C , D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co - IP assay . Arabidopsis protoplasts expressing HF / NMT1 - Myc and HF - EGR2 / NMT1 - Myc were treated with or without 4 ° C for 2 h . Total proteins were immunoprecipated by anti - HA agarose beads . Anti - Myc and anti - HA were used to detect NMT1 - Myc and HF - EGR2 , respectively . In ( C ) , relative band intensity is shown below the co - IP blot . In ( D ) , each bar represents the mean ± SEM of three biological experiments ( * * P < 0 . 01 , two - tailed t - test ) .

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

C, D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co-IP assay. Arabidopsis protoplasts expressing HF/NMT1-Myc and HF-EGR2/NMT1-Myc were treated with or without 4°C for 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc and anti-HA were used to detect NMT1-Myc and HF-EGR2, respectively. In (C), relative band intensity is shown below the co-IP blot. In (D), each bar represents the mean ± SEM of three biological experiments (**P < 0.01, two-tailed t-test).


E In vitro pull - down assay showing decreased interaction intensity of EGR2 ( 4 ° C ) and NMT1 compared with that of EGR2 ( 22 ° C ) and NMT1 . Total proteins prepared from two - week - old EGR2 - Myc plants grown at 4 ° C for 0 h and 6 h were immunoprecipated by anti - Myc agarose beads and incubated with GST - NMT1 in pull down buffer for 2 h at 4 ° C . Anti - Myc and anti - GST antibodies were used to detect EGR2 and NMT1 proteins . Relative band intensity is shown below the output blot .

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

E In vitro pull-down assay showing decreased interaction intensity of EGR2 (4°C) and NMT1 compared with that of EGR2 (22°C) and NMT1. Total proteins prepared from two-week-old EGR2-Myc plants grown at 4°C for 0 h and 6 h were immunoprecipated by anti-Myc agarose beads and incubated with GST-NMT1 in pull down buffer for 2 h at 4°C. Anti-Myc and anti-GST antibodies were used to detect EGR2 and NMT1 proteins. Relative band intensity is shown below the output blot.


F Expression of NMT1 in NMT1 - Myc overexpression plants . EF1α was used as a loading control .

F Expression of NMT1 in NMT1-Myc overexpression plants. EF1α was used as a loading control.


Freezing phenotype ( G ) of NMT1 - Myc overexpression transgenic plants . Two - week - old Super : NMT1 - Myc plants were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) .

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

Freezing phenotype (G) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA).


survival rate ( H ) of NMT1 - Myc overexpression transgenic plants . Two - week - old Super : NMT1 - Myc plants were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . In ( H ) , each bar represents the mean ± SEM of three biological experiments , and asterisks indicate significant differences compared with the wild type for the same treatment ( * P < 0 . 05 , two tailed t - test ) .

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

survival rate (H) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). In (H), each bar represents the mean ± SEM of three biological experiments, and asterisks indicate significant differences compared with the wild type for the same treatment (*P < 0.05, two tailed t-test).


OST1 kinase activity in NMT1 - Myc overexpression plants under cold stress . Top , autoradiograph ; bottom , CBB staining . Representative pictures are shown in ( I ) In ( I ) : the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 .

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

OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. Top, autoradiograph; bottom, CBB staining. Representative pictures are shown in (I) In (I): the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1.00.


OST1 kinase activity in NMT1 - Myc overexpression plants under cold stress . the relative kinase activity is shown in ( J ) . In ( J ) : each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two tailed t - test ) .

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

OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. the relative kinase activity is shown in (J). In (J): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two tailed t-test).


A , B Pull - down assay . Total proteins prepared from EGR2 - Myc or EGR2G2A - Myc transgenic plants was immunoprecipated with anti - Myc agarose beads , and then incubated with GST - OST1 . Anti - Myc and anti - GST antibody were used to detect GST - OST1 and EGE2 - Myc . Representative pictures are shown in ( A ) . Relative interaction is shown in ( B ) . Data information : In ( A Relative band intensities are shown . In ( B each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) .

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

A, B Pull-down assay. Total proteins prepared from EGR2-Myc or EGR2G2A-Myc transgenic plants was immunoprecipated with anti-Myc agarose beads, and then incubated with GST-OST1. Anti-Myc and anti-GST antibody were used to detect GST-OST1 and EGE2-Myc. Representative pictures are shown in (A). Relative interaction is shown in (B). Data information: In (A Relative band intensities are shown. In (B each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


C LCI of EGR2 and EGR2G2A with OST1 in N . benthamiana leaves . EGR2 - cLuc / OST1 - nLuc and EGR2G2A - cLuc / OST1 - nLuc were expressed in N . benthamiana leaves for 48 h . EGR2 - cLuc / nLuc , EGR2G2A - cLuc / nLuc , and cLuc / OST1 - nLuc were used as negative controls . Scale bar : 2 cm .

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

C LCI of EGR2 and EGR2G2A with OST1 in N. benthamiana leaves. EGR2-cLuc/OST1-nLuc and EGR2G2A-cLuc/OST1-nLuc were expressed in N. benthamiana leaves for 48 h. EGR2-cLuc/nLuc, EGR2G2A-cLuc/nLuc, and cLuc/OST1-nLuc were used as negative controls. Scale bar: 2 cm.


D , E Co - IP assay . EGR2 - Myc / HF - OST1 , EGR2G2A - Myc / HF - OST1 , HF / EGR2 - Myc and HF / EGR2G2A - Myc were expressed in N . benthamiana leaves . Total proteins were immunoprecipated by anti - HA agarose beads . Anti - HA and anti - Myc antibodies were used to detect HF - OST1 , EGR2 - Myc and EGR2G2A - Myc , respectively . Representative pictures are shown in ( D ) , and relative interaction is shown in ( E ) . Data information : In : Relative band intensities are shown . In each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) .

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

D, E Co-IP assay. EGR2-Myc/HF-OST1, EGR2G2A-Myc/HF-OST1, HF/EGR2-Myc and HF/EGR2G2A-Myc were expressed in N. benthamiana leaves. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-HA and anti-Myc antibodies were used to detect HF-OST1, EGR2-Myc and EGR2G2A-Myc, respectively. Representative pictures are shown in (D), and relative interaction is shown in (E). Data information: In : Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


F , G Effect of EGR2G2A on the interaction of OST1 and EGR2 in Arabidopsis protoplasts expressing HF - OST1 / EGR2 - GFP / EGR2 - Myc , HF - OST1 / EGR2 - GFP / EGR2G2A - Myc , or HF - OST1 / EGR2 - GFP . Total proteins were immunoprecipated by anti - HA agarose beads . Anti - Myc , anti - GFP , and anti - HA antibodies were used to detect EGR2 - Myc , EGR2 - GFP , and HF - OST1 , respectively . Representative pictures are shown in ( F ) , and relative interaction is shown in ( G ) . Data information In Relative band intensities are shown . In each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) .

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

F, G Effect of EGR2G2A on the interaction of OST1 and EGR2 in Arabidopsis protoplasts expressing HF-OST1/EGR2-GFP/EGR2-Myc, HF-OST1/EGR2-GFP/EGR2G2A-Myc, or HF-OST1/EGR2-GFP. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc, anti-GFP, and anti-HA antibodies were used to detect EGR2-Myc, EGR2-GFP, and HF-OST1, respectively. Representative pictures are shown in (F), and relative interaction is shown in (G). Data information In Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


H , I Effect of cold stress on the interaction of OST1 and EGR2 . Arabidopsis protoplasts expressing EGR2 - Myc / HF - OST1 and EGR2 - Myc / HF were treated at 4 ° C for 0 and 2 h . Total proteins were immunoprecipated by anti - HA agarose beads . The co - immunoprecipitated products were detected by anti - HA and anti - Myc antibodies , respectively . Representative pictures are shown in ( H ) , and relative interaction is shown in ( I ) . Data information : In H ) Relative band intensities are shown . In I ) : each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) .

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

H, I Effect of cold stress on the interaction of OST1 and EGR2. Arabidopsis protoplasts expressing EGR2-Myc/HF-OST1 and EGR2-Myc/HF were treated at 4°C for 0 and 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. The co-immunoprecipitated products were detected by anti-HA and anti-Myc antibodies, respectively. Representative pictures are shown in (H), and relative interaction is shown in (I). Data information: In H) Relative band intensities are shown. In I): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


Freezing phenotype ( A ) of wild type , ost1 - 3 , egr2 - 3 , and ost1 egr2 mutants . Two - week - old seedlings grown on MS medium were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) .

Freezing phenotype (A) of wild type, ost1-3, egr2-3, and ost1 egr2 mutants. Two-week-old seedlings grown on MS medium were treated at −5°C (NA) or −8°C for 0.5 h (CA).


survival rate and ion leakage ( B ) of wild type , ost1 - 3 , egr2 - 3 , and ost1 egr2 mutants . Two - week - old seedlings grown on MS medium were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

survival rate and ion leakage (B) of wild type, ost1-3, egr2-3, and ost1 egr2 mutants. Two-week-old seedlings grown on MS medium were treated at −5°C (NA) or −8°C for 0.5 h (CA). Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


C Expression of CBFs in wild type , ost1 - 3 , egr2 - 3 , and ost1 egr2 - 3 plants under cold stress . Two - week - old seedlings grown at 22 ° C were treated at 4 ° C for 0 h and 3 h . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

C Expression of CBFs in wild type, ost1-3, egr2-3, and ost1 egr2-3 plants under cold stress. Two-week-old seedlings grown at 22°C were treated at 4°C for 0 h and 3 h. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


( C ) 293T cells were transfected with empty vector ( EV ) , a vector encoding FLAG - tagged full length CYLD ( wt ) , or constructs encoding FLAG - tagged CYLD fragments 1 - 581 ( F1 ) lacking the C - terminus , or 581 - 956 ( F2 ) lacking the N - terminus , as indicated , followed by FLAG - IP . The blot was probed with antibodies recognizing SPATA2 , FLAG and tubulin . Endogenous SPATA2 was co - immunoprecipitated to comparable levels by CYLD and the C - terminal CYLD protein fragment , containing the USP domain .

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

(C) 293T cells were transfected with empty vector (EV), a vector encoding FLAG-tagged full length CYLD (wt), or constructs encoding FLAG-tagged CYLD fragments 1-581 (F1) lacking the C-terminus, or 581-956 (F2) lacking the N-terminus, as indicated, followed by FLAG-IP. The blot was probed with antibodies recognizing SPATA2, FLAG and tubulin. Endogenous SPATA2 was co-immunoprecipitated to comparable levels by CYLD and the C-terminal CYLD protein fragment, containing the USP domain.


( D ) CYLD - / - MEF were infected with retrovirus encoding FLAG - CYLD as indicated and CYLD was purified by FLAG - IP . The blot was probed with antibodies recognizing CYLD , SPATA2 and actin .

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

(D) CYLD-/- MEF were infected with retrovirus encoding FLAG-CYLD as indicated and CYLD was purified by FLAG-IP. The blot was probed with antibodies recognizing CYLD, SPATA2 and actin.


( E ) 293T cells were transfected with empty vector ( EV ) , a vector encoding FLAG - tagged full length SPATA2 or a construct encoding the FLAG - tagged N - terminal part of SPATA2 ( NT ) , as indicated , followed by FLAG - IP . The blot was probed with antibodies recognizing CYLD , SPATA2 and tubulin . M , marker lane .

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

(E) 293T cells were transfected with empty vector (EV), a vector encoding FLAG-tagged full length SPATA2 or a construct encoding the FLAG-tagged N-terminal part of SPATA2 (NT), as indicated, followed by FLAG-IP. The blot was probed with antibodies recognizing CYLD, SPATA2 and tubulin. M, marker lane.


( A ) 293T cells were transfected with a vector encoding V5 - tagged full length SPATA2 , along with empty vector ( EV ) a vector encoding FLAG - tagged HOIP wild - type ( wt ) or a vector encoding a FLAG - tagged PUB domain mutant of HOIP ( N102A ) . After FLAG - IP , the blot was probed with antibodies recognizing HOIP , CYLD , V5 and actin .

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

(A) 293T cells were transfected with a vector encoding V5-tagged full length SPATA2, along with empty vector (EV) a vector encoding FLAG-tagged HOIP wild-type (wt) or a vector encoding a FLAG-tagged PUB domain mutant of HOIP (N102A). After FLAG-IP, the blot was probed with antibodies recognizing HOIP, CYLD, V5 and actin.


( B ) 293T cells were transfected with empty vector or a vector encoding FLAG - tagged HOIP along with either V5 - tagged SPATA2 ( wt ) or a V5 - tagged PIM mutant ( PIMmut ) of SPATA2 . After V5 - IP , the blot was probed with antibodies recognizing FLAG , CYLD , V5 and tubulin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were transfected with empty vector or a vector encoding FLAG-tagged HOIP along with either V5-tagged SPATA2 (wt) or a V5-tagged PIM mutant (PIMmut) of SPATA2. After V5-IP, the blot was probed with antibodies recognizing FLAG, CYLD, V5 and tubulin.


( C ) 293T cells were transfected with a vector encoding FLAG - tagged HOIP and increasing amounts of V5 - tagged SPATA2 . After FLAG - IP , the blot was sequentially probed with antibodies recognizing OTULIN , HOIP , V5 and tubulin .

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

(C) 293T cells were transfected with a vector encoding FLAG-tagged HOIP and increasing amounts of V5-tagged SPATA2. After FLAG-IP, the blot was sequentially probed with antibodies recognizing OTULIN, HOIP, V5 and tubulin.


( D ) 293T cells were transfected with empty vector , FLAG - SPATA2 or FLAG - HOIP . After FLAG - IP , the blot was probed with antibodies recognizing OTULIN , CYLD , FLAG and actin .

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

(D) 293T cells were transfected with empty vector, FLAG-SPATA2 or FLAG-HOIP. After FLAG-IP, the blot was probed with antibodies recognizing OTULIN, CYLD, FLAG and actin.


( E ) 293T cells were transfected with empty vector , V5 - tagged SPATA2 ( wt ) or a V5 - tagged PUB domain mutant PUBmut of SPATA2 . After V5 - IP , the blot was probed with antibodies recognizing CYLD , FLAG , V5 and tubulin .

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

(E) 293T cells were transfected with empty vector, V5-tagged SPATA2 (wt) or a V5-tagged PUB domain mutant PUBmut of SPATA2. After V5-IP, the blot was probed with antibodies recognizing CYLD, FLAG, V5 and tubulin.


( F ) A549 cells were treated with FLAG - TNF ( 2µg / ml ) for the indicated time . The purified TNF - RSC was sequentially probed with antibodies recognizing SPATA2 , CYLD , RIPK1 , TRADD , SHARPIN , TNF and tubulin as indicated .

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

(F) A549 cells were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, RIPK1, TRADD, SHARPIN, TNF and tubulin as indicated.


( A ) A549 cells were infected to express a CRISPR / Cas9 system targeting luciferase ( as control , indicated wt ) or the spata2 gene ( ko ) . Mixed cell cultures were treated with FLAG - huTNF ( 2µg / ml ) for the indicated time . The purified TNF - RSC was sequentially probed with antibodies recognizing SPATA2 , CYLD , HOIP , SHARPIN , TRADD , TNF and tubulin as indicated .

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

(A) A549 cells were infected to express a CRISPR/Cas9 system targeting luciferase (as control, indicated wt) or the spata2 gene (ko). Mixed cell cultures were treated with FLAG-huTNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, HOIP, SHARPIN, TRADD, TNF and tubulin as indicated.


( B ) MEF were infected to express a CRISPR / Cas9 system targeting luciferase ( wt ) or the spata2 gene ( ko ) . Single clones were generated , which were treated with FLAG - TNF ( 2µg / ml ) for the indicated time . The purified TNF - RSC was sequentially probed with antibodies recognizing M1 - linked ubiquitin , CYLD , RIPK1 , and tubulin as indicated .

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(B) MEF were infected to express a CRISPR/Cas9 system targeting luciferase (wt) or the spata2 gene (ko). Single clones were generated, which were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing M1-linked ubiquitin, CYLD, RIPK1, and tubulin as indicated.


FLAG - tagged CYLD or catalytically inactive FLAG - CYLD ( C598A ) , respectively , were stably expressed in CYLD - / - MEF , followed by infection with control retrovirus , or retrovirus encoding SPATA2 , and purified by FLAG - IP . Purified CYLD or the inactive mutant were added to recombinant di - ubiquitin for 0 , 20 or 40 min at 37 ° C as indicated . After electrophoresis , the gel was cut and the lower part was silver stained to visualize mono - and di - ubiquitin , while the upper part was transferred to a membrane and probed for FLAG and V5 . In ( A ) , the experiment was done with M1 - linked di - ubiquitin substrate , in ( B ) K63 - linked di - ubiquitin substrate was subjected to the assay , and in ( C ) K48 - linked di - ubiquitin substrate was analyzed .

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FLAG-tagged CYLD or catalytically inactive FLAG-CYLD (C598A), respectively, were stably expressed in CYLD-/-MEF, followed by infection with control retrovirus, or retrovirus encoding SPATA2, and purified by FLAG-IP. Purified CYLD or the inactive mutant were added to recombinant di-ubiquitin for 0, 20 or 40 min at 37°C as indicated. After electrophoresis, the gel was cut and the lower part was silver stained to visualize mono-and di-ubiquitin, while the upper part was transferred to a membrane and probed for FLAG and V5. In (A), the experiment was done with M1-linked di-ubiquitin substrate, in (B) K63-linked di-ubiquitin substrate was subjected to the assay, and in (C) K48-linked di-ubiquitin substrate was analyzed.


( A ) MEF expressing CRISPR / Cas9 targeting luciferase ( wt ) or single clones generated from MEF expressing CRISPR / Cas9 targeting the spata2 gene ( ko ) were treated with mTNF ( 10 ng / ml ) as indicated . The blots were probed with antibodies recognizing P - IkBαIkBα , P - p38 , p38 , P - JNK , JNK , P - p65 , p65 and tubulin .

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

(A) MEF expressing CRISPR/Cas9 targeting luciferase (wt) or single clones generated from MEF expressing CRISPR/Cas9 targeting the spata2 gene (ko) were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBαIkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin.


( B ) 15P - 1 Sertoli cells expressing CRISPR / Cas9 targeting luciferase ( as control ) or single clones generated from 15P - 1 Sertoli cells expressing CRISPR / Cas9 targeting the spata2 gene were treated with mTNF ( 10 ng / ml ) as indicated . The blots were probed with antibodies recognizing P - IkBα , IkBα , P - p38 , p38 , P - JNK , JNK , P - p65 , p65 and tubulin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting luciferase (as control) or single clones generated from 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting the spata2 gene were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBα, IkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin.


( C ) TAK1 - / - MEF were infected to express a CRISPR / Cas9 system targeting luciferase or the spata2 gene , from which single clones were generated . The cells were treated with mTNF ( 10 ng / ml ) or mTNF along with Necrostatin - 1 ( Nec - 1 ) for 1 - 3 h as indicated . The blot was sequentially probed with antibodies recognizing PARP , cleaved caspase - 3 and tubulin .

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

(C) TAK1-/-MEF were infected to express a CRISPR/Cas9 system targeting luciferase or the spata2 gene, from which single clones were generated. The cells were treated with mTNF (10 ng/ml) or mTNF along with Necrostatin-1 (Nec-1) for 1-3 h as indicated. The blot was sequentially probed with antibodies recognizing PARP, cleaved caspase-3 and tubulin.


( D ) . The same cells as used in ( C ) were treated as before with TNF ( 10 ng / ml ) or TNF along with Necrostatin - 1 ( Nec - 1 ) for one and 2 h as indicated . Caspase activity was determined using the fluorogenic substrate DEVD - AMC .

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

(D). The same cells as used in (C) were treated as before with TNF (10 ng/ml) or TNF along with Necrostatin-1 (Nec-1) for one and 2 h as indicated. Caspase activity was determined using the fluorogenic substrate DEVD-AMC.


( E ) Cells from the experiment as shown in ( D ) were analyzed for apoptosis by Annexin V staining after 2 h . In ( D ) and ( E ) , a representative experiment is shown with error bars referring to technical replicates .

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

(E) Cells from the experiment as shown in (D) were analyzed for apoptosis by Annexin V staining after 2 h. In (D) and (E), a representative experiment is shown with error bars referring to technical replicates.


( F ) Cells as described before were treated with mTNF ( 10 ng / ml ) for the indicated time and lysates were subjected to immunoprecipitation with an anti - FADD antibody . The immunoprecipitates were subjected to Western blotting and probed for RIPK1 , FADD and actin .

(F) Cells as described before were treated with mTNF (10 ng/ml) for the indicated time and lysates were subjected to immunoprecipitation with an anti-FADD antibody. The immunoprecipitates were subjected to Western blotting and probed for RIPK1, FADD and actin.


Primary 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 .

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

Primary 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 .

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

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

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.


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

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

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


Immunostaining with anti - PEX1 ( red ) or PEX14 ( red ) , anti - PMP70 ( green ) , anti - ninein ( blue ) , 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 ) .

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

Immunostaining with anti-PEX1 (red) or PEX14 (red), anti-PMP70 (green), anti-ninein (blue), and anti-acetylated tubulin (white) in wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells in quiescent G0 phase. Arrowheads indicate primary cilia. Scale bar, 10 μm. Quantification of the number of PMP70-positive peroxisomes per cell from (B). Peroxisome formation in PEX1−/− and PEX14−/− hTERT-RPE1 cells was significantly impaired compared with that of the parental cells (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 20-25 cells per experiment). Quantification of proportion of ciliated cells from (B). Ciliogenesis in PEX1−/− and PEX14−/− hTERT-RPE1 cells was not significantly altered compared with that of the parental cells (mean ± s.d.: one-way ANOVA with Tukey's HSD, n=3: 190-200 cells per experiment).


Quiescent G0 - phase wild - type , PEX1 − / − , 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 .

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

Quiescent G0-phase wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells transfected with AcGFP1-tagged D4 as a cholesterol probe were immunostained with anti-pericentrin (white) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (red). Arrows and arrowheads indicate primary cilia and cytosolic accumulations of AcGFP1-tagged D4, respectively. Scale bar, 5 μm. Quantification of the Filipin III intensity at primary cilia from (E). PEX1−/− and PEX14−/− hTERT-RPE1 cells had significant reductions in the ciliary signal of Filipin III (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively. Quantification of the AcGFP1-tagged D4 intensity at primary cilia from (E). The ciliary signal of AcGFP1-tagged D4 in PEX1−/− and PEX14−/− hTERT-RPE1 cells was significantly diminished (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 25-30 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Quiescent G0 - phase wild - type , PEX1 − / − , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quiescent G0-phase wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells were treated with or without 1.5% methyl-β-cyclodextrin for 45 min, and then incubated with or without cholesterol (cholesterol/methyl-β-cyclodextrin complex) for 1 h. After removing exogenous cholesterol, they were stimulated with 50 nM Shh-N for 24 h in the presence of pravastatin, and then immunostained with anti-Smo (green), anti-acetylated-tubulin (blue), and anti-γ-tubulin (red) antibodies. For the alternative cholesterol complementation, LDL (0.06 mg/ml) was co-incubated with Shh-N and pravastatin for 24 h after methyl-β-cyclodextrin-mediated cholesterol depletion. Scale bar, 2.5 μm. Quantification of the Smo intensity at primary cilia in wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells from (H). PEX1−/− and PEX14−/− hTERT-RPE1 cells exhibited the dampened Shh-N ligand-induced ciliary accumulation of Smo. The complementation of exogenous cholesterol (cholesterol/methyl-β-cyclodextrin complex) restored the ciliary accumulation of Smo in both PEX-knockout cells, while the LDL complementation did not rescue the ciliary phenotypes efficiently (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 90-100 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Quiescent G0 - phase hTERT - RPE1 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 .

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

Quiescent G0-phase hTERT-RPE1 cells were immunostained with anti-PEX14 (red), anti-ninein (white), and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (Green). Magnified images of the boxed regions showing peroxisome accompanied by cholesterol (arrows). Three-dimensional reconstitution of the same cell indicates that Filipin III stains the membrane regions of ciliary axonemes and peroxisomes. The scale bars indicate 2.5 μm and 1.25 μm in lower- and higher-magnified images, respectively.


Quiescent G0 - phase wild - type hTERT - RPE1 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 ) .

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

Quiescent G0-phase wild-type hTERT-RPE1 cells treated with Cytochalasin-D (200 nM), colcemid (50 nM), or Ciliobrevin-D (10 μM) for 6 h were immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ninein (green), and anti-PMP70 (white) antibodies. Arrows indicate the peroxisomes interacting with the ciliary pocket. Scale bar, 5 μm. Quantification of proportion of primary cilia interacting with peroxisomes from (B). Colcemid (50 nM) and Ciliobrevin-D (10 μM) significantly inhibited the spatial interaction between peroxisomes and primary cilia (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment).


Quiescent G0 - phase wild - type hTERT - RPE1 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 .

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

Quiescent G0-phase wild-type hTERT-RPE1 cells were treated with Cytochalasin-D (200 nM), Colcemid (50 nM), or Ciliobrevin-D (10 μM) for 6 h and then immunostained with anti-acetylated tubulin (blue) and anti-pericentrin (green) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 2.5 μm. Quantification of the Filipin III intensity at primary cilia from (D). Colcemid and Ciliobrevin-D interfered with the distribution of cholesterol in the ciliary membrane (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


hTERT - RPE1 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 .

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

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 .

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

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


Recombinant GST ( 0 . 2 μg ) or GST fused to PEX14 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 .

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

3D reconstitution of the quiescent G0-phase hTERT-RPE1 cells transfected with AcGFP1-tagged EHD3 (white) immunostained with anti-acetylated-tubulin (blue) and anti-PEX14 (red) antibodies. Cholesterol was stained with Filipin III (green). Cholesterol-containing peroxisome interacted with the ciliary pocket (arrow). The scale bars indicate 2 μm.


Maximum projection of z - stack sections of hTERT - RPE1 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 .

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


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 .

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


Single FIB - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


ORP3 + / + 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 .

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

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 ) .

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 .

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

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

ORP3−/− hTERT-RPE1 cells were transfected with AcGFP1-tagged ORP3, PH domain deleted-ORP3 mutant (ΔPH), FFAT motif-deleted-ORP3 mutant (ΔFFAT), or OHD domain deleted-ORP3 mutant (ΔOHD) and cultured without serum for 24 h before Filipin-III (green)-mediated cholesterol staining and immunostaining with anti-GFP (red), anti-pericentrin (white), and anti-acetylated-tubulin (blue) antibodies. Arrows represent AcGFP1-tagged ORP3 or the mutants localized to the ciliary pocket. AcGFP1-tagged ORP3ΔPH mutant mis-localized to the ciliary pocket. The scale bars indicate 5 μm. Quantification of the Filipin III intensity at primary cilia from (H). AcGFP1-tagged ORP3 deletion mutants did not restore the ciliary cholesterol insufficiency in the ORP3−/− hTERT-RPE1 cells (*p<0.05, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Rabin8 + / + 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 .

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

Rabin8+/+ and Rabin8−/− hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Scale bar, 5 μm. Quantification of the Filipin III intensity at primary cilia from (A). Rabin8−/− hTERT-RPE1 cells exhibited a significant reduction of ciliary cholesterol (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Quiescent G0 - phase Rabin8 + / + 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 ) .

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

Quiescent G0-phase 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 .

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

Quiescent G0-phase hTERT-RPE1 cells 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 .

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

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

Rabin8−/− hTERT-RPE1 cells were transfected with AcGFP1-tagged Rabin8, GFP-tagged Rab8A, GFP-tagged Rab8A-Q67L (constitutively active form), GFP-Rab8B, GFP-Rab8B-Q67L (constitutively active form), AcGFP1-tagged Rab10, or AcGFP1-tagged Rab10-Q68L (constitutively active form), and then immunostained with anti-GFP (red), anti-acetylated-tubulin (blue), and anti-ninein (white) antibodies. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm. Quantification of (I) showing that AcGFP1-tagged Rabin8 and AcGFP1-tagged Rab10-Q68L effectively restored the defect in ciliary enrichment of cholesterol in Rabin8−/− hTERT-RPE1 cells (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Whole - 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 .

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

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


Whole - 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 .

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

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 .

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 .

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

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 .

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

Quiescent G0-phase hTERT-RPE1 cells 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 .

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 .

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

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 ) .

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

KIFC3−/− hTERT-RPE1 cells were transfected with AcGFP1, AcGFP1-tagged KIFC3, rod domain-deleted KIFC3 mutant (ΔRod), or motor domain-deleted KIFC3 mutant (ΔMotor) and cultured without serum for 24 h before Filipin-III (green)-mediated cholesterol staining and immunostaining with anti-GFP (red), anti-pericentrin (white), and anti-acetylated-tubulin (blue) antibodies. Arrows represent ciliary localization of cholesterol. The scale bars indicate 5 μm. Quantification of the Filipin III intensity at primary cilia from (K). AcGFP1-tagged KIFC3 deletion mutants did not restore the ciliary cholesterol insufficiency in the KIFC3−/− hTERT-RPE1 cells (mean +/- s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Quiescent G0 - phase wild - type , Rab10 − / − , KIFC3 − / − , ORP3 − / − , 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 .

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

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


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

Quantification of the Filipin III intensity at primary cilia from (A). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10−/−and KIFC3−/−hTERT-RPE1 cells, but not in ORP3−/−and EHD3−/− hTERT-RPE1 cells (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


( B ) Example of bead survival distributions for 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 )

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


( D ) Off - rates under force for additional pMHCs interacting with 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 )

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

(D) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( A ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( B ) 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 .

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

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


( C ) 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 ) .

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


( F ) Experimental value of P ( symbols ) over antigen affinity fitted by 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 O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


F . Influence of mutating σ4 on promoter recognition .

F. Influence of mutating σ4 on promoter recognition.


B , C . Effects of deleting β ′ ZBD on transcriptional activities of RNAP on different promoters . 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 .

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


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

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


E . Binding of σ28 - RNAP with fliCp as detected by EMSA ( left ) . 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 ) .

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

E. 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 .

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


B , C . Promoter activities of tarp ( B ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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

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


E . Relative mRNA 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 .

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


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

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