C ) Z - score distribution per experimental group for genes within APPtg - Blue module . Boxplots : center , median ; box limits , 25th - 75th quartiles ; whiskers , 1 . 5x interquartile range . Empirical p - values are Bonferroni adjusted ( * * * pbonf < 0 . 001 ) indicate significant shift in z - score

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C) Z-score distribution per experimental group for all genes within the APPtg-Blue module. Boxplots: center line, median; box limits, 25th-75th quartiles; whiskers, 1.5x interquartile range. Empirical p-values are Bonferroni adjusted (***pbonf<0.001) and indicate significant shift in z-score distribution


A ) UMAP clustering of sorted Cd11b + / Cd45 + myeloid from mouse hippocampus . ARM : activated response microglia ; CAM : CNS - associated macrophages ; CPM : cycling proliferating microglia ; HM . 1 : homeostatic microglia cluster 1 ; HM . 2 : homeostatic microglia cluster 2 ; IRM : interferon - response microglia ; MHC . high : high MHC - expressing microglia ; Mnc : monocytes ; TRM : transitioning microglia .

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A) UMAP clustering of sorted Cd11b+/Cd45+ myeloid cells from mouse hippocampus. ARM: activated response microglia; CAM: CNS-associated macrophages; CPM: cycling and proliferating microglia; HM.1: homeostatic microglia cluster 1; HM.2: homeostatic microglia cluster 2; IRM: interferon-response microglia; MHC.high: high MHC-expressing microglia; Mnc: monocytes; TRM: transitioning microglia.


B ) Distribution of across different clusters per experimental group ( n = 2 mice pooled for experimental groups , except for TAUtg - 11M TAUwt - 11M with n = 3 mice pooled ) .

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B) Distribution of cells across the different clusters per experimental group (n=2 mice pooled for all experimental groups, except for TAUtg-11M and TAUwt-11M with n=3 mice pooled).


C ) Distribution of per experimental group over different clusters , expressed in percentages as stated on top of each bar . E . g . of within APPtg - 11M , 56 . 7 % of are ARM , while 12 . 5 % are HM . 1 .

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C) Distribution of cells per experimental group over the different clusters, expressed in percentages as stated on top of each bar. E.g. of all cells within APPtg-11M, 56.7% of cells are ARM, while 12.5% are HM.1 cells.


E ) Differential expression of ARM vs HM . 1 microglia for APPtg TAUtg mice separately . Genes with a positive log ( fold change ) ( LFC ) are higher expressed in ARMs compared to HM . 1 microglia . As can be seen which genes are more highly expressed in ARMs vs HM . 1 are highly similar among APPtg TAUtg mice ( Spearman correlation r = 0 . 91 , p = 2 . 2e - 16 ) . LFC > | 0 . 2 | with padj < 0 . 05 are considered significant .

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E) Differential expression of ARM vs HM.1 microglia for APPtg and TAUtg mice separately. Genes with a positive log(fold change) (LFC) are higher expressed in ARMs compared to HM.1 microglia. As can be seen which genes are more highly expressed in ARMs vs HM.1 cells are highly similar among APPtg and TAUtg mice (Spearman correlation r=0.91, p=2.2e-16). LFC > |0.2| with padj<0.05 are considered significant.


A , B In - gel kinase of OST1 activity in ABA receptor mutants under cold stress . Ten - day - old wild type , ost1 - 3 , pyr1 pyl1 , 4 , pyr1 pyl1 , 2 , 4 , pyr1 pyl4 , 5 , 8 pyr1 pyl1 , 4 , 5 , 8 mutants were treated at 4 ° C for 2 h . Total extracts were prepared separated on SDS - PAGE gel containing 0 . 1 mg / mL GST - ∆ ABF2 as a substrate , incubated with 60 μCi of [ γ - 32P ] ATP . Representative pictures are shown in ( A ) relative kinase activity is shown in ( B ) . Data information : In ( A , Top , gel autoradiograph ; bottom , Coomassie Brilliant Blue ( CBB ) of RuBisCO large subunit was used as a loading control . of band of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 . In ( B , each bar represents mean ± of three biological repeats . * P < 0 . 05 ( two - t - ) .

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A, B In-gel kinase assay of OST1 activity in ABA receptor mutants under cold stress. Ten-day-old wild type, ost1-3, pyr1 pyl1,4, pyr1 pyl1,2,4, pyr1 pyl4,5,8 and pyr1 pyl1,4,5,8 mutants were treated at 4°C for 2 h. Total protein extracts were prepared and separated on SDS-PAGE gel containing 0.1 mg/mL GST-∆ABF2 as a substrate, and incubated with 60 μCi of [γ-32P]ATP. Representative pictures are shown in (A) and relative kinase activity is shown in (B). Data information: In (A , Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (B , each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).


C , D In - gel kinase of OST1 activity in ABI1 - OE14 abi1 abi2 hab1 mutants under cold stress . Representative pictures are shown in ( C ) relative kinase activity is shown in ( D ) . Data information : In C ) , Top , gel autoradiograph ; bottom , Coomassie Brilliant Blue ( CBB ) of RuBisCO large subunit was used as a loading control . of band of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 . In D ) , each bar represents mean ± of three biological repeats . * P < 0 . 05 ( two - t - ) .

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C, D In-gel kinase assay of OST1 activity in ABI1-OE14 and abi1 abi2 hab1 mutants under cold stress. Representative pictures are shown in (C) and relative kinase activity is shown in (D). Data information: In C), Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In D), each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).


A Interaction of OST1 EGR2 in yeast . Yeast harboring different vector combinations were grown on SC / − Leu / − Trp medium for 3 d or SC / − Leu / − Trp / − His / − Ade medium for 6 d . OST1 - BD / AD BD / EGR2 - AD were used as negative controls .

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A Interaction of OST1 and EGR2 in yeast. Yeast cells harboring different vector combinations were grown on SC/−Leu/−Trp medium for 3 d or SC/−Leu/−Trp/−His/−Ade medium for 6 d. OST1-BD/AD and BD/EGR2-AD were used as negative controls.


B OST1 interacts with EGR2 in vitro . Purified recombinant MBP - His - EGR2 or MBP - His from E . coli were immunoprecipitated with MBP beads , then incubated with purified recombinant GST - OST1 . Precipitated were detected with anti - His anti - GST antibodies .

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B OST1 interacts with EGR2 in vitro. Purified recombinant MBP-His-EGR2 or MBP-His proteins from E. coli were immunoprecipitated with MBP beads, and then incubated with purified recombinant GST-OST1. Precipitated proteins were detected with anti-His and anti-GST antibodies.


C Co - IP of OST1 with EGR2 in vivo . 35S : HF - OST1 / Super : EGR2 - Myc or 35S : HF - OST1 / Super : Myc was expressed in N . benthamiana leaves . Total were immunoprecipitated with anti - Myc agarose beads , co - immunoprecipitation products were subjected to immunoblot . EGR2 - Myc HF - OST1 were detected with anti - Myc anti - HA antibodies , respectively .

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C Co-IP assay of OST1 with EGR2 in vivo. 35S:HF-OST1/Super:EGR2-Myc or 35S:HF-OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-Myc agarose beads, and the co-immunoprecipitation products were subjected to immunoblot analysis. EGR2-Myc and HF-OST1 were detected with anti-Myc and anti-HA antibodies, respectively.


D Bimolecular complementation ( BiFC ) . Wild - type Arabidopsis protoplasts transformed with OST1 - YFPC EGR2 - YFPN were incubated for 18 h . interaction signal was detected by confocal microcopy . combinations of OST1 - YFPC / GUS - YFPN GUS - YFPC / EGR2 - YFPN were used as negative controls . Scale bars : 100 μm .

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D Bimolecular fluorescence complementation (BiFC) assay. Wild-type Arabidopsis protoplasts transformed with OST1-YFPC and EGR2-YFPN proteins were incubated for 18 h. The interaction signal was detected by confocal microcopy. The combinations of OST1-YFPC/GUS-YFPN and GUS-YFPC/EGR2-YFPN were used as negative controls. Scale bars: 100 μm.


E EGR2 inhibits OST1 kinase activity in vitro . were incubated with 1 μCi [ γ - 32P ] ATP in kinase reaction buffer for 30 min at 30 ° C , then separated by SDS - PAGE . Top , autoradiograph ; bottom , CBB .

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E EGR2 inhibits OST1 kinase activity in vitro. Proteins were incubated with 1 μCi [γ-32P]ATP in kinase reaction buffer for 30 min at 30°C, and then separated by SDS-PAGE. Top, autoradiograph; bottom, CBB staining.


F , G In - gel of OST1 activity in 10 - d - old wild type , egr1 egr2 single mutants , egr1 egr2 double mutant under cold stress . Data information : of band of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 . In ( F representative pictures are shown : Top , autoradiograph ; bottom , CBB . In ( G relative kinase activity is shown . Each bar represents mean ± of three biological repeats . * * P < 0 . 01 ( two - t - ) .

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F, G In-gel assay of OST1 activity in 10-d-old wild type, egr1 and egr2 single mutants, and the egr1 egr2 double mutant under cold stress. Data information: the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (F representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In (G relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


H , I In - gel of OST1 activity in 10 - d - old wild - type EGR2 - overexpressing under cold stress . Data information of band of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 . In H ) , representative pictures are shown : Top , autoradiograph ; bottom , CBB . In I ) , relative kinase activity is shown . Each bar represents mean ± of three biological repeats . * * P < 0 . 01 ( two - t - ) .

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H, I In-gel assay of OST1 activity in 10-d-old wild-type and EGR2-overexpressing plants under cold stress. Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


Freezing phenotype ( A ) of egr2 - 3 mutant egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) .

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Freezing phenotype (A) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA).


survival rate ( B ) of egr2 - 3 mutant egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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survival rate (B) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (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).


ion leakage ( C ) of egr2 - 3 mutant egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

ion leakage (C) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (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).


Freezing phenotype of EGR2 - overexpressing . Two - week - old Super : EGR2 - Myc

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Freezing phenotype of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants


survival rate ( E ) of EGR2 - overexpressing . Two - week - old Super : EGR2 - Myc Data information : each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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survival rate (E) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants 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).


ion leakage ( F ) of EGR2 - overexpressing . Two - week - old Super : EGR2 - Myc Data information : each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

ion leakage (F) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants 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).


Expression of CBFs ( G in egr2 - 3 mutant . Two - week - old seedlings were treated at 4 ° C for indicated period subjected to qRT - PCR . Data information : each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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Expression of CBFs (G in egr2-3 mutant plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. 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).


Expression of CBFs target genes ( H in egr2 - 3 mutant . Two - week - old seedlings were treated at 4 ° C for indicated period subjected to qRT - PCR . Data information : each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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Expression of CBFs target genes (H in egr2-3 mutant plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. 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).


Expression of CBFs in EGR2 - overexpressing . Two - week - old seedlings were treated at 4 ° C for indicated period subjected to qRT - PCR . Data information : each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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Expression of CBFs in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. 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).


Expression of CBFs target genes J ) in EGR2 - overexpressing . Two - week - old seedlings were treated at 4 ° C for indicated period subjected to qRT - PCR . Data information : each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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Expression of CBFs target genes J) in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. 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).


N - myristoylation of EGR2 EGR2G2A in N . benthamiana leaves . extracts prepared from N . benthamiana leaves expressing EGR2 - Myc or EGR2G2A - Myc were immunoprecipated by anti - Myc agarose beads . Precipitated were detected by anti - Myc anti - myristic acid antibodies .

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N-myristoylation of EGR2 and EGR2G2A in N. benthamiana leaves. Protein extracts prepared from N. benthamiana leaves expressing EGR2-Myc or EGR2G2A-Myc were immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies.


D Immunoblot showing that total level of myristoylated in wild type . Total were prepared from two - week - old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22 ° C for 72 h then subjected to click - reaction with biotin azide . reaction was protected from at room for 3 h . products were detected by anti - streptavidin - HRP antibody .

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D Immunoblot analysis showing that the total level of myristoylated proteins in wild type plants. Total proteins were prepared from two-week-old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22°C for 72 h and then subjected to click-reaction with the biotin azide. The reaction was protected from light at room temperature for 3 h. The products were detected by anti-streptavidin-HRP antibody.


E N - myristoylation of EGR2 in . Total were prepared from two - week - old EGR2 - Myc seedlings products were immunoprecipated by anti - Myc agarose beads detected by anti - streptavidin - HRP antibody . EGR2 - Myc detected by anti - Myc antibody was used as a loading control .

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E N-myristoylation of EGR2 in plants. Total proteins were prepared from two-week-old EGR2-Myc seedlings The products were immunoprecipated by anti-Myc agarose beads and detected by anti-streptavidin-HRP antibody. EGR2-Myc detected by anti-Myc antibody was used as a loading control.


F Immunoblot of myristoylated EGR2 under cold stress . Total were prepared form EGR2 - Myc treated with 4 ° C for indicated period , immunoprecipated by anti - Myc agarose beads . Precipitated were detected by anti - Myc anti - myristic acid antibodies . of band detected by anti - myristic acid antibody to anti - Myc antibody without cold treatment was set to 1 . 00 . Representative pictures are shown ( left ) quantitative is shown ( right ) . Each bar represents mean ± of three biological repeats . * * P < 0 . 01 ( two - t - ) .

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F Immunoblot assay of myristoylated EGR2 under cold stress. Total proteins were prepared form EGR2-Myc plants treated with 4°C for the indicated period, and immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies. The ratio of band intensity detected by anti-myristic acid antibody to anti-Myc antibody without cold treatment was set to 1.00. Representative pictures are shown (left) and quantitative analysis is shown (right). Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


G Expression of EGR2 in egr2 - 3 EGR2 : EGR2G2A - Myc complementation transgenic . Total RNAs were extracted from 2 - week - old seedlings subjected to RT - PCR . EF1α was used as a loading control .

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G Expression of EGR2 in egr2-3 EGR2:EGR2G2A-Myc complementation transgenic plants. Total RNAs were extracted from 2-week-old seedlings and subjected to RT-PCR analysis. EF1α was used as a loading control.


Freezing tolerance assays of egr2 - 3 mutant EGR2 : EGR2G2A - Myc transgenic in egr2 - 3 mutant . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar at 22 ° C were treated at − 5 ° C for 0 - 1 . 5 h ( NA ) or − 8 ° C for 0 - 1 . 5 h ( CA ) . Representative ( - 5oC , 0 . 5 h ; - 8oC , 0 . 5 h ) are shown in ( H )

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Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). Representative photographs (-5oC, 0.5 h; -8oC, 0.5 h) are shown in (H)


Freezing tolerance assays of egr2 - 3 mutant EGR2 : EGR2G2A - Myc transgenic in egr2 - 3 mutant . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar at 22 ° C were treated at − 5 ° C for 0 - 1 . 5 h ( NA ) or − 8 ° C for 0 - 1 . 5 h ( CA ) . survival rate is shown in ( I ) . Data shown in ( I ) are mean values ± of three biological replicates , each of which has three technical replicates . Asterisks indicate significant differences compared with wild type for same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) ; n . s . , not significant .

O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). survival rate is shown in (I). Data shown in (I) are mean values ± SEM of three biological replicates, each of which has three technical replicates. Asterisks indicate significant differences compared with wild type for the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test); n. s., not significant.


A In vitro pull - down showing interaction of NMT1 EGR2 . Recombinant GST - NMT1 was incubated with immunoprecipated MBP - His - EGR2 or MBP - His at 4 ° C for 2 h . Anti - GST anti - MBP were used to detect NMT1 , EGR2 MBP - His , respectively .

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A In vitro pull-down assay showing the interaction of NMT1 and EGR2. Recombinant GST-NMT1 was incubated with immunoprecipated MBP-His-EGR2 or MBP-His at 4°C for 2 h. Anti-GST and anti-MBP were used to detect NMT1, EGR2 and MBP-His, respectively.


Co - localization of NMT1 EGR2 in N . benthamiana leaves . Scale bar : 50

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Co-localization of NMT1 and EGR2 in N. benthamiana leaves. Scale bar: 50 μm


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

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

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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 . EF1α was used as a loading control .

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


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

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

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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 under cold stress . Top , autoradiograph ; bottom , CBB . Representative pictures are shown in ( I ) In ( I ) : of band of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 .

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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 under cold stress . relative kinase activity is shown in ( J ) . In ( J ) : each bar represents mean ± of three independent experiments ( * * P < 0 . 01 , two t - ) .

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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 . Total prepared from EGR2 - Myc or EGR2G2A - Myc transgenic was immunoprecipated with anti - Myc agarose beads , then incubated with GST - OST1 . Anti - Myc anti - GST antibody were used to detect GST - OST1 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 mean ± of three independent experiments ( * * P < 0 . 01 , two - t - ) .

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

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

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

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

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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 , 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 ) .

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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 ion leakage ( B ) of wild type , ost1 - 3 , egr2 - 3 , 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 mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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

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

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(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 CYLD was purified by FLAG - IP . blot was probed with antibodies recognizing CYLD , SPATA2 actin .

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

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(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 were transfected with a vector encoding V5 - tagged full 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 , blot was probed with antibodies recognizing HOIP , CYLD , V5 actin .

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(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 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 , blot was probed with antibodies recognizing FLAG , CYLD , V5 tubulin .

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

O O O O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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 were transfected with empty vector , FLAG - SPATA2 or FLAG - HOIP . After FLAG - IP , blot was probed with antibodies recognizing OTULIN , CYLD , FLAG actin .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 were transfected with empty vector , V5 - tagged SPATA2 ( wt ) or a V5 - tagged PUB domain mutant PUBmut of SPATA2 . After V5 - IP , blot was probed with antibodies recognizing CYLD , FLAG , V5 tubulin .

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 B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR 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 were treated with FLAG - TNF ( 2µg / ml ) for indicated time . purified TNF - RSC was sequentially probed with antibodies recognizing SPATA2 , CYLD , RIPK1 , TRADD , SHARPIN , TNF tubulin as indicated .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR 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 were infected to express a CRISPR / Cas9 system targeting luciferase ( as control , indicated wt ) or spata2 gene ( ko ) . Mixed cultures were treated with FLAG - huTNF ( 2µg / ml ) for indicated time . purified TNF - RSC was sequentially probed with antibodies recognizing SPATA2 , CYLD , HOIP , SHARPIN , TRADD , TNF 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 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 B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR 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 spata2 gene ( ko ) . Single clones were generated , which were treated with FLAG - TNF ( 2µg / ml ) for indicated time . purified TNF - RSC was sequentially probed with antibodies recognizing M1 - linked ubiquitin , CYLD , RIPK1 , tubulin as indicated .

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

(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 , purified by FLAG - IP . Purified CYLD or inactive mutant were added to recombinant di - ubiquitin for 0 , 20 or 40 min at 37 ° C as indicated . After electrophoresis , gel was cut lower part was silver stained to visualize mono - di - ubiquitin , while upper part was transferred to a probed for FLAG V5 . In ( A ) , experiment was done with M1 - linked di - ubiquitin substrate , in ( B ) K63 - linked di - ubiquitin substrate was subjected to , in ( C ) K48 - linked di - ubiquitin substrate was analyzed .

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

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 spata2 gene ( ko ) were treated with mTNF ( 10 ng / ml ) as indicated . blots were probed with antibodies recognizing P - IkBαIkBα , P - p38 , p38 , P - JNK , JNK , P - p65 , p65 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 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 B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR 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 expressing CRISPR / Cas9 targeting luciferase ( as control ) or single clones generated from 15P - 1 Sertoli expressing CRISPR / Cas9 targeting spata2 gene were treated with mTNF ( 10 ng / ml ) as indicated . blots were probed with antibodies recognizing P - IkBα , IkBα , P - p38 , p38 , P - JNK , JNK , P - p65 , p65 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 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 B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR 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 spata2 gene , from which single clones were generated . were treated with mTNF ( 10 ng / ml ) or mTNF along with Necrostatin - 1 ( Nec - 1 ) for 1 - 3 h as indicated . blot was sequentially probed with antibodies recognizing PARP , caspase - 3 tubulin .

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

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 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

(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 ) from experiment as shown in ( D ) were analyzed for apoptosis by Annexin V after 2 h . In ( D ) ( 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

(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 ) as described before were treated with mTNF ( 10 ng / ml ) for indicated time lysates were subjected to immunoprecipitation with an anti - FADD antibody . immunoprecipitates were subjected to Western blotting probed for RIPK1 , FADD 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 B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O

(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.


skin fibroblasts from a normal , SLOS , ZS , X - ALD , NPC incubated for 24 h without serum were immunostained with anti - pericentrin ( red ) anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( green ) . Arrows indicate cilia . Scale bar , 5 μm . of Filipin III signal at cilia from ( a ) was remarkably reduced in SLOS ZS ( * * p < 0 . 01 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of skin fibroblasts in quiescent G0 phase treated with 50 nM Shh - N for 24 h using anti - Smo ( green ) , anti - acetylated - tubulin ( blue ) , anti - γ - tubulin ( red ) antibodies . Arrows indicate cilia . Scale bar , 2 . 5 μm . of proportion of Smo - positive ciliated upon Shh - N stimulation from ( C ) . populations of Smo - positive ciliated fibroblasts from SLOS ZS were significantly reduced ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 100 per experiment ) . Smo at cilia in fibroblasts from ( C ) . ciliary localization of Smo upon Shh - N stimulation was significantly impaired in fibroblasts from SLOS ZS ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 90 - 100 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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

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.


fibroblasts from a normal , SLOS ZS were treated with 1 . 5 % methyl - β - cyclodextrin for 45 min to remove cholesterol , then incubated with or without cholesterol ( cholesterol / methyl - β - cyclodextrin ) for 1 h . After washing out exogenous cholesterol , fibroblasts were stimulated with 50 nM Shh - N for 24 h in presence of pravastatin , then immunostained with anti - Smo ( green ) , anti - acetylated - tubulin ( blue ) , anti - pericentrin ( red ) antibodies . For alternative cholesterol complementation , LDL ( 0 . 06 mg / ml ) was co - incubated with Shh - N pravastatin for 24 h after methyl - β - cyclodextrin - mediated cholesterol depletion . Scale bar , 2 . 5 μm . of Smo at cilia in fibroblasts from ( F ) . ciliary accumulation of Smo upon Shh - N stimulation of fibroblasts from an SLO was restored by complementation of both exogenous cholesterol / methyl - β - cyclodextrin LDL , while that of from ZS was efficiently rescued by not LDL but cholesterol / methyl - β - cyclodextrin ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 90 - 100 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 showing depletion of PEX1 PEX14 normal processing of Shh in PEX1 − / − PEX14 − / − hTERT - RPE1 clones . GAPDH served as a loading control .

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

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


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

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

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


Quiescent G0 - phase wild - type , PEX1 − / − , PEX14 − / − hTERT - RPE1 transfected with AcGFP1 - tagged D4 as a cholesterol probe were immunostained with anti - pericentrin ( ) anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( red ) . Arrows arrowheads indicate cilia accumulations of AcGFP1 - tagged D4 , respectively . Scale bar , 5 μm . of Filipin III at cilia from ( E ) . PEX1 − / − PEX14 − / − hTERT - RPE1 had significant reductions in ciliary signal of Filipin III ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively . of AcGFP1 - tagged D4 at cilia from ( E ) . ciliary signal of AcGFP1 - tagged D4 in PEX1 − / − PEX14 − / − hTERT - RPE1 was significantly diminished ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 25 - 30 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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

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


Quiescent G0 - phase wild - type , PEX1 − / − , PEX14 − / − hTERT - RPE1 were treated with or without 1 . 5 % methyl - β - cyclodextrin for 45 min , then incubated with or without cholesterol ( cholesterol / methyl - β - cyclodextrin ) for 1 h . After removing exogenous cholesterol , they were stimulated with 50 nM Shh - N for 24 h in presence of pravastatin , then immunostained with anti - Smo ( green ) , anti - acetylated - tubulin ( blue ) , anti - γ - tubulin ( red ) antibodies . For alternative cholesterol complementation , LDL ( 0 . 06 mg / ml ) was co - incubated with Shh - N pravastatin for 24 h after methyl - β - cyclodextrin - mediated cholesterol depletion . Scale bar , 2 . 5 μm . of Smo at cilia in wild - type , PEX1 − / − , PEX14 − / − hTERT - RPE1 from ( H ) . PEX1 − / − PEX14 − / − hTERT - RPE1 exhibited dampened Shh - N ligand - induced ciliary accumulation of Smo . complementation of exogenous cholesterol ( cholesterol / methyl - β - cyclodextrin ) restored ciliary accumulation of Smo in both PEX - knockout , while LDL complementation did not rescue ciliary efficiently ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 90 - 100 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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

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


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

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

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


Quiescent G0 - phase wild - type hTERT - RPE1 treated with Cytochalasin - D ( 200 nM ) , colcemid ( 50 nM ) , or Ciliobrevin - D ( 10 μM ) for 6 h were immunostained with anti - ARL13B ( blue ) , anti - phospho - S473 - Akt ( red ) , anti - ninein ( green ) , anti - PMP70 ( ) antibodies . Arrows indicate peroxisomes interacting with ciliary pocket . Scale bar , 5 μm . of proportion of cilia interacting with peroxisomes from ( B ) . Colcemid ( 50 nM ) Ciliobrevin - D ( 10 μM ) significantly inhibited spatial interaction between peroxisomes cilia ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 per experiment ) .

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

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


Quiescent G0 - phase wild - type hTERT - RPE1 were treated with Cytochalasin - D ( 200 nM ) , Colcemid ( 50 nM ) , or Ciliobrevin - D ( 10 μM ) for 6 h then immunostained with anti - acetylated tubulin ( blue ) anti - pericentrin ( green ) antibodies . Cholesterol was stained with Filipin III ( green ) . Arrows indicate cilia . Scale bar , 2 . 5 μm . of Filipin III at cilia from ( D ) . Colcemid Ciliobrevin - D interfered with distribution of cholesterol in ciliary ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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

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


hTERT - RPE1 were transfected with AcGFP1 - tagged EHD3 cultured without serum for 24 h before immunostaining with anti - GFP ( green ) , anti - ARL13B ( blue ) , anti - PMP70 ( ) , anti - phospho - S473 - Akt ( red ) antibodies . scale bars represent 2 . 5 μm .

O O O O 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 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

hTERT-RPE1 cells were transfected with AcGFP1-tagged EHD3 and cultured without serum for 24 h before immunostaining with anti-GFP (green), anti-ARL13B (blue), anti-PMP70 (white), and anti-phospho-S473-Akt (red) antibodies. The scale bars represent 2.5 μm.


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

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

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


Recombinant GST ( 0 . 2 μg ) or GST fused to PEX14 ( 1 μg ) 6 × His - EHD3 ( 1 μg ) were pulled down using glutathione - Sepharose beads . GST - tagged PEX14 6 × His - tagged EHD3 in pull - down fractions inputs were detected by western blotting .

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

Recombinant GST (0.2 μg) or GST fused to PEX14 proteins (1 μg) and 6×His-EHD3 protein (1 μg) were pulled down using glutathione-Sepharose beads. GST-tagged PEX14 and 6×His-tagged EHD3 proteins in the pull-down fractions and inputs were detected by western blotting.


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

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O 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 expressing MCHR1 - GFP ( blue ) , DsRed2 - PACT ( red ) ECFP - SKL ( green ) 48 h after serum starvation . ECFP - SKL - positive with cilia , indicated by red box , was analyzed by FIB - . Top , differential interference contrast ( DIC ) ; bottom , merged image . Scale bar , 10 µm .

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

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


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

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


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

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

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

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


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

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

ORP3+/+ and ORP3−/− hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 5 μm. Quantification of the Filipin III intensity at primary cilia from (C). ORP3−/− hTERT-RPE1 cells exhibited a significant reduction of ciliary cholesterol (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


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

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

Three-dimensional reconstitution of the quiescent G0-phase ORP3+/+ and ORP3−/− hTERT-RPE1 cells immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ORP3 (green), and anti-PMP70 (white) antibodies indicates that ORP3 at the ciliary pocket (arrow and arrowhead) mediates the membrane regions of ciliary pocket and peroxisomes (arrowhead). The scale bars indicate 5 μm. Quantification of proportion of primary cilia interacting with peroxisomes from (E). Depletion of ORP3 significantly interfered with the spatial interaction between peroxisomes and primary cilia (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment).


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

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

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

B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 + / + Rabin8 − / − hTERT - RPE1 were immunostained with anti - ARL13B ( blue ) , anti - phospho - S473 - Akt ( red ) , anti - ninein ( green ) , anti - PMP70 ( ) antibodies . Arrow indicates peroxisomes contacting ciliary pocket . Scale bar , 5 μm . of ( C ) indicating that disruption of Rabin8 gene significantly inhibited spatial interaction between peroxisomes cilia ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 per experiment ) .

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

Quiescent G0-phase Rabin8+/+ and Rabin8−/− hTERT-RPE1 cells were immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ninein (green), and anti-PMP70 (white) antibodies. Arrow indicates the peroxisomes contacting the ciliary pocket. Scale bar, 5 μm. Quantification of (C) indicating that disruption of the Rabin8 gene significantly inhibited the spatial interaction between peroxisomes and primary cilia (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment).


Quiescent G0 - phase hTERT - RPE1 transiently expressing PEX3 - GFP - FRB ( green ) tdTomato - BicD2 - FKBP ( red ) treated with rapamycin were imaged live for 10 min by confocal microscopy ( Movie EV10 ) . Scale bar , 5 μm .

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

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

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.