is shown as " RPc " . Percentages of RNAP - promoter open ( RPo ) closed ( RPc ) in EMSA performed using σ28 - RNAP fliCp ( right ) .

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E. Binding of σ28-RNAP with fliCp as detected by EMSA (left). The heparin-resistant RNAP-promoter open complex is indicated as "RPo", and the heparin-sensitive RNAP-promoter closed complex is shown as "RPc". Percentages of RNAP-promoter open complex (RPo) and closed complex (RPc) in EMSA performed using σ28-RNAP and fliCp (right).


F . DNase I footprinting of fliCp with σ28 - RNAP . Promoter was labeled on non - template strand . promoter region protected by RNAP is indicated by a red box at bottom panel .

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F. DNase I footprinting of the fliCp complex with σ28-RNAP. Promoter DNA was labeled on the non-template strand. The promoter region protected by RNAP is indicated by a red box at the bottom panel.


B , C . Promoter activities of tarp ( B ) fliCp ( C ) in E . coli wild - type ( Ec - WT ) its mutants expressing ZBD - mutated RNAP ( Ec - K74A , Ec - K87A Ec - K74A - K87A ) shown as mean ± SD from three replicates .

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B, C. Promoter activities of tarp (B) and fliCp (C) in E. coli wild-type (Ec-WT) and its mutants expressing ZBD-mutated RNAP (Ec-K74A, Ec-K87A and Ec-K74A-K87A) shown as mean ± SD from three replicates.


D . Volcano plot showing log2 - fold change of mRNA abundance as a of Ec - K74A - K87A Ec - WT strains . Blue dots represent genes with significant difference between these two strains . Red dots represent flagellar genes . Data are shown as mean values of three independent colonies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Volcano plot showing log2-fold change of mRNA abundance as a ratio of Ec-K74A-K87A and Ec-WT strains. Blue dots represent genes with significant difference between these two strains. Red dots represent flagellar genes. Data are shown as mean values of three independent colonies.


E . Relative mRNA of flagellar genes in Ec - K74A - K87A comparing with that in Ec - WT ( normalized to 1 for each gene ) . Diagram for role of σ28 ( fliA gene ) in flagella regulon hierarchy Clarke & Sperandio , 2005Fitzgerald , 2014 ( ; ) is shown in left panel . Data are mean ± from three replicates .

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E. Relative mRNA levels of flagellar genes in Ec-K74A-K87A strain comparing with that in Ec-WT (normalized to 1 for each gene). Diagram for the role of σ28 (fliA gene) in flagella regulon hierarchy Clarke & Sperandio, 2005Fitzgerald, 2014(; ) is shown in the left panel. Data are mean ± SEM from three replicates.


F . Motilities of Ec - K74A - K87A strains with over - expression of wild - type rpoC gene ( Ec - K74A - K87A - C ) or carrying control plasmid ( Ec - K74A - K87A - V ) .

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F. Motilities of Ec-K74A-K87A strains with over-expression of wild-type rpoC gene (Ec-K74A-K87A-C) or carrying the control plasmid (Ec-K74A-K87A-V).


G . Activities of fliCp in Ec - K74A - K87A - C Ec - K74A - K87A - V strains . L - arabinose was used at final of 0 . 002 % . Data are mean ± SD from three colonies .

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G. Activities of fliCp in Ec-K74A-K87A-C and Ec-K74A-K87A-V strains. L-arabinose was used at final concentration of 0.002%. Data are mean ± SD from three colonies.


B . Activities of chimeric σ28 - R4m in initiating transcription from tarp - 35m , when reconstituted with wild - type ( WT ) or ZBD mutated RNAP enzyme .

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B. Activities of chimeric σ28-R4m in initiating transcription from tarp-35m, when reconstituted with wild-type (WT) or ZBD mutated RNAP core enzyme.


Effects of mutating β ′ ZBD on transcriptional activities of σ38 - RNAP on osmYp .

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Effects of mutating β′ ZBD on transcriptional activities of σ38-RNAP on osmYp.


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Effects of mutating β ′ ZBD on transcriptional activities of σ70 - RNAP on osmYp .

Effects of mutating β′ ZBD on transcriptional activities of σ70-RNAP on osmYp.


B . Characterization of various B progenitor fractions as described by [ 38 ] in bone marrow of 4 wildtype 4 Shld2 − / − littermate controls .

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B. Characterization of the various B cell progenitor fractions as described by [38] in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.


C . Characterization of indicated thymic T populations in bone marrow of 4 wildtype 4 Shld2 − / − littermate controls .

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C. Characterization of the indicated thymic T cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.


D . Characterization of indicated immature mature splenic B populations in bone marrow of 4 wildtype 4 Shld2 − / − littermate controls .

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D. Characterization of the indicated immature and mature splenic B cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.


E . Characterization of indicated peritoneal B populations in bone marrow of 4 wildtype 4 Shld2 − / − littermate controls .

E. Characterization of the indicated peritoneal B cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.


F . Left panel : Schematic of A70 . 2 INV - 4 strategy to induce RAG1 / 2 - mediated recombination using imatinib . Right panel : A70 . 2 INV - 4 lines were transduced with lentiviruses encoding lentiCRISPRv2 expressing sgRNAs against 53bp1 , Shld1 , Shld2 , Shld3 , Lig4 genes . Guide targeting chicken AID was used as a negative control ( Ctrl ) . were selected with puromycin treated with 3 μM imatinib for 4 days after which GFP frequency was measured ( mean ± SD of 3 biological replicates ) . insertion - deletion ( indel ) penetrance as measured by TIDE [ 39 ] of for each of these sgRNA constructs is shown in Figure EV1E , baseline GFP frequency prior to imatinib stimulation is shown in Figure EV1F . sgRNA sequences used are shown in Table EV2 .

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F. Left panel: Schematic of the A70.2 INV-4 cell line strategy to induce RAG1/2-mediated recombination using imatinib. Right panel: A70.2 INV-4 cell lines were transduced with lentiviruses encoding the lentiCRISPRv2 expressing sgRNAs against the 53bp1, Shld1, Shld2, Shld3, and Lig4 genes. Guide RNA targeting chicken AID was used as a negative control (Ctrl). Cells were selected with puromycin and treated with 3 μM imatinib for 4 days after which GFP frequency was measured (mean ± SD of 3 biological replicates). The insertion-deletion (indel) penetrance as measured by TIDE analysis [39] of sequence for each of these sgRNA constructs is shown in Figure EV1E, and the baseline GFP frequency prior to imatinib stimulation is shown in Figure EV1F. sgRNA sequences used are shown in Table EV2.


A . of various indicated isotypes in serum of 6 - 8 week old unimmunized WT Shld2 − / − mice . Values are mean ± SD of 4 biological replicates ; * * P ≤ 0 . 01 , unpaired two - t - .

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A. Concentration of the various indicated isotypes in the serum of 6-8 week old unimmunized WT and Shld2−/− mice. Values are mean concentration ± SD of 4 biological replicates; ** P ≤ 0.01, unpaired two-tailed t-test.


B . B were purified from spleens from WT Shld2 − / − mice , stimulated to undergo CSR to various indicated isotypes using different stimulation cocktails [ 33 ] . were then analyzed by flow cytometry for expression of various indicated isotypes expression of each isotype are reported . Values are mean frequency ± SD of 4 biological replicates ; * * * * P ≤ 0 . 0001 , unpaired two - t - .

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B. B cells were purified from spleens from WT and Shld2−/− mice, and stimulated to undergo CSR to the various indicated isotypes using different stimulation cocktails [33]. Cells were then analyzed by flow cytometry for expression of the various indicated isotypes and the percent expression of each isotype are reported. Values are mean frequency ± SD of 4 biological replicates; **** P ≤ 0.0001, unpaired two-tailed t-test.


C . WT , 53bp1 − / − , Shld2 − / − mice were immunized with NP - CGG serum was withdrawn 2 post immunization serial dilutions were subjected to ELISA for NP - specific antibodies of indicated isotypes . Values are mean absorbance ± SD of 4 biological replicates .

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C. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG and the serum was withdrawn 2 weeks post immunization and serial dilutions were subjected to ELISA analysis for NP-specific antibodies of the indicated isotypes. Values are mean absorbance ± SD of 4 biological replicates.


D . WT , 53bp1 − / − , Shld2 − / − mice were immunized with NP - CGG , spleens were isolated anti - IgG secreting were enumerated by ELISPOT . Values are mean frequency ± SD of 4 biological replicates , except for Shld2 - / - ( n = 3 ) ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , unpaired two - t .

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D. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG, spleens were isolated and anti-IgG secreting cells were enumerated by the ELISPOT assay. Values are mean frequency ± SD of 4 biological replicates, except for Shld2-/- (n=3); * P ≤ 0.05, ** P ≤ 0.01, unpaired two-tailed t test.


A . WT Shld2 − / − B were purified from spleens stimulated with LPS + IL4 examined for IgM IgG1 expression 3 , 6 , 9 days post - stimulation by flow cytometry . Representative plots are shown for both WT Shld2 − / − B 6 - days post stimulation . graph plots show proportion of IgG1 + Iglo , mean ± SD from 6 biological replicates ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , two - way ANOVA with post hoc Dunnett ' s .

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A. WT and Shld2−/− B cells were purified from spleens and stimulated with LPS + IL4 and examined for IgM and IgG1 expression 3, 6, and 9 days post-stimulation by flow cytometry. Representative plots are shown for both WT and Shld2−/− B cells 6-days post stimulation. The graph plots show proportion of IgG1+ and Iglo cells, mean ± SD from 6 biological replicates; ** P ≤ 0.01, *** P ≤ 0.001, two-way ANOVA with post hoc Dunnett's test.


B . WT CH12 , as well as two each of 53BP1 - , SHLD1 - , SHLD2 - , SHLD3 - deficient clones generated previously [ 21 ] , as well as a LIG4 - deficient CH12 clone were subjected to CSR induction with CIT cocktail measured for both IgM IgA expression by flow cytometry . Representative flow plots for WT 53bp1 − / − CH12 are shown at day 3 post CIT stimulation . graph plots show proportion of IgA + Iglo CH12 , mean ± SD from 3 biological replicates ; * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 , two - way ANOVA with post hoc Dunnett ' s . letters below x - axis represent clone codes for 53BP1 , SHLD1 , SHLD2 , SHLD3 - deficient CH12 clones . One LIG4 - deficient CH12 clone was also used .

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B. WT CH12 cells, as well as two each of 53BP1-, SHLD1-, SHLD2-, and SHLD3-deficient clones generated previously [21], as well as a LIG4-deficient CH12 clone were subjected to CSR induction with the CIT cocktail and measured for both IgM and IgA expression by flow cytometry. Representative flow plots for WT and 53bp1−/− CH12 cells are shown at day 3 post CIT stimulation. The graph plots show proportion of IgA+ and Iglo CH12 cells, mean ± SD from 3 biological replicates; * P ≤ 0.05, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. The letters below the x-axis represent the clone codes for the 53BP1, SHLD1, SHLD2, and SHLD3-deficient CH12 clones. One LIG4-deficient CH12 clone was also used.


C . Xlf , Ku70 , Ku80 , Xrcc4 , Paxx genes were knocked out in CH12 by CRISPR , two independent clones each were analyzed as in Figure 3B with mean ± 3 biological replicates ; * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 , two - way ANOVA with post hoc Dunnett ' s .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. The Xlf, Ku70, Ku80, Xrcc4, and Paxx genes were knocked out in CH12 cells by CRISPR, and two independent clones each were analyzed as in Figure 3B with mean ± 3 biological replicates; * P ≤ 0.05, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test.


A . WT , two independent clones each of 53bp1 − / − , Shld2 − / − / − , Shld3 − / − CH12 were stimulated with CIT analyzed by flow cytometry for IgM IgA expression . Iglo population were reduced after 7 days in culture . Values are mean ± SD from 3 biological replicates ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 , two - way ANOVA with post hoc Dunnett ' s .

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A. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 cells were stimulated with CIT and analyzed by flow cytometry for IgM and IgA expression. The Iglo population were reduced after 7 days in culture. Values are mean ± SD from 3 biological replicates; * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test.


B . WT , two independent clones each of 53bp1 − / − , Shld2 − / − / − , Shld3 − / − CH12 clones were stimulated with CIT for 3 days . IgM + , IgA + , Iglo populations were sorted reanalyzed for expression of IgM IgA 5 days post sort as well as 12 days post sort ( see Figure EV4B ) . Shown on bar graphs are sorted IgM + , IgA + , Iglo populations ( each column , 1 technical replicate ) from WT mutant CH12 clones , of expressing IgM , IgA , or low for both isotypes ( Iglo ) after 5 days of culture post - sort . A representative flow plot of 53bp1 − / − CH12 3 days post CIT treatment is shown .

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B. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 clones were stimulated with CIT for 3 days. The IgM+, IgA+, and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 5 days post sort as well as 12 days post sort (see Figure EV4B). Shown on bar graphs are sorted IgM+, IgA+, and Iglo populations (each column, 1 technical replicate) from WT and mutant CH12 clones, and the percent of cells expressing IgM, IgA, or low for both isotypes (Iglo) after 5 days of culture post-sort. A representative flow plot of 53bp1−/− CH12 3 days post CIT treatment is shown.


A . mRNA expression of IgM IgA was carried out by RT - PCR for Iglo subclones from WT two independent 53bp1 − / − CH12 ( TA T1 ) clones . Iglo subclones derived from 53bp1 − / − CH12 TA clone are listed as D01 - D18 , while Iglo subclones derived from 53bp1 − / − CH12 T1 clone are listed as F03 - F24 . Gels show RT - PCR for IgM cDNA ( top ) , IgA cDNA ( middle ) , AID cDNA ( bottom ) as control .

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A. mRNA expression analysis of IgM and IgA was carried out by RT-PCR for Iglo subclones from WT and two independent 53bp1−/− CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show the RT-PCR analysis for IgM cDNA (top), IgA cDNA (middle), and AID cDNA (bottom) as control.


B . mRNA expression as in A of Iglo subclones of two independent Shld2 - / - / - ( F2 F3 ) clones , IgA + subclones from Shld2 - / - / - clone F3 . Iglo subclones derived from Shld2 - / - / - CH12 F2 clone are listed as H03 - H40 , while Iglo subclones derived from Shld2 - / - / - CH12 F3 clone are listed as J02 - J109 .

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B. mRNA expression analysis as in A of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones, and IgA+ subclones from Shld2-/-/- clone F3. The Iglo subclones derived from the Shld2-/-/- CH12 F2 clone are listed as H03-H40, while the Iglo subclones derived from the Shld2-/-/-CH12 F3 clone are listed as J02-J109.


Long - range PCR of Iglo subclones from WT two independent 53bp1 - / - CH12 ( TA T1 ) clones . Iglo subclones derived from 53bp1 − / − CH12 TA clone are listed as D01 - D18 , while Iglo subclones derived from 53bp1 − / − CH12 T1 clone are listed as F03 - F24 . Gels show long - range amplicons ( top ) AID genomic PCR control ( bottom ) .

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Long-range PCR of Iglo subclones from WT and two independent 53bp1-/- CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show long-range amplicons (top) and AID genomic DNA PCR control (bottom).


Long - range PCR as in B of Iglo subclones of two independent Shld2 - / - / - ( F2 F3 ) clones ( left 2 panels ) , IgA + subclones from Shld2 - / - / - clone F3 .

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Long-range PCR as in B of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones (left 2 panels), and IgA+ subclones from Shld2-/-/- clone F3.


of a representative subset of long range - PCR amplicons . Symbol annotations : ( * ) inverted , ( ◊ ) rearranged fragment order , ( † ) amplified with EμF primer rather than IμF , ( ‡ ) undamaged Cα coding .

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Sequence analysis of a representative subset of long range-PCR amplicons. Symbol annotations: (*) inverted sequence, (◊) rearranged fragment order, (†) amplified with the EμF primer rather than IμF, (‡) undamaged Cα coding sequence.


A . of nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine . In this subsequent similar figures , number of mice is indicated .

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A. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine. In this and subsequent similar figures, the number of mice is indicated.


B . Averaged intracellular Ca2 + signals in cultured mouse DRG neurons in response to consecutive applications of 0 . 25 ppm iodine , 30 μM AITC 2 μM capsaicin . of iodine responsive neurons ( n = 332 ) from 6 adult mice were included in .

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B. Averaged intracellular Ca2+ signals in cultured mouse DRG neurons in response to consecutive applications of 0.25 ppm iodine, 30 μM AITC and 2 μM capsaicin. All of the iodine responsive neurons (n = 332) from 6 adult mice were included in the analysis.


C . of nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine , following intraperitoneal injection of HC030031 ( HC ) ( C ) or vehicle .

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C. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of HC030031 (HC) (C) or vehicle.


D . of nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine , following intraperitoneal injection of AMG517 ( D ) or vehicle .

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D. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of AMG517 (D) or vehicle.


E . of nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT TRPA1 - / - mice .

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E. Quantification of the nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT and TRPA1-/-mice.


F . of nociceptive responses in TRPA1 - / - mice within 5 min after intraplantar injection of control saline or 1 , 000 ppm iodine , following intraperitoneal injection of saline or vehicle .

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F. Quantification of the nociceptive responses in TRPA1-/- mice within 5 min after intraplantar injection of control saline or 1,000 ppm iodine, following intraperitoneal injection of saline or vehicle.


A . Left , time course of ear swelling elicited by topical application of 5 % PVP - I solution in 0 . 15 % Oxa - challenged WT TRPA1 - / - mice , or 5 % povidone solution in Oxa - challenged WT mice . In this subsequent similar figures , number of mice is indicated . Right , bar graph highlighting response at 24 h shown at left .

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A. Left, time course of ear swelling elicited by topical application of 5% PVP-I solution in 0.15% Oxa-challenged WT and TRPA1-/-mice, or 5% povidone solution in Oxa-challenged WT mice. In this and subsequent similar figures, the number of mice is indicated. Right, bar graph highlighting the response at 24 h shown at left.


B . Time course of ear swelling elicited by topical application of indicated concentrations of iodine in 0 . 15 % Oxa - challenged mice , a bar graph highlighting response at 24 h .

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B. Time course of ear swelling elicited by topical application of the indicated concentrations of iodine in 0.15% Oxa-challenged mice, and a bar graph highlighting the response at 24 h.


C . Time course of ear swelling elicited by topical application of 3 % iodine in 0 . 15 % Oxa - challenged WT TRPA1 - / - mice , bar graph highlighting response at 24 h .

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C. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged WT and TRPA1-/-mice, and bar graph highlighting the response at 24 h.


D . Hematoxylin eosin stained tissue sections of iodine treated ear in 0 . 15 % Oxa - challenged WT TRPA1 - / - mice ( n ≥ 3 ) .

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D. Hematoxylin and eosin stained tissue sections of iodine treated ear in 0.15% Oxa-challenged WT and TRPA1-/-mice (n ≥ 3).


E . Time course of ear swelling elicited by topical application of 3 % iodine in 0 . 15 % Oxa - challenged TRPA1 - / - mice , following intraperitoneal injection of AMG517 or vehicle , bar graph highlighting response at 24 h .

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E. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged TRPA1-/-mice, following intraperitoneal injection of AMG517 or vehicle, and bar graph highlighting the response at 24 h.


F . Time course of ear swelling elicited by topical application of 3 % iodine in 0 . 15 % Oxa - challenged mice , following intraperitoneal injection of RP67580 or vehicle , bar graph highlighting response at 24 h .

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F. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged mice, following intraperitoneal injection of RP67580 or vehicle, and bar graph highlighting the response at 24 h.


A . Representative intracellular Ca2 + signals in hTRPA1 - expressing HEK293 in response to different concentrations of iodine . 2 - APB , a TRPA1 agonist , was subsequently applied to fully activate TRPA1 . RFU : relative unit .

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A. Representative intracellular Ca2+ signals in hTRPA1-expressing HEK293 cells in response to different concentrations of iodine. 2-APB, a TRPA1 agonist, was subsequently applied to fully activate TRPA1. RFU: relative fluorescence unit.


B . - response relationships of iodine - induced intracellular Ca2 + increase in HEK293 expressing WT or mutant TRPA1 channels . Data are presented as mean ± s . e . m . n ≥ 8 for each construct at each . smooth curves are fits to Hill equation .

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B. Concentration-response relationships of iodine-induced intracellular Ca2+ increase in HEK293 cells expressing WT or mutant TRPA1 channels. Data are presented as mean ± s.e.m. n ≥ 8 for each construct at each concentration. The smooth curves are fits to the Hill equation.


C . Time course of iodine - induced currents in hTRPA1 - expressing Xenopus oocytes . HC :

C. Time course of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. HC: HC030031


D . - response relationship of iodine - induced currents in hTRPA1 - expressing Xenopus oocytes . Data are presented as mean ± s . e . m . n ≥ 8 for each . smooth curve is a fit to Hill equation .

D. Concentration-response relationship of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. Data are presented as mean ± s.e.m. n ≥ 8 for each concentration. The smooth curve is a fit to the Hill equation.


E . Time course of intracellular iodine - induced macroscopic currents in an inside - out patch from hTRPA1 - expressing HEK293 ( n = 3 ) .

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E. Time course of intracellular iodine-induced macroscopic currents in an inside-out patch from hTRPA1-expressing HEK293 cell (n = 3).


F . Time course of iodine - induced currents in a hTRPA1 - expressing Xenopus oocyte , which presents current reduction upon DTT treatment ( n = 3 ) .

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F. Time course of iodine-induced currents in a hTRPA1-expressing Xenopus oocyte, which presents the current reduction upon DTT treatment (n = 3).


G , H . Comparisons of iodine - induced whole - current in HEK293 expressing WT hTRPA1 ( G ) or triple mutant channel ( C421S / K710R / C856S ) ( H ) ( n ≥ 4 ) .

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G, H. Comparisons of the iodine-induced whole-cell current in HEK293 cells expressing WT hTRPA1 (G) or the triple mutant channel (C421S/K710R/C856S) (H) (n ≥ 4).


I . Representative whole - currents in HEK293 expressing human TRPV1 in response to iodine subsequently applied capsaicin ( n = 3 ) .

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I. Representative whole-cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin (n = 3).


A Growth curves show proliferation reduction in ATRX depleted U87 . Data are expressed as means ± standard deviation ( s . d . ) , N = 2 . 500 / well were plated .

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A Growth curves show proliferation reduction in ATRX depleted U87 cells. Data are expressed as means ± standard deviation (s.d.), N = 2. 500 cells/well were plated.


B β - gal revealed increased senescent population in ATRX - deleted U87 . Upper panel , of control ATRX - deleted U87 processed for ATRX immunofluorescence ( IF ) β - gal senescence . Scale bar , 200 µm . D , DAPI . Lower panel , of senescent . Data are expressed as means ± s . d . , N = 3 , unpaired t .

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B β-gal staining revealed the increased senescent cell population in ATRX-deleted U87 cells. Upper panel, micrographs of control and ATRX-deleted U87 cells processed for ATRX immunofluorescence (IF) and β-gal senescence assay. Scale bar, 200 µm. D, DAPI. Lower panel, percentage of senescent cells. Data are expressed as means ± s.d., N = 3, unpaired t test.


C , D PML / TelG immuno - FISH ( C ) shows increased APB formation in ATRX - deleted U87 ( D ) . Scale bar , 10 µm . ( D ) Percentages of APB positive are expressed as means ± s . d . , N = 3 , unpaired t .

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C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87 cells (D). Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.


E C - circle assays revealed enhanced C - circles formation in ATRX - deleted U87 . Reactions lacking or ɸ29 serve as negative controls . 30 ng of indicated template was used in each reaction .

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E C-circle assays revealed enhanced C-circles formation in ATRX-deleted U87 cells. Reactions lacking DNA or ɸ29 serve as negative controls. 30 ng of indicated template DNA was used in each reaction.


G 53BP1 / TelG immuno - FISH shows increased TIF formation in ATRX - deleted U87 ( G ) . Arrows denote telomeres positive for 53BP1 signals . Scale bar , 10 µm . ( G ) Percentages of containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t .

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G 53BP1/TelG immuno-FISH shows increased TIF formation in ATRX-deleted U87 cells (G). Arrows denote telomeres positive for 53BP1 signals. Scale bar, 10 µm. (G) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


H Growth curves revealed increase of proliferation in ATRX - deleted U87 ( A - 1 A - 2 ) after hTERT transduction . Data are expressed as means ± s . d . , N = 2 .

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H Growth curves revealed increase of cell proliferation in ATRX-deleted U87 cells (A-1 and A-2) after hTERT transduction. Data are expressed as means ± s.d., N = 2.


I Western blots show depletion of ATRX expression in hTERT - transduced ATRX - deleted A - 1 A - 2 U87 .

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I Western blots show depletion of ATRX protein expression in hTERT-transduced and ATRX-deleted A-1 and A-2 U87 cells.


A Growth curves revealed that deletion of ATRX had minor effect on proliferation of TERT - overexpressed U87 - T , but further removal of exogenous floxed TERT by Ad - Cre induced rapid cycle arrest . Data are expressed as means ± s . d . , N = 3 .

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A Growth curves revealed that deletion of ATRX had minor effect on proliferation of TERT-overexpressed U87-T cells, but further removal of the exogenous floxed TERT by Ad-Cre induced rapid cell cycle arrest. Data are expressed as means ± s.d., N = 3.


B Telomere restriction fragment ( TRF ) shows increased telomeric heterogeneities in ATRX - deleted U87 - T further enhancement following Ad - Cre treatment . Genomic DNAs ( EtBr , left panel ) prepared from control ATRX - deleted U87 - T at day 9 post control or Ad - Cre infection ( middle panel ) were assayed by hybridization with 32P - labelled ( TTAGGG ) 4 probe , followed by re - hybridization with an oligonucleotide probe specific for centromere region ( right panel ) . Genomic from U2OS was used as an ALT positive control .

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B Telomere restriction fragment (TRF) analysis shows increased telomeric length heterogeneities in ATRX-deleted U87-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (middle panel) were assayed by hybridization with 32P-labelled (TTAGGG)4 probe, followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). Genomic DNA from U2OS cells was used as an ALT positive control.


C , D PML / TelG immuno - FISH ( C ) shows increased APB formation in ATRX - deleted U87 - T ( D ) at day 9 post control or Ad - Cre infection . are maximum - projections of ~ 10 stacks captured with a 63x lens with 2x zoom . Scale bar , 10 µm . ( D ) Percentages of APB positive are expressed as means ± s . d . , N = 3 , unpaired t .

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C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87-T cells (D) at day 9 post control or Ad-Cre infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.


E 2D gel revealed increased T - circle formation in ATRX - deleted U87 - T without or with Ad - Cre infection . Shown are gels stained with EtBr blots hybridized with a TelG probe . Indicated are linear ( lin ) open ( oc ) T - circle forms of telomeric .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E 2D gel analysis revealed increased T-circle formation in ATRX-deleted U87-T cells without or with Ad-Cre infection. Shown are gels stained with EtBr and blots hybridized with a TelG probe. Indicated are linear (lin) and open (oc) T-circle forms of telomeric DNA.


F C - circle assays shows increased C - circle formation in ATRX - deleted U87 - T at day 9 post control or Ad - Cre infection .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

F C-circle assays shows increased C-circle formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection.


G , H 53BP1 / TelG immuno - FISH ( G ) shows increased TIF formation in ATRX - deleted U87 - T at day 9 post control or Ad - Cre infection ( H ) . Scale bar , 10 µm . ( H ) Percentages of containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t .

O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H 53BP1/TelG immuno-FISH (G) shows increased TIF formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (H). Scale bar, 10 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


B Western blots of extracts from U87 infected with Lenti - CRISPR ( puro ) encoding ATRX - targeted sgRNAs . extracts were isolated after puromycin selection , assayed for ATRX depletion using antibody against ATRX or ACTIN control . asterisks ( * ) denote two sgATRXs ( sgA # 10 sgA # 19 ) used in following experiments .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR 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 Western blots of extracts from U87 cells infected with Lenti-CRISPR(puro) encoding individual ATRX-targeted sgRNAs. The extracts were isolated after puromycin selection, and assayed for ATRX depletion levels using antibody against ATRX or ACTIN control. The asterisks (*) denote the two sgATRXs (sgA#10 and sgA#19) used in following experiments.


C ATRX immunofluorescence of U87 infected with lentiviral control sgRosa , sgA # 10 , or sgA # 19 . D , DAPI . Scale bar , 50 µm .

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C ATRX immunofluorescence staining of U87 cells infected with lentiviral control sgRosa, sgA#10, or sgA#19. D, DAPI. Scale bar, 50 µm.