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 2 ( 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 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 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 ) .

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 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

Figure 2(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 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 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).


Figure 3 ( 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 .

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

Figure 3(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.


Figure 4 ( 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 ) 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 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 )

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

Figure 4(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) 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 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


Figure 2F . Influence of mutating σ4 on promoter recognition .

Figure 2F. Influence of mutating σ4 on promoter recognition.


Figure 3 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 . RNA products indicated by solid triangles were quantified . 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 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. RNA products indicated by solid triangles were quantified.  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 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.


Figure 4 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 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 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 the control plasmid ( Ec - K74A - K87A - V ) . 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 .

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

Figure 4 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 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 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 the control plasmid (Ec-K74A-K87A-V). 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.


Figure 5 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 . Effects of mutating β ′ ZBD on transcriptional activities of σ38 - RNAP on osmYp . Effects of mutating β ′ ZBD on transcriptional activities of σ70 - RNAP on osmYp .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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.  Effects of mutating β′ ZBD on transcriptional activities of σ38-RNAP on osmYp.  Effects of mutating β′ ZBD on transcriptional activities of σ70-RNAP on osmYp.


Figure 1B . 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 the indicated thymic T cell populations in the bone marrow of 4 wildtype and 4 Shld2 − / − littermate controls . 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 the indicated peritoneal B cell populations in the bone marrow of 4 wildtype and 4 Shld2 − / − littermate controls . 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 .

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 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 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 O O O O O O O

Figure 1B. 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 the indicated thymic T cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.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 the indicated peritoneal B cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.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.


Figure 2A . 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 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 − / − , 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 − / − , 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 .

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

Figure 2A. 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 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−/−, 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−/−, 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.


Figure 3A . 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 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 . 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 .

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 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 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 3A. 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 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. 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.


Figure 4A . 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 , 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 .

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 O O O O O O O

Figure 4A. 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, 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.


Figure 5A . 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 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 .

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

Figure 5A. 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 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.


Figure 6Long - 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 and F3 ) clones ( left 2 panels ) , and IgA + subclones from Shld2 - / - / - clone F3 . 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 .

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 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 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

Figure 6Long-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 and F3) clones (left 2 panels), and IgA+ subclones from Shld2-/-/- clone F3.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.


Figure 1A . 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 and 2 μM capsaicin . All of the iodine responsive neurons ( n = 332 ) from 6 adult mice were included in the analysis . 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 . 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 . 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 . 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 .

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Figure 1A. 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 and 2 μM capsaicin. All of the iodine responsive neurons (n = 332) from 6 adult mice were included in the analysis.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. 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. 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. 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.


Figure 2A . 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 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 and TRPA1 - / - mice , and bar graph highlighting the response at 24 h . 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 , 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 , and bar graph highlighting the response at 24 h .

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Figure 2A. 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 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 and TRPA1-/-mice, and bar graph highlighting the response at 24 h.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, 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, and bar graph highlighting the response at 24 h.


Figure 3A . 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 . 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 : HC030031D . 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 cell ( n = 3 ) . 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 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 - cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin ( n = 3 ) .

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Figure 3A. 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. 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: HC030031D. 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 cell (n = 3).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 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-cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin (n = 3).