Anti - Ly6G treatment led to impaired fracture healing as shown by the reduced % callus mineralization at day 28 after surgery and representative micro - CT images . Anti - Ly6G treatment led to delayed mineralization and remodeling of the fracture callus compared to control . Data are presented as mean ± SEM . * * * P = 0 . 0006 by unpaired two - sided t - test . Scale Bar , 2 mm .

Anti-Ly6G treatment led to impaired fracture healing as shown by the reduced % callus mineralization at day 28 after surgery and representative micro-CT images. Anti-Ly6G treatment led to delayed mineralization and remodeling of the fracture callus compared to control. Data are presented as mean ± SEM. ***P = 0.0006 by unpaired two-sided t-test. Scale Bar, 2 mm.


Anti - Ly6G treatment depletes local monocytes / macrophages at the fracture site . Counts of positively stained infiltrative F4 / 80 + cells in the adjacent muscle to the fracture site following treatment with control or anti - Ly6G at day 1 and day 7 . Data are presented as mean ± SEM . * * P = 0 . 005 at day 1 and * * P = 0 . 002 at day 7 , and * P = 0 . 013 for control at day 1 versus control at day 7 , by unpaired two - sided t - test .

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Anti-Ly6G treatment depletes local monocytes/macrophages at the fracture site. Counts of positively stained infiltrative F4/80+ cells in the adjacent muscle to the fracture site following treatment with control or anti-Ly6G at day 1 and day 7. Data are presented as mean ± SEM. **P = 0.005 at day 1 and **P = 0.002 at day 7, and *P = 0.013 for control at day 1 versus control at day 7, by unpaired two-sided t-test.


Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils ( Ly6G + , CD11b + cells ) and monocytes / macrophages ( Ly6G − , CD11b + , CD115 + cells ) in the cellular infiltrate . These effects were abrogated by the addition of anti - CCL2 . Neutrophils : FS versus FS + TNF * P = 0 . 015 , FS + TNF versus FS + TNF + anti - CCL2 * P = 0 . 028 , and FS + TNF + anti - CCL2 versus FS + TNF + IgG * P = 0 . 042 by one - way ANOVA followed by Bonferroni ' s multiple comparison test . Monocytes / macrophages : FS versus FS + TNF * * P = 0 . 0063 , FS + TNF versus FS + TNF + anti - CCL2 * * * * P < 0 . 0001 , and FS + TNF + anti - CCL2 v FS + TNF + IgG * P = 0 . 015 by one - way ANOVA followed by Bonferroni ' s multiple comparisons test .

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Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) in the cellular infiltrate. These effects were abrogated by the addition of anti-CCL2. Neutrophils: FS versus FS + TNF *P = 0.015, FS + TNF versus FS + TNF + anti-CCL2 *P = 0.028, and FS + TNF + anti-CCL2 versus FS + TNF + IgG *P = 0.042 by one-way ANOVA followed by Bonferroni's multiple comparison test. Monocytes/macrophages: FS versus FS + TNF **P = 0.0063, FS + TNF versus FS + TNF + anti-CCL2 ****P < 0.0001, and FS + TNF + anti-CCL2 v FS + TNF + IgG *P = 0.015 by one-way ANOVA followed by Bonferroni's multiple comparisons test.


Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch . " Neat " indicates level of CCL2 in fracture supernatant before injection into air pouch . Data are presented as mean ± SEM . FS versus FS + TNF * * P = 0 . 0020 by one - way ANOVA with Bonferroni ' s multiple comparisons test .

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Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch. "Neat" indicates level of CCL2 in fracture supernatant before injection into air pouch. Data are presented as mean ± SEM. FS versus FS + TNF **P = 0.0020 by one-way ANOVA with Bonferroni's multiple comparisons test.


Addition of rhTNF to enriched bone marrow - derived murine neutrophils pre - exposed to fracture supernatant in vitro led to up - regulation of CCL2 production at 1 h . Data are presented as mean ± SEM . No TNF versus TNF 0 . 01 ng / ml * P = 0 . 012 , TNF 0 . 1 ng / ml * * * P < 0 . 0001 , TNF 1 . 0 ng * * * P = 0 . 0001 , TNF 10 ng / ml * * * P < 0 . 0001 , and TNF 100 ng / ml * P = 0 . 023 by one - way ANOVA with Dunnett ' s multiple comparisons test .

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Addition of rhTNF to enriched bone marrow-derived murine neutrophils pre-exposed to fracture supernatant in vitro led to up-regulation of CCL2 production at 1 h. Data are presented as mean ± SEM. No TNF versus TNF 0.01 ng/ml *P = 0.012, TNF 0.1 ng/ml ***P < 0.0001, TNF 1.0 ng ***P = 0.0001, TNF 10 ng/ml ***P < 0.0001, and TNF 100 ng/ml *P = 0.023 by one-way ANOVA with Dunnett's multiple comparisons test.


Treatment with CCR2 antagonist , INCB3344 , led to impaired fracture healing compared to vehicle control as shown by the reduced % callus mineralization at day 28 after operation . Data are presented as mean ± SEM . * P = 0 . 011 unpaired two - sided t - test . Representative micro - CT images are shown . Scale bar , 2 mm .

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Treatment with CCR2 antagonist, INCB3344, led to impaired fracture healing compared to vehicle control as shown by the reduced % callus mineralization at day 28 after operation. Data are presented as mean ± SEM. *P = 0.011 unpaired two-sided t-test. Representative micro-CT images are shown. Scale bar, 2 mm.


rhTNF treatment augmented early phase fracture healing in osteoporotic mice . Treatment with 1 ng rhTNF at the fracture site on days 0 and 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but the same final result at day 28 . Data are presented as mean ± SEM . At day 14 , PBS control versus TNF , * * P = 0 . 0099 ; at day 28 , PBS control versus TNF , nsP = 0 . 37 , by unpaired two - sided t - test . Representative micro - CT images at day 14 are shown . rhTNF treatment led to mature callus bridging across the fracture which was absent in the PBS - treated control group . Scale bar , 2 mm .

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rhTNF treatment augmented early phase fracture healing in osteoporotic mice. Treatment with 1 ng rhTNF at the fracture site on days 0 and 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but the same final result at day 28. Data are presented as mean ± SEM. At day 14, PBS control versus TNF, **P = 0.0099; at day 28, PBS control versus TNF, nsP = 0.37, by unpaired two-sided t-test. Representative micro-CT images at day 14 are shown. rhTNF treatment led to mature callus bridging across the fracture which was absent in the PBS-treated control group. Scale bar, 2 mm.


pH - dependent dissociation of E and M . TBEV preparations treated at pH 8 ( left ) and pH 6 ( right ) were solubilized with Triton X - 100 and subjected to sucrose gradient centrifugation . The fractions were analyzed by SDS - PAGE and densitometry . Representative examples of at least two independent experiments are shown . Positions of E dimers ( D ) and E trimers ( T ) in the gradients are indicated .

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pH-dependent dissociation of E and M. TBEV preparations treated at pH 8 (left) and pH 6 (right) were solubilized with Triton X-100 and subjected to sucrose gradient centrifugation. The fractions were analyzed by SDS-PAGE and densitometry. Representative examples of at least two independent experiments are shown. Positions of E dimers (D) and E trimers (T) in the gradients are indicated.


Trimer stability expressed as percentage of the fraction detected in the trimer peak and normalized with respect to the corresponding Ev fraction in sedimentation analyses after incubation at 70 ° C relative to 37 ° C ( 100 % ) . Data are from at least three independent experiments .

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Trimer stability expressed as percentage of the fraction detected in the trimer peak and normalized with respect to the corresponding Ev fraction in sedimentation analyses after incubation at 70°C relative to 37°C (100%). Data are from at least three independent experiments.


Difference in deuterium uptake by sE404r and sE419r trimers ( D404 - D419 ) . Left panel , deuteration difference for each peptide reported against the 1 - p value obtained via Student ' s t - test between the two different constructs . In the right panel , peptides are reported as short horizontal lines , with length and position corresponding to the peptide length and location along the protein sequence ( x - axis ) and with their deuteration delta on the y - axis . In both panels , red ( positive ) and blue ( negative ) indicate changes above the Student ' s t - test threshold , and in grey those not reaching this threshold . Four peptides , termed HDX - a , HDX - b , HDX - c and HDX - d , were identified as showing a strong increase in deuteration in sE404r compared to sE419r . The same pattern was observed in the comparison between sE404r and sE428r , whereas no difference deemed " significant " by the same test was seen when comparing sE419r with sE428r ( see Fig EV3 ) .

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

Difference in deuterium uptake by sE404r and sE419r trimers (D404-D419). Left panel, deuteration difference for each peptide reported against the 1-p value obtained via Student's t-test between the two different constructs. In the right panel, peptides are reported as short horizontal lines, with length and position corresponding to the peptide length and location along the protein sequence (x-axis) and with their deuteration delta on the y-axis. In both panels, red (positive) and blue (negative) indicate changes above the Student's t-test threshold, and in grey those not reaching this threshold. Four peptides, termed HDX-a, HDX-b, HDX-c and HDX-d, were identified as showing a strong increase in deuteration in sE404r compared to sE419r. The same pattern was observed in the comparison between sE404r and sE428r, whereas no difference deemed "significant" by the same test was seen when comparing sE419r with sE428r (see Fig EV3).


Blocking ELISA with mabs A1 , A2 , A3 and B4 . Serial dilutions of the trimers were pre - incubated with a predetermined concentration of the respective mabs . The fraction of mab not blocked by the antigen was detected in ELISA with TBEV - coated plates ( Materials and Methods ) . The bar charts show the results expressed relative to the reactivity with the sE401v trimer . Data are from at least three independent experiments .

Blocking ELISA with mabs A1, A2, A3 and B4. Serial dilutions of the trimers were pre-incubated with a predetermined concentration of the respective mabs. The fraction of mab not blocked by the antigen was detected in ELISA with TBEV-coated plates (Materials and Methods). The bar charts show the results expressed relative to the reactivity with the sE401v trimer. Data are from at least three independent experiments.


A . Top : DDX42 KO and CTRL KO U87 - MG / CD4 / CXCR4 / Cas9 / Firefly cells were generated using 3 sgRNAs and 4 non - targeting sgRNAs , respectively ( for CTRL , the average of data obtained with 4 cell populations is shown ) . Cells were treated or not with IFN 24 h prior to infection with HIV - 1 Renilla ( NL4 - 3 / Nef - IRES - Renilla ) . Relative luminescence results for IFN - treated and - untreated conditions are shown . Two - way ANOVA on log - transformed data with Sidak ' s test . Bottom : Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42 - depleted populations ; Actin served as a loading control .

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A. Top: DDX42 KO and CTRL KO U87-MG/CD4/CXCR4/Cas9/Firefly cells were generated using 3 sgRNAs and 4 non-targeting sgRNAs, respectively (for CTRL, the average of data obtained with 4 cell populations is shown). Cells were treated or not with IFN 24 h prior to infection with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla). Relative luminescence results for IFN-treated and -untreated conditions are shown. Two-way ANOVA on log-transformed data with Sidak's test. Bottom: Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42-depleted populations; Actin served as a loading control.


C . DDX42 silencing efficiency measured by RT - qPCR ( top ) and immunoblot ( bottom ) in parallel samples from B .

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C. DDX42 silencing efficiency measured by RT-qPCR (top) and immunoblot (bottom) in parallel samples from B.


G . Primary CD4 + T cells were electroporated with Cas9 - sgRNA RNPs using 2 non - targeting sgRNAs ( sgCTRL1 and 2 ) and 5 sgRNAs targeting DDX42 . Top : Cells were then infected with HIV - 1 Renilla ( NL4 - 3 / Nef - IRES - Renilla ) and relative infection efficiencies obtained with cells from 3 donors are shown . Two - way ANOVA on log - transformed data with Dunnett ' s test . Bottom : DDX42 protein levels were determined by immunoblot , Actin served as a loading control . A representative immunoblot is shown .

G. Primary CD4+ T cells were electroporated with Cas9-sgRNA RNPs using 2 non-targeting sgRNAs (sgCTRL1 and 2) and 5 sgRNAs targeting DDX42. Top: Cells were then infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla) and relative infection efficiencies obtained with cells from 3 donors are shown. Two-way ANOVA on log-transformed data with Dunnett's test. Bottom: DDX42 protein levels were determined by immunoblot, Actin served as a loading control. A representative immunoblot is shown.


C . Heat - annealed ODN / RNA 1 - 294 complex was incubated with HIV - 1 RT and increasing concentrations of recombinant DDX42 . Reverse transcription was initiated by addition of the four dNTPs . Extension was for 1 , 5 , 20 or 60 min and samples were analysed by PAGE 8 % ( P / T : primer / template ; SSDNA : Strong - Stop DNA ) . A representative autoradiograph is shown .

C. Heat-annealed ODN/RNA 1-294 complex was incubated with HIV-1 RT and increasing concentrations of recombinant DDX42. Reverse transcription was initiated by addition of the four dNTPs. Extension was for 1, 5, 20 or 60 min and samples were analysed by PAGE 8% (P/T: primer/template; SSDNA: Strong-Stop DNA). A representative autoradiograph is shown.


D . Quantification of 3 biological replicates performed as in C . Two - way ANOVA on log - transformed data with Dunnett ' s test .

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D. Quantification of 3 biological replicates performed as in C. Two-way ANOVA on log-transformed data with Dunnett's test.


E . PLAs were performed in MDMs infected with HIV - 1 or not ( N . I . CTRL ) , using anti - Capsid and anti - DDX42 antibodies ( nuclei stained with Hoechst ) . Images were acquired using a LSM880 Airyscan microscope . Left : representative images , scale - bar : 10 μm . Right : Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD ( n > 65 cells per condition ) .

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E. PLAs were performed in MDMs infected with HIV-1 or not (N.I. CTRL), using anti-Capsid and anti-DDX42 antibodies (nuclei stained with Hoechst). Images were acquired using a LSM880 Airyscan microscope. Left: representative images, scale-bar: 10 μm. Right: Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD (n>65 cells per condition).


K . HEK293T were co - transfected with pRPS - GFP and a Flag - Firefly - ( negative control ) or Flag - DDX42 - coding plasmid , followed by Flag immunoprecipitation and immunoblot analysis . A representative immunoblot is shown .

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K. HEK293T were co-transfected with pRPS-GFP and a Flag-Firefly- (negative control) or Flag-DDX42-coding plasmid, followed by Flag immunoprecipitation and immunoblot analysis. A representative immunoblot is shown.


( A ) Left , A549 - ACE2 cells were infected or not with SARS - CoV - 2 for 24 h prior to PLA using mouse anti - dsRNA ( J2 ) and rabbit anti - DDX42 antibodies , followed by additional immunofluorescence ( IF ) staining with anti - mouse Alexa Fluor 546 antibody ( PLA in green , IF in magenta ) . Representative Z - stack projection images are shown ; scale bar : 15 μm . Right , Average punctae were quantified in 3 biological replicates with mean ± SD ( n > 75 cells per condition ) .

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(A) Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA using mouse anti-dsRNA (J2) and rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition).


Left , A549 - ACE2 cells were infected or not with SARS - CoV - 2 for 24 h prior to PLA rabbit anti - DDX42 antibodies , followed by additional immunofluorescence ( IF ) staining with anti - mouse Alexa Fluor 546 antibody ( PLA in green , IF in magenta ) . Representative Z - stack projection images are shown ; scale bar : 15 μm . Right , Average punctae were quantified in 3 biological replicates with mean ± SD ( n > 75 cells per condition ) . using an anti - N

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Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition). using an anti-N antibody


F . The following biotinylated RNAs were used to pull - down recombinant DDX42 poly ( I : C ) , CHIKV G4 , TRF2 G4 and a mutated TRF2 G4 sequence ( Mut . TRF2 G4 ) , and DDX42 was revealed by an immunoblot .

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F. The following biotinylated RNAs were used to pull-down recombinant DDX42 poly(I:C), CHIKV G4, TRF2 G4 and a mutated TRF2 G4 sequence (Mut. TRF2 G4), and DDX42 was revealed by an immunoblot.


A CUEDC2protein levels in mouse tissues . Adult ( 12 - week - old ) mouse was sacrifices and total protein was extracted from the liver , kidney , lung , spleen , heart , stomach , intestine , prostate , testis , brain , and thymus .

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A CUEDC2protein levels in mouse tissues. Adult (12-week-old) mouse was sacrifices and total protein was extracted from the liver, kidney, lung, spleen, heart, stomach, intestine, prostate, testis, brain, and thymus.


B ( top ) In vivo cardiac ischemia - reperfusion model protocol ( 30 - minute ischemia followed by indicated times of reperfusion ) . ( bottom ) Mice were subjected to reversible ischemia in vivo for 30 minutes and reperfused for different time period . Protein was extracted from the area at risk of heart at the indicated time points and subjected to immunoblot analysis of CUEDC2 and CUEDC1 . Representative results were shown from 2 mice at each time point , and experiments were repeated for three times .

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B (top) In vivo cardiac ischemia-reperfusion model protocol (30-minute ischemia followed by indicated times of reperfusion). (bottom) Mice were subjected to reversible ischemia in vivo for 30 minutes and reperfused for different time period. Protein was extracted from the area at risk of heart at the indicated time points and subjected to immunoblot analysis of CUEDC2 and CUEDC1. Representative results were shown from 2 mice at each time point, and experiments were repeated for three times.


C Isolated primary ratneonatal cardiomyocytes were treated to hypoxia with serum free - medium for 6h and reoxygenated accompanying adding serum back for different time period in vitro and the protein level of CUEDC2 was tested at different time points during reoxygenation . Each experiment was repeated for three times .

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C Isolated primary ratneonatal cardiomyocytes were treated to hypoxia with serum free-medium for 6h and reoxygenated accompanying adding serum back for different time period in vitro and the protein level of CUEDC2 was tested at different time points during reoxygenation. Each experiment was repeated for three times.


D CUEDC2 protein level was plotted using the immunohistochemical scores as described in methods . ( left ) Plot of CUEDC2 scores in each BZ and DZ ( n = 9 patients ) . ( right ) Representative images from immunohistochemistry staining for CUEDC2 in tissues from patients who suffered acute myocardial infarctions . The boxed areas in the left images are magnified in the middle and right images . DZ , distant zone ; BZ , border zone . Scale bars , 250 μm ( left ) , 50 μm ( middle and right ) . * p = 0 . 001 .

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D CUEDC2 protein level was plotted using the immunohistochemical scores as described in methods. (left) Plot of CUEDC2 scores in each BZ and DZ (n = 9 patients). (right) Representative images from immunohistochemistry staining for CUEDC2 in tissues from patients who suffered acute myocardial infarctions. The boxed areas in the left images are magnified in the middle and right images. DZ, distant zone; BZ, border zone. Scale bars, 250 μm (left), 50 μm (middle and right). * p = 0.001.


A Primary mousecardiomyocytes were treated with H2O2 ( 1 mM ) for 3 hours and cardiomyocyteviability was determined by flow cytometry . Representative results of flow cytometry analysis .

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A Primary mousecardiomyocytes were treated with H2O2 (1 mM) for 3 hours and cardiomyocyteviability was determined by flow cytometry. Representative results of flow cytometry analysis.


B Quantitative analysis of PI - positive cells treated with H2O2 was shown ( 42 . 9 ± 3 . 1 % in wild - type group vs . 10 . 1 ± 4 . 4 % in Cuedc2 - / - group , * p = 0 . 0004 , n = 5 wells per group and repeated for three times ) .

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B Quantitative analysis of PI-positive cells treated with H2O2 was shown (42.9 ± 3.1% in wild-type group vs. 10.1 ± 4.4% in Cuedc2-/- group, * p = 0.0004, n = 5 wells per group and repeated for three times).


C Mouse neonatal cardiomyocytes were subjected to hypoxia for 6 hours and then reoxygenation for 30 minutes . Cardiomyocytes were then stained with 5 μM CellROX ® Deep Red Reagent and analyzed by flow cytometry . Representative results of flow cytometry analysis are shown , and all experiments were repeated for three times . D ROS levels were quantitated and summarized . * p = 0 . 0108 , n = 3 wells per group and repeated for three times .

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C Mouse neonatal cardiomyocytes were subjected to hypoxia for 6 hours and then reoxygenation for 30 minutes. Cardiomyocytes were then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. Representative results of flow cytometry analysis are shown, and all experiments were repeated for three times.D ROS levels were quantitated and summarized. * p = 0.0108, n = 3 wells per group and repeated for three times.


E HPLC detection of a superoxide probe oxidized dihydroethidium ( DHE ) product in sham and I / R injury heart tissue ( 30 - min ischemia followed by 30 - min reperfusion ) , 2 - hydroxyethidium ( EOH ) , a specific product for superoxide anion radical . * p < 0 . 0001 , n = 5 in each group . F In the same samples of ( E ) , HPLC detection of ethidium ( E ) , oxidized by other reactive oxygen species such as H2O2 ( mainly ) and ONOO . * p = 0 . 0001 , n = 5 in each group .

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E HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in sham and I/R injury heart tissue (30-min ischemia followed by 30-min reperfusion), 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. *p < 0.0001, n = 5 in each group.F In the same samples of (E), HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. *p = 0.0001, n = 5 in each group.


A Protein was extracted from the area at risk of left ventricle from wild - type or Cuedc2 - / - mice with 30 - minute ischemia followed by 30 - minute reperfusion and subjected to immunoblot analysis . Representative results from 2 mice are shown , and each experiment was repeated for three times .

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A Protein was extracted from the area at risk of left ventricle from wild-type or Cuedc2-/- mice with 30-minute ischemia followed by 30-minute reperfusion and subjected to immunoblot analysis. Representative results from 2 mice are shown, and each experiment was repeated for three times.


B Neonatal mousecardiomyocytes were subjected to hypoxia for 6 hours , followed by the indicated times of reoxygenation , before protein was extracted and subjected to immunoblot analysis .

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B Neonatal mousecardiomyocytes were subjected to hypoxia for 6 hours, followed by the indicated times of reoxygenation, before protein was extracted and subjected to immunoblot analysis.


C Luciferase assay of WT or Cuedc2 - / - MEFs transiently transfected with an NF - κB - responsive luciferase reporter and then , 1 d later , treated for 6 h with TNF ( 10 ng / ml ; TNF ) or for H / R . * p = 0 . 0032 , n = 3 per group , * * p = 0 . 013 , n = 3 per group .

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C Luciferase assay of WT or Cuedc2-/- MEFs transiently transfected with an NF-κB-responsive luciferase reporter and then, 1 d later, treated for 6 h with TNF (10 ng/ml; TNF) or for H/R. * p = 0.0032, n = 3 per group, ** p = 0.013, n = 3 per group.


D Wild - type and Cuedc2 - / - mice were treated with a sham or I / R operation in vivo . Three hours after the onset of reperfusion , the mRNA levels of tumor necrosis factor α ( TNFα ) , interleukin 6 ( IL - 6 ) and interleukin 23 ( IL - 23 ) were tested . * p = 0 . 0005 , * * p = 0 . 0001 , n = 3 per group .

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D Wild-type and Cuedc2-/- mice were treated with a sham or I/R operation in vivo. Three hours after the onset of reperfusion, the mRNA levels of tumor necrosis factor α (TNFα), interleukin 6 (IL-6) and interleukin 23 (IL-23) were tested. * p = 0.0005, ** p = 0.0001, n = 3 per group.


A Protein was extracted from the left ventricle of wild - type or Cuedc2 - / - adult mice ( 12 week - old ) and subjected to immunoblot analysis ( 3 mice per group ) .

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A Protein was extracted from the left ventricle of wild-type or Cuedc2-/- adult mice (12 week-old) and subjected to immunoblot analysis (3 mice per group).


B Wild - type or Cuedc2 - / - mice were subjected to a heart ischemia for 30 - minute in vivo , followed by reperfusion for the indicated times . Protein was extracted from the area at risk of left ventricle and subjected to immunoblot analysis .

B Wild-type or Cuedc2-/- mice were subjected to a heart ischemia for 30-minute in vivo, followed by reperfusion for the indicated times. Protein was extracted from the area at risk of left ventricle and subjected to immunoblot analysis.


C GPX1 protein level was plotted using the immunohistochemical scores as described in methods . ( left ) representative images from immunohistochemistry staining for GPX1 in tissues from patients who suffered acute myocardial infarctions . ( right ) plot of GPX1 scores in each BZ and DZ ( n = 9 patients ) . DZ , distant area ; BZ , border zone . Scale bars , 250 μm ( left ) . * p = 0 . 0039 .

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C GPX1 protein level was plotted using the immunohistochemical scores as described in methods. (left) representative images from immunohistochemistry staining for GPX1 in tissues from patients who suffered acute myocardial infarctions. (right) plot of GPX1 scores in each BZ and DZ (n = 9 patients). DZ, distant area; BZ, border zone. Scale bars, 250 μm (left). * p = 0.0039.


D Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were treated with H2O2 ( 1 mM ) for 3 hours . Cell viability was detected by FACS . * p = 0 . 0001 ; * * p = 0 . 0275 , n = 3 wells per group . NS , not significant .

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D Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were treated with H2O2 (1 mM) for 3 hours. Cell viability was detected by FACS. * p = 0.0001; ** p = 0.0275, n = 3 wells per group. NS, not significant.


E Primary neonatal mousecardiomyocytes were transfected with lentivirus carrying control or GPX1 shRNA , and the efficiency of GPX1 knockdown was tested by immunoblotting .

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E Primary neonatal mousecardiomyocytes were transfected with lentivirus carrying control or GPX1 shRNA, and the efficiency of GPX1 knockdown was tested by immunoblotting.


F Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were subjected to H2O2 ( 1 mM ) for 30 minutes and then stained with 5 μM CellROX ® Deep Red Reagent and analyzed by flow cytometry . * p = 0 . 0056 ; * * p = 0 . 0218 , n = 3 wells per group , n = 3 wells per group . NS , not significant .

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F Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were subjected to H2O2 (1 mM) for 30 minutes and then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. * p = 0.0056; ** p = 0.0218, n = 3 wells per group, n = 3 wells per group. NS, not significant.


A Lysates from MEF - WT or MEF - Cuedc2 - / - cells treated with 20μM cycloheximide ( CHX ) for the indicated times were subjected to immunoblotting ( left ) . Relative GPX1 levels were quantified by densitometry ( right ) .

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A Lysates from MEF-WT or MEF-Cuedc2-/- cells treated with 20μM cycloheximide (CHX) for the indicated times were subjected to immunoblotting (left). Relative GPX1 levels were quantified by densitometry (right).


B MEF - Cuedc2 - / - cells were transfected with increasing amounts of FLAG - CUEDC2 ( mouse ) plasmids ( 1 μg and 2 μg ) . At 24 hours after transfection , the cells were treated with the proteasome inhibitor MG - 132 ( 10 μM ) . Cells were cultured for additional 6 hours and subjected to immunoblotting .

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B MEF-Cuedc2-/- cells were transfected with increasing amounts of FLAG-CUEDC2 (mouse) plasmids (1 μg and 2 μg). At 24 hours after transfection, the cells were treated with the proteasome inhibitor MG-132 (10 μM). Cells were cultured for additional 6 hours and subjected to immunoblotting.


C HEK293T cells were transfected with the FLAG - GPX1 , HA - CUEDC2 , and Myc - ubiquitin plasmids as indicated and treated with MG - 132 ( 10 µM ) for 6 hours before harvest . Cell lysates were immunoprecipitated ( IP ) with anti - FLAG ( M2 ) . The immunoprecipitates were analyzed by western blot using an anti - GPX1 antibody . Whole - cell lysates ( WCL ) were analyzed by Western blots with anti - FLAG or anti - HA antibody to determine the protein of Flag - GPX1 and HA - CUEDC2 .

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C HEK293T cells were transfected with the FLAG-GPX1, HA-CUEDC2, and Myc-ubiquitin plasmids as indicated and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-FLAG (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-FLAG or anti-HA antibody to determine the protein of Flag-GPX1 and HA-CUEDC2.


D The heart tissue lysates from wide - type and Cuedc2 - / - mice were incubated with IgG or anti - GPX1 antibody , and then captured on protein G - Sepharose beads . The immunoprecipitates were analyzed by immunoblotting with an anti - ubiquitin antibody .

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D The heart tissue lysates from wide-type and Cuedc2-/- mice were incubated with IgG or anti-GPX1 antibody, and then captured on protein G-Sepharose beads. The immunoprecipitates were analyzed by immunoblotting with an anti-ubiquitin antibody.


E HEK293T cells were transfected with the Flag - CUEDC2 or Flag - CUEDC2 △ CUE ( deletion of the CUE domain of CUEDC2 ) , and treated with MG - 132 ( 10 µM ) for 6 hours before harvest . Cell lysates were immunoprecipitated ( IP ) with anti - Flag ( M2 ) . The immunoprecipitates were analyzed by western blot using an anti - GPX1 antibody . Whole - cell lysates ( WCL ) were analyzed by Western blots with anti - Flag or anti - GPX1 antibody to determine the protein of Flag - CUEDC2 and GPX1 . v : vector .

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E HEK293T cells were transfected with the Flag-CUEDC2 or Flag-CUEDC2△CUE (deletion of the CUE domain of CUEDC2), and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-Flag (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-Flag or anti-GPX1 antibody to determine the protein of Flag-CUEDC2 and GPX1. v: vector.


A HEK293 cells were transfected with increasing amounts of Flag - TRIM33 expression vectors and 48 hours after transfection , cell lysates were subjected to immunoblotting ( IB ) using antibodies as indicated .

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A HEK293 cells were transfected with increasing amounts of Flag-TRIM33 expression vectors and 48 hours after transfection, cell lysates were subjected to immunoblotting (IB) using antibodies as indicated.


B HEK293 cells were transfected with increasing amounts of Flag - FBXW7 expression vectors and 48 hours after transfection , cell lysates were subjected to immunoblotting ( IB ) .

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B HEK293 cells were transfected with increasing amounts of Flag-FBXW7 expression vectors and 48 hours after transfection, cell lysates were subjected to immunoblotting (IB).


C HEK293T cells were transfected as indicated , at 18 h after transfection , the cells were treated with the proteasome inhibitor MG132 ( 20μM ) for 6 hours and then harvested . Cell lysates were immunoprecipitated by Flag - antibody ( M2 beads ) and Ubiquitin - conjugated GPX1 was detected by Western blotting with anti - Myc antibody .

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C HEK293T cells were transfected as indicated, at 18 h after transfection, the cells were treated with the proteasome inhibitor MG132 (20μM) for 6 hours and then harvested. Cell lysates were immunoprecipitated by Flag-antibody (M2 beads) and Ubiquitin-conjugated GPX1 was detected by Western blotting with anti-Myc antibody.


D HEK293 cells were transfected with either control or CUEDC2 siRNA and increasing amounts of Flag - TRIM33 expression vectors , cell lysates were subjected to immunoblotting ( IB ) using antibodies as indicated .

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D HEK293 cells were transfected with either control or CUEDC2 siRNA and increasing amounts of Flag-TRIM33 expression vectors, cell lysates were subjected to immunoblotting (IB) using antibodies as indicated.


A CUEDC2 ablation reduced I / R injury , which was induced by 30 minutes of ischemia followed by 24 hours reperfusion . AAR , area at risk ; LV , left ventricular area , IRI , area of I / R injury . The ratios of AAR to LV , IRI to AAR and IRI to LV are shown . * p < 0 . 0001 compared with WT , n = 15 mice per group . ( top panel ) Representative cross - sectional slices derived from the hearts stained by TTC . Area of I / R injury is indicated in light pink .

A CUEDC2 ablation reduced I/R injury, which was induced by 30 minutes of ischemia followed by 24 hours reperfusion. AAR, area at risk; LV, left ventricular area, IRI, area of I/R injury. The ratios of AAR to LV, IRI to AAR and IRI to LV are shown. * p < 0.0001 compared with WT, n = 15 mice per group. (top panel) Representative cross-sectional slices derived from the hearts stained by TTC. Area of I/R injury is indicated in light pink.


B Representative images of ventricular myocardium sections from WT and Cuedc2 - / - mice exposed to sham operation or I / R injury ( 2 hours after the onset of reperfusion following 30 - min ischemia ) . Green , TUNEL - positive myocytenuclei ; red , Troponin I - stained cardiomyocytes ; blue , DAPI - stained nuclei . Scale bar , 50 μm .

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B Representative images of ventricular myocardium sections from WT and Cuedc2-/- mice exposed to sham operation or I/R injury (2 hours after the onset of reperfusion following 30-min ischemia). Green, TUNEL-positive myocytenuclei; red, Troponin I-stained cardiomyocytes; blue, DAPI-stained nuclei. Scale bar, 50 μm.


C Quantitative analysis of apoptosis . * p < 0 . 0001 , n = 8 mice per group .

C Quantitative analysis of apoptosis. * p < 0.0001, n = 8 mice per group.


D SerumLDH levels in sham - operated mice or those subjected to ischemia and a 4 - hour reperfusion . Data from 3 separate experiments was analyzed by one - way analysis of variance . * p < 0 . 0001 , n = 13 mice per group .

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D SerumLDH levels in sham-operated mice or those subjected to ischemia and a 4-hour reperfusion. Data from 3 separate experiments was analyzed by one-way analysis of variance. * p < 0.0001, n = 13 mice per group.


E Echocardiography analysis of left ventricular dimensions and cardiac function at 1 week post - I / R in mice . * * p < 0 . 0001 , n = 15 mice in the sham - operated group and n = 10 mice in the I / R group .

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E Echocardiography analysis of left ventricular dimensions and cardiac function at 1 week post-I/R in mice. ** p < 0.0001, n = 15 mice in the sham-operated group and n = 10 mice in the I/R group.


A Immunoblotting for CUEDC2 protein level . 4 weeks after tail - vein injection of rAAV9 - CUEDC2 , indicated mice tissues were harvested and subjected to immunoblotting to detect CUEDC2 protein level . Ctl : heart of rAAV9 - GFP injected mice .

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A Immunoblotting for CUEDC2 protein level. 4 weeks after tail-vein injection of rAAV9-CUEDC2, indicated mice tissues were harvested and subjected to immunoblotting to detect CUEDC2 protein level. Ctl: heart of rAAV9-GFP injected mice.


B Animals were injected by rAAV9 - CUEDC2 in a different amount of viral genomes ( VG ) . Heart tissues were then analyzed by immunoblotting to detect CUEDC2 transduction . Tubulin protein level was examined for normalization purposes .

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B Animals were injected by rAAV9-CUEDC2 in a different amount of viral genomes (VG). Heart tissues were then analyzed by immunoblotting to detect CUEDC2 transduction. Tubulin protein level was examined for normalization purposes.


C The ratios of AAR to LV , IRI to AAR and IRI to LV are shown . n = 10 mice in WT + rAAV - GFP group and Cuedc2 - / - + rAAV - GFP group ; n = 12 mice in Cuedc2 - / - + rAAV - CUEDC2 ( 1 × 1010 VG ) group and Cuedc2 - / - + rAAV - CUEDC2 ( 1 × 1011 VG ) group . * p = 0 . 0055 , * * p = 0 . 0146 , * * * p = 0 . 0200 , * * * * p = 0 . 0377 .

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C The ratios of AAR to LV, IRI to AAR and IRI to LV are shown. n = 10 mice in WT + rAAV-GFP group and Cuedc2-/- + rAAV-GFP group; n = 12 mice in Cuedc2-/- + rAAV-CUEDC2 (1×1010 VG) group and Cuedc2-/- + rAAV-CUEDC2 (1×1011 VG) group. * p = 0.0055, ** p = 0.0146, *** p = 0.0200, **** p = 0.0377.


D Effect of rAAV9 - CUEDC2 treatment on echocardiography indices of left ventricle function recovery post - I / R measured by fractional shortening ( FS ) and ejection fraction ( EF ) . WT + rAAV - GFP ( n = 10 ) ; Cuedc2 - / - + rAAV - GFP ( n = 10 ) ; Cuedc2 - / - + rAAV - CUEDC2 ( Low dose ) ( n = 12 ) ; Cuedc2 - / - + rAAV - CUEDC2 ( High dose ) ( n = 12 ) . * * p < 0 . 0001 .

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D Effect of rAAV9-CUEDC2 treatment on echocardiography indices of left ventricle function recovery post-I/R measured by fractional shortening (FS) and ejection fraction (EF). WT+ rAAV-GFP (n = 10); Cuedc2-/- + rAAV-GFP (n = 10); Cuedc2-/- + rAAV-CUEDC2 (Low dose) (n = 12); Cuedc2-/- + rAAV-CUEDC2 (High dose) (n = 12). ** p < 0.0001.


A HPLC detection of a superoxide probe oxidized dihydroethidium ( DHE ) product in young ( 8 - week - old ) and old mouse ( 20 - month - old ) heart tissue , 2 - hydroxyethidium ( EOH ) , a specific product for superoxide anion radical . * p = 0 . 0087 , n = 5 in each group .

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A HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in young (8-week-old) and old mouse (20-month-old) heart tissue, 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. * p = 0.0087, n = 5 in each group.


B HPLC detection of ethidium ( E ) , oxidized by other reactive oxygen species such as H2O2 ( mainly ) and ONOO . * p = 0 . 0021 , n = 5 in each group .

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B HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. * p = 0.0021, n = 5 in each group.


C Comparison of 8 - hydroxy - 2 ' - deoxyguanosine ( 8 - OHdG ) in young and old mice . * p = 0 . 0164 , n = 6 mice per group .

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C Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in young and old mice. * p = 0.0164, n = 6 mice per group.


D Comparison of heart weight / body weight ( HW / BW ) in WT and Cuedc2 - / - mice . * * p = 0 . 036 , n = 15 in young WT and Cuedc2 - / - mice , n = 10 in old WT mice and n = 12 in old Cuedc2 - / - mice .

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D Comparison of heart weight / body weight (HW/BW) in WT and Cuedc2-/- mice. ** p = 0.036, n = 15 in young WT and Cuedc2-/- mice, n = 10 in old WT mice and n = 12 in old Cuedc2-/- mice.


E Comparison of the left ventricle function in WT and Cuedc2 - / - mice by transthoracic echocardiography . * p = 0 . 0384 , * * p = 0 . 0389 . n = 15 in young WT and Cuedc2 - / - mice , n = 10 in old WT mice and n = 12 in old Cuedc2 - / - mice .

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E Comparison of the left ventricle function in WT and Cuedc2-/- mice by transthoracic echocardiography. *p = 0.0384, **p = 0.0389. n = 15 in young WT and Cuedc2-/- mice, n = 10 in old WT mice and n = 12 in old Cuedc2-/- mice.


F Left ventriclecardiomyocytes apoptosis was tested by TUNEL staining . * p = 0 . 0017 , n = 5 in each group .

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F Left ventriclecardiomyocytes apoptosis was tested by TUNEL staining. *p = 0.0017, n = 5 in each group.


G Left ventricle myocardial fibrosis was tested by Masson staining in WT and Cuedc2 - / - mice . The represent results were shown in ( G ) and the quantitative analysis of myocardial fibrosis was shown in ( H ) . * p = 0 . 0177 , n = 5 in each group .

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G Left ventricle myocardial fibrosis was tested by Masson staining in WT and Cuedc2-/- mice. The represent results were shown in (G) and the quantitative analysis of myocardial fibrosis was shown in (H). *p = 0.0177, n = 5 in each group.


A - D ) Confocal image analysis of cryostat sections from adult CX3CR1 + / GFP ; Sfrp1 + / βgal ( A , B ) ; Sfrp1 + / βgal ( C ) and CX3CR1 + / GFP ; Sfrp1 - / - ( D ) and mouse brains three days after intra - cortical infusion of saline or LPS . Sections were immunostained for βgal ( magenta , green in C ) and Iba1 ( green in A ; red in C ) or Sfrp1 ( magenta ) and GFP ( green , B and D ) , and GFAP ( cyan in A ; B , D , red in C ) . Arrowheads indicate βgal / GFAP ( A ) and Sfrp1 / GFP co - localization ( B ) . No Sfrp1 protein is detected in the null mice independently of the treatment ( two bottom lines ) . Scale bar 25μm . E ) ELISA determination of Sfrp1 levels in brain extracts from 3 months - old wt and Sfrp1 - / - mice . WT mice were injected either with saline or LPS . Three days after injection the region around the injected side ( 10 mm3 cortical cube ) was isolated and SFRP1 content compared with that present in similar region of non - injected or Sfrp1 - / - mice used as negative control ( n = 5 mice for each group ) . Error bars represent Standard Error . Statistical significance : ns P > 0 . 5 * * * * P < 0 . 0001 ; One - way ANOVA followed by Bonferroni multiple comparisons 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 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 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

A-D) Confocal image analysis of cryostat sections from adult CX3CR1+/GFP;Sfrp1+/βgal (A,B); Sfrp1+/βgal (C) and CX3CR1+/GFP;Sfrp1-/- (D) and mouse brains three days after intra-cortical infusion of saline or LPS. Sections were immunostained for βgal (magenta, green in C) and Iba1 (green in A; red in C) or Sfrp1 (magenta) and GFP (green, B and D), and GFAP (cyan in A;B, D, red in C). Arrowheads indicate βgal/GFAP (A) and Sfrp1/GFP co-localization (B). No Sfrp1 protein is detected in the null mice independently of the treatment (two bottom lines). Scale bar 25μm. E) ELISA determination of Sfrp1 levels in brain extracts from 3 months-old wt and Sfrp1-/- mice. WT mice were injected either with saline or LPS. Three days after injection the region around the injected side (10 mm3 cortical cube) was isolated and SFRP1 content compared with that present in similar region of non-injected or Sfrp1-/- mice used as negative control (n=5 mice for each group). Error bars represent Standard Error. Statistical significance: ns P>0.5 ****P<0.0001; One-way ANOVA followed by Bonferroni multiple comparisons test.


A ) Coronal cryostat sections of LV - IRES - Gfp or LV - Sfrp1 - IRES - Gfp infected brains 1 - month post - infusion , immunostained for SFRP1 ( magenta ) Iba1 ( green ) GFAP ( cyan ) , or TauP ( red ) . Lv , lateral ventricle . Scale bar 100μm . B ) The graph shows the normalised level of GFAP , Iba1 and CD45 immunoreactivity ( IR ) and the area occupied by TauP signal ( n = 24 acquisitions , white dots ; N = 3 mice , black dots , for each group ) , normalized to LV - IRES - Gfp infected brains . Error bars represent Standard Error . Statistical significance : * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 by two - sided Student ' s 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 O O O O O O O O O

A) Coronal cryostat sections of LV-IRES-Gfp or LV-Sfrp1-IRES-Gfp infected brains 1-month post-infusion, immunostained for SFRP1 (magenta) Iba1 (green) GFAP (cyan), or TauP (red). Lv, lateral ventricle. Scale bar 100μm. B) The graph shows the normalised level of GFAP, Iba1 and CD45 immunoreactivity (IR) and the area occupied by TauP signal (n=24 acquisitions, white dots; N=3 mice, black dots, for each group), normalized to LV-IRES-Gfp infected brains. Error bars represent Standard Error. Statistical significance: *P<0.05; **P<0.01; ****P<0.0001 by two-sided Student's t-test.


C , D ) Coronal sections from wt and Sfrp1 - / - mouse brains three days after infusion of saline or LPS , immunostained for GFAP ( cyan , C ) or CD45 ( magenta , D ) . The images are high power views of the somatosensory cortex ( lower power view in Fig EV1D ) . Scale bar 60μm . E , F ) The graphs show the normalised levels of immunoreactivity ( IR ) for GFAP ( E ) and CD45 ( F , P = 0 , 006 ) present in cortical sections ( n = 24 acquisitions white dots ; from N = 3 animals , black dots , per group ) from wt and Sfrp1 - / - animals infused with saline or LPS . Bars represent Standard Error . Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black . * * or # # P < 0 . 01 ; * * * or # # # P < 0 . 001 ; * * * * or # # # # P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons test . * and # indicate significance between genotypes and treatments , respectively .

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

C, D) Coronal sections from wt and Sfrp1-/- mouse brains three days after infusion of saline or LPS, immunostained for GFAP (cyan, C) or CD45 (magenta, D). The images are high power views of the somatosensory cortex (lower power view in Fig EV1D). Scale bar 60μm. E, F) The graphs show the normalised levels of immunoreactivity (IR) for GFAP (E) and CD45 (F, P=0,006) present in cortical sections (n=24 acquisitions white dots; from N=3 animals, black dots, per group) from wt and Sfrp1-/- animals infused with saline or LPS. Bars represent Standard Error. Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black. ** or ## P<0.01; *** or ### P<0.001; **** or #### P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test. * and # indicate significance between genotypes and treatments, respectively.


C ) Wt and Sfrp1 - / - mice immunized with MOG and sacrificed 16 days post immunization . Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4 , Iba1 , GFAP and MBP . Images show the region dorsal fasciculus . White arrowheads point to the disruption of the pia surface in wt . Scale bar 100μm . D ) Quantification of CD4 + infiltrated lymphocytes , Iba1 normalised immunoreactivity , MBP normalised immunoreactivity and MBP immune - reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1 - / - mice 16 days after immunization ( n = 16 acquisitions , white dots ; from N = 4 animals , black dots , per genotype ) . Error bars represent Standard Error . Statistic refers to number of acquisitions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wt and Sfrp1-/- mice immunized with MOG and sacrificed 16 days post immunization. Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4, Iba1, GFAP and MBP. Images show the region dorsal fasciculus. White arrowheads point to the disruption of the pia surface in wt. Scale bar 100μm. D) Quantification of CD4+ infiltrated lymphocytes, Iba1 normalised immunoreactivity, MBP normalised immunoreactivity and MBP immune-reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1-/- mice 16 days after immunization (n=16 acquisitions, white dots; from N=4 animals, black dots, per genotype). Error bars represent Standard Error. Statistic refers to number of acquisitions.


D ) ELISA determination of SFRP1 levels present in the media of microglia ( n = 4 cultures per genotype ) , astrocytes ( n = 4 cultures per genotype ) or mixed astrocytes and microglial ( 2 : 1 , n = 7 cultures per genotype ) cultures derived from wt or Sfrp1 - / - pups exposed for 24h to saline or LPS ( 1μg / ml ) . Error bars represent Standard Error . Statistical significance : * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; One - way ANOVA followed by Bonferroni 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 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

D) ELISA determination of SFRP1 levels present in the media of microglia (n=4 cultures per genotype), astrocytes (n=4 cultures per genotype) or mixed astrocytes and microglial (2:1, n=7 cultures per genotype) cultures derived from wt or Sfrp1-/- pups exposed for 24h to saline or LPS (1μg/ml). Error bars represent Standard Error. Statistical significance: *P<0.05; ***P<0.001; ****P<0.0001; One-way ANOVA followed by Bonferroni Test.


Secretory profile of cytokines present in the medium of microglia , astrocytes or mixed astrocytes and microglial ( 2 : 1 ) culture as above ( n = 5 - 6 cultures type - microglia , astrocytes or mixed cultures - and genotype ) . The content of IL1β , IL10 was determined by Multiplex ELISA . Values are represented in absolute concentration . Error bars represent Standard Error . Statistical significance : ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons 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

Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IL1β, IL10 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.


F ) Secretory profile of cytokines present in the medium of microglia , astrocytes or mixed astrocytes and microglial ( 2 : 1 ) culture as above ( n = 5 - 6 cultures type - microglia , astrocytes or mixed cultures - and genotype ) . The content of IFNγ , TNFα , IL4 , IL6 was determined by Multiplex ELISA . Values are represented in absolute concentration . Error bars represent Standard Error . Statistical significance : ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons 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

F) Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IFNγ, TNFα, IL4, IL6 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.


B , C ) Volcano plots of differential gene expression from CX3CR1GFP / + ( B ) or CX3CR1GFP / + ; Sfrp1 - / - ( C ) microglial cell in response to LPS . Data are represented as Log2 Fold Change vs - Log10 adjusted p - value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method . Blue vertical lines represent an increase of 75 and 400 % in the expression levels respectively . Green horizontal lines represent a 0 . 05 or 0 . 01 adjusted p - value of statistical significance respectively . Genes with an expression change higher than 75 % and an adjusted p - value lower than 0 . 05 are highlighted in dark red ( + FC ) and dark blue ( - FC ) . Genes with an expression change higher than 400 % and an adjusted p - value lower than 0 . 01 are coloured in light red ( + FC ) and light blue ( - FC ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plots of differential gene expression from CX3CR1GFP/+ (B) or CX3CR1GFP/+;Sfrp1-/- (C) microglial cell in response to LPS. Data are represented as Log2 Fold Change vs -Log10 adjusted p-value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method. Blue vertical lines represent an increase of 75 and 400% in the expression levels respectively. Green horizontal lines represent a 0.05 or 0.01 adjusted p-value of statistical significance respectively. Genes with an expression change higher than 75% and an adjusted p-value lower than 0.05 are highlighted in dark red (+FC) and dark blue (-FC). Genes with an expression change higher than 400% and an adjusted p-value lower than 0.01 are coloured in light red (+FC) and light blue (-FC).


B ) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP / + and CX3CR1GFP / + ; Sfrp1 - / - mouse brains . Scale bar : 2 Kbp .

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) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- mouse brains. Scale bar: 2 Kbp.


B - D . Examples of Min protein patterns emerging from nearly homogeneous initial conditions in E . coli cells of different sizes . Lateral dimensions ( in µm ) from top to bottom : 2x6 . 5 , 2x8 . 8 , and 5 . 2x8 . 8 respectively . The gray - scale images show cytosolic near - infrared fluorescence emitted by the protein eqFP670 at the first ( left ) and last ( right ) time points . The color montages show the sfGFP - MinD intensity ( indicated by the color scale at the bottom right ) over time . The scale bar in panel B corresponds to 5 μm . Red arrows show the oscillation mode at the respective time point .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. Examples of Min protein patterns emerging from nearly homogeneous initial conditions in E. coli cells of different sizes. Lateral dimensions (in µm) from top to bottom: 2x6.5, 2x8.8, and 5.2x8.8 respectively. The gray-scale images show cytosolic near-infrared fluorescence emitted by the protein eqFP670 at the first (left) and last (right) time points. The color montages show the sfGFP-MinD intensity (indicated by the color scale at the bottom right) over time. The scale bar in panel B corresponds to 5 μm. Red arrows show the oscillation mode at the respective time point.


E . Two early and two late frames depicting sfGFP - MinD patterns in a cell exhibiting stable transverse oscillations . The images share the scale bar in B .

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E. Two early and two late frames depicting sfGFP-MinD patterns in a cell exhibiting stable transverse oscillations. The images share the scale bar in B.


F . Difference in sfGFP - MinD intensity between the top half and bottom half of the cell plotted against time .

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F. Difference in sfGFP-MinD intensity between the top half and bottom half of the cell plotted against time.


Figure 3 . Computing stability in multistability regimes . A . Two plots that show the non - degeneracy of even and odd modes in an elliptical geometry for varying cell geometry and MinD recruitment rate . The degeneracy ( light blue area ) increases with the MinD recruitment rate .

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

Figure 3. Computing stability in multistability regimes. A. Two plots that show the non-degeneracy of even and odd modes in an elliptical geometry for varying cell geometry and MinD recruitment rate. The degeneracy (light blue area) increases with the MinD recruitment rate.


Figure 4 . Basins of attraction predicted from systematic perturbations of patterns with shallow attachment gradients . A . Relative distribution of the final patterns ( indicated on the right ) observed after sampling all alignment angles of the MinD attachment template from 0 to 90 degrees . The MinD recruitment rate was set to a constant value kdD = 0 . 1 . The data shows the increase in the incidence of multistability as the cell size is increased beyond minimal values for cell length and cell width . B . Fractions of the final patterns in cells of 9 - and 10 - µm length after sampling all alignment angles of the MinD attachment template from 0 to 90 degrees . The data shows that increasing the MinD recruitment rate facilitates multistability .

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Figure 4. Basins of attraction predicted from systematic perturbations of patterns with shallow attachment gradients. A. Relative distribution of the final patterns (indicated on the right) observed after sampling all alignment angles of the MinD attachment template from 0 to 90 degrees. The MinD recruitment rate was set to a constant value kdD = 0.1. The data shows the increase in the incidence of multistability as the cell size is increased beyond minimal values for cell length and cell width. B. Fractions of the final patterns in cells of 9- and 10-µm length after sampling all alignment angles of the MinD attachment template from 0 to 90 degrees. The data shows that increasing the MinD recruitment rate facilitates multistability.


Figure 5 . The effect of cell - shape change during growth on the stability of Min protein patterns . A . Cytosolic fluorescence during growth of a cell from a small elliptical form into a large rectangular shape . Numbers in red indicate time in hours . Illustrations show the positions and orientations of the cell in the first and last time frames . Green and blue lines indicate the maximum and minimum Feret diameters , respectively . B . sfGFP - MinD patterns during the growth of the cell shown in A . Illustrations indicate the cell boundaries and oscillation angles observed in the first and last frames ( not to scale ) . C . Quantitative data obtained from the cell shown in A and B . The maximum and minimum Feret diameters ( green and blue ) , and the measured MinD oscillations ( red ) were expressed in terms of length ( top ) and angle ( bottom ) and plotted against time . The number of cells that fit this category was 41 / 97 . Arrows indicate the time when cell width reached the chamber width of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5. The effect of cell-shape change during growth on the stability of Min protein patterns. A. Cytosolic fluorescence during growth of a cell from a small elliptical form into a large rectangular shape. Numbers in red indicate time in hours. Illustrations show the positions and orientations of the cell in the first and last time frames. Green and blue lines indicate the maximum and minimum Feret diameters, respectively. B. sfGFP-MinD patterns during the growth of the cell shown in A. Illustrations indicate the cell boundaries and oscillation angles observed in the first and last frames (not to scale). C. Quantitative data obtained from the cell shown in A and B. The maximum and minimum Feret diameters (green and blue), and the measured MinD oscillations (red) were expressed in terms of length (top) and angle (bottom) and plotted against time. The number of cells that fit this category was 41/97. Arrows indicate the time when cell width reached the chamber width of 5 μm.


D - F . Data are presented as in A - C for another cell that showed persistent oscillations along the horizontal axis throughout growth . The number of cells that fit this category was 28 / 97 .

O O O O O O O O O O O O O O O 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-F. Data are presented as in A-C for another cell that showed persistent oscillations along the horizontal axis throughout growth. The number of cells that fit this category was 28/97.


G . Time - lapse images of sfGFP - MinD that reveal stochastic switching of patterns in a cell with an asymmetric shape and a low aspect ratio . White arrows indicate the oscillation axes .

O O O O O O O O O O O O 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. Time-lapse images of sfGFP-MinD that reveal stochastic switching of patterns in a cell with an asymmetric shape and a low aspect ratio. White arrows indicate the oscillation axes.


H . The angles of the maximum and minimum Feret diameters ( green and blue ) , and the measured MinD clusters ( red ) for the cell shown in panel G plotted against time . The number of cells that fit this category was 10 / 97 . All scale bars correspond to 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

H. The angles of the maximum and minimum Feret diameters (green and blue), and the measured MinD clusters (red) for the cell shown in panel G plotted against time. The number of cells that fit this category was 10/97. All scale bars correspond to 5 μm.


Figure 6 . Transitions between various modes of Min protein patterns . A . Time - lapse images showing the transition from a longitudinal pole - to - pole mode to the transverse mode . Scale bar , 5 μm . B . Time - lapse images showing the transition from a transverse mode to a longitudinal pole - to - pole mode .

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

Figure 6. Transitions between various modes of Min protein patterns. A. Time-lapse images showing the transition from a longitudinal pole-to-pole mode to the transverse mode. Scale bar, 5 μm. B. Time-lapse images showing the transition from a transverse mode to a longitudinal pole-to-pole mode.


C . Bar plot showing the distribution of the number of transitions . Inset : Data from experiments carried out under nutrient - poor conditions in which growth rates are reduced .

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C. Bar plot showing the distribution of the number of transitions. Inset: Data from experiments carried out under nutrient-poor conditions in which growth rates are reduced.


D . Representative time - course of a change in the mode of sfGFP - MinD oscillation . The black line is a sigmoidal fit . The dashed black lines indicate 15 ° and 75 ° and the dashed red line indicates 45 ° .

O O O O O O O O O O O O O O O O O O O O O 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. Representative time-course of a change in the mode of sfGFP-MinD oscillation. The black line is a sigmoidal fit. The dashed black lines indicate 15° and 75° and the dashed red line indicates 45°.


E . Bar plot showing the time scale of the switch in the oscillations . Inset : Data from experiments carried out in nutrient - poor conditions .

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E. Bar plot showing the time scale of the switch in the oscillations. Inset: Data from experiments carried out in nutrient-poor conditions.


F . Bar plots showing the relative numbers of the indicated transitions that occur at different cell lengths . All cells have a width of 5 μm .

F. Bar plots showing the relative numbers of the indicated transitions that occur at different cell lengths. All cells have a width of 5 μm.


G . Distribution of final patterns in cells of the indicated widths as indicates , and lengths of 9 - 10 μm .

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G. Distribution of final patterns in cells of the indicated widths as indicates, and lengths of 9-10 μm.


H . Time - lapse images of various modes of transitions between patterns . Cell sizes from top to bottom are respectively 10x2x1 , 10x6x1 , 9x5x1 , 10x4x1 μm3 . Note that the cells are scaled differently . On the right is an illustration showing Min pattern transitions through intermediate states .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time-lapse images of various modes of transitions between patterns. Cell sizes from top to bottom are respectively 10x2x1, 10x6x1, 9x5x1, 10x4x1 μm3. Note that the cells are scaled differently. On the right is an illustration showing Min pattern transitions through intermediate states.


U2Os cells co - transfected with YFP - FKBP and PCM1F2 - Ce3 - FRB , PACT - Ce3 - FRB , or Ce3 - FRB - Centrin2 were treated with rapamycin ( 100 nM ) . The translocation of YFP - FKBP onto PCM1F2 - Ce3 - FRB - labeled centriolar satellites , the PACT - Ce3 - FRB - labeled pericentriolar matrix , and the Ce3 - FRB - Centrin2 - labeled centriolar lumen was monitored . The normalized fluorescence intensity of YFP - FKBP accumulation at centrosomes upon rapamycin ( 100 nM ; blue ) and DMSO ( 0 . 1 % , vehicle control ; red ) treatment . Data are shown as the mean ± S . E . M . The graphs show immediate translocation of YFP - FKBP after rapamycin induction at centriolar satellites ( left , n = 24 cells ) , the pericentriolar matrix ( middle , n = 20 cells ) , and the centriolar lumen ( right , n = 23 cells ) from seven independent experiments .

O O O O O O O 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 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 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

U2Os cells co-transfected with YFP-FKBP and PCM1F2-Ce3-FRB, PACT-Ce3-FRB, or Ce3-FRB-Centrin2 were treated with rapamycin (100 nM). The translocation of YFP-FKBP onto PCM1F2- Ce3-FRB-labeled centriolar satellites, the PACT-Ce3-FRB-labeled pericentriolar matrix, and the Ce3-FRB-Centrin2-labeled centriolar lumen was monitored. The normalized fluorescence intensity of YFP-FKBP accumulation at centrosomes upon rapamycin (100 nM; blue) and DMSO (0.1%, vehicle control; red) treatment. Data are shown as the mean ± S.E.M. The graphs show immediate translocation of YFP-FKBP after rapamycin induction at centriolar satellites (left, n = 24 cells), the pericentriolar matrix (middle, n = 20 cells), and the centriolar lumen (right, n = 23 cells) from seven independent experiments.


U2Os cells co - transfected with each YFP - FKBP - labeled varisized probe ( YFP - FKBP , YFP - FKBP - Grp1 , YFP - FKBP - Luciferase , YFP - PSD95 - FKBP , YFP - FKBP - β - Gals , YFP - FKBP - β - Galm , YFP - FKBP - ΔNβ - Gal , and YFP - FKBP - β - Gal ) and Ce3 - FRB with labels specific for centrosomal sub - compartments were treated with rapamycin ( 100 nM ) . The images of cerulean channel show centrosome sub - compartment from the untreated cells . Arrowheads indicate sites of centrosomes . Insets show higher - magnification images of the centrosomal regions . Dotted lines indicate the cell boundaries . The normalized fluorescence intensity of each probe at centriolar satellites ( left , n = 14 , 19 , 17 , 13 , 11 , 22 , 14 , and 9 cells from small to large probes ) , pericentriolar matrix ( middle , n = 20 , 19 , 18 , 16 , 10 , 18 , 10 , and 12 cells from small to large probes ) , and centriolar lumen ( right , n = 10 , 13 , 13 , 6 , 3 , 19 , 13 , and 9 cells from small to large probes ) from 3 - 6 independent experiments .

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

U2Os cells co-transfected with each YFP-FKBP-labeled varisized probe (YFP-FKBP, YFP-FKBP-Grp1, YFP-FKBP-Luciferase, YFP-PSD95-FKBP, YFP-FKBP-β-Gals, YFP-FKBP-β-Galm, YFP-FKBP-ΔNβ-Gal, and YFP-FKBP-β-Gal) and Ce3-FRB with labels specific for centrosomal sub-compartments were treated with rapamycin (100 nM). The images of cerulean channel show centrosome sub-compartment from the untreated cells. Arrowheads indicate sites of centrosomes. Insets show higher-magnification images of the centrosomal regions. Dotted lines indicate the cell boundaries. The normalized fluorescence intensity of each probe at centriolar satellites (left, n = 14, 19, 17, 13, 11, 22, 14, and 9 cells from small to large probes), pericentriolar matrix (middle, n = 20, 19, 18, 16, 10, 18, 10, and 12 cells from small to large probes), and centriolar lumen (right, n = 10, 13, 13, 6, 3, 19, 13, and 9 cells from small to large probes) from 3-6 independent experiments.


Depolymerization of microtubules by Nocodazole ( 10 µM ) treatment . U2Os cells that expressed PACT - Ce3 - FRB ( blue , a marker of centrosomes ) were incubated with antibodies against α - tubulin to label microtubules ( red ) in the absence ( 0 . 1 % DMSO ) or presence of Nocodazole treatment for 20 min . Lower panels show magnified images of the centrosomal regions . The normalized intensity of α - tubulin around centrosomes in the absence or presence of Nocodazole treatment for 20 min . The circular ROIs in diameter of 3 µm , centering PACT - labeled centrosomes , were used for measurement of the level of microtubule around centrosomes . Data ( black ) are shown as the mean ± SD . ( n = 196 and 156 cells from left to right ; 3 independent experiments ) .

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 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 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

Depolymerization of microtubules by Nocodazole (10 µM) treatment. U2Os cells that expressed PACT-Ce3-FRB (blue, a marker of centrosomes) were incubated with antibodies against α-tubulin to label microtubules (red) in the absence (0.1% DMSO) or presence of Nocodazole treatment for 20 min. Lower panels show magnified images of the centrosomal regions. The normalized intensity of α-tubulin around centrosomes in the absence or presence of Nocodazole treatment for 20 min. The circular ROIs in diameter of 3 µm, centering PACT-labeled centrosomes, were used for measurement of the level of microtubule around centrosomes. Data (black) are shown as the mean ± SD. (n = 196 and 156 cells from left to right; 3 independent experiments).


U2Os cells expressing PACT - Ce3 - FRB ( blue , a marker of centrosomes ) were incubated with phalloidin to label the F - actin ( red ) with DMSO ( 0 . 1 % ) , CK666 ( 0 . 4 mM ) for 2 hr , or Latrunculin A ( LatA , 0 . 5 mM ) for 1 hr . Lower panels show the magnified images of the centrosomal regions . The normalized intensity of actin filaments around centrosomes in the absence or presence of CK666 or LatA treatment . The circular ROIs in diameter of 3 µm , centering PACT - labeled centrosomes , were used for measurement of the level of actin filaments around centrosomes .

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

U2Os cells expressing PACT-Ce3-FRB (blue, a marker of centrosomes) were incubated with phalloidin to label the F-actin (red) with DMSO (0.1%), CK666 (0.4 mM) for 2 hr, or Latrunculin A (LatA, 0.5 mM) for 1 hr. Lower panels show the magnified images of the centrosomal regions. The normalized intensity of actin filaments around centrosomes in the absence or presence of CK666 or LatA treatment. The circular ROIs in diameter of 3 µm, centering PACT-labeled centrosomes, were used for measurement of the level of actin filaments around centrosomes.


HeLa cells co - transfected with H2B - RFP ( a chromosome marker ) , PACT - Ce3 - FRB , and YFP - FKBP - Luciferase were sequentially incubated with thymidine ( 2 mM ) for 16 - 18 hr and RO3306 ( 2 . 5 ng / mL ) for 12 hr to synchronize cells in G2 / M phase ( Interphase cells in left panel ) . The probe trapping experiment in interphase cells was performed right after RO3306 washout . Anaphase cells were chosen for probe trapping experiment 45 - 60 min after RO3306 washout . Transfected cells in interphase or anaphase were treated with 100 nM rapamycin ( Rapa ) . The centrosome regions are shown in lower panels . The normalized fluorescence intensity of YFP - FKBP - Luciferase accumulation at centrosomes in interphase ( Green , n = 12 cells ) or anaphase ( Red , n = 10 cells ) cells upon rapamycin ( 100 nM ) treatment .

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

HeLa cells co-transfected with H2B-RFP (a chromosome marker), PACT-Ce3-FRB, and YFP-FKBP-Luciferase were sequentially incubated with thymidine (2 mM) for 16-18 hr and RO3306 (2.5 ng/mL) for 12 hr to synchronize cells in G2/M phase (Interphase cells in left panel). The probe trapping experiment in interphase cells was performed right after RO3306 washout. Anaphase cells were chosen for probe trapping experiment 45-60 min after RO3306 washout. Transfected cells in interphase or anaphase were treated with 100 nM rapamycin (Rapa). The centrosome regions are shown in lower panels. The normalized fluorescence intensity of YFP-FKBP-Luciferase accumulation at centrosomes in interphase (Green, n = 12 cells) or anaphase (Red, n =10 cells) cells upon rapamycin (100 nM) treatment.


HeLa cells co - transfected with H2B - RFP ( a chromosome marker ) , PACT - Ce3 - FRB , and YFP - FKBP - Luciferase were synchronized in anaphase with or without CK666 treatment ( 0 . 4 mM ) . Rapamycin ( 100 nM ) was added for 5 min to trigger the YFP - FKBP - Luciferase trapping at centrosomes . The right panel is the enlarged images of centrosome regions and arrows indicated centrosome sites . The percentage of cells in D exhibiting probe translocation . Data are shown as mean ± S . E . M . ( n = 47 and 31 cells in DMSO and CK666 treated groups , respectively ; 3 - 4 independent experiments ) .

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

HeLa cells co-transfected with H2B-RFP (a chromosome marker), PACT-Ce3-FRB, and YFP-FKBP-Luciferase were synchronized in anaphase with or without CK666 treatment (0.4 mM). Rapamycin (100 nM) was added for 5 min to trigger the YFP-FKBP-Luciferase trapping at centrosomes. The right panel is the enlarged images of centrosome regions and arrows indicated centrosome sites. The percentage of cells in D exhibiting probe translocation. Data are shown as mean ± S.E.M. (n = 47 and 31 cells in DMSO and CK666 treated groups, respectively; 3-4 independent experiments).


U2Os cells were incubated with antibodies against 𝑟 - tubulin ( green ; a marker of 𝑟 - TuRC ) or Pericentrin ( red ; a marker of centrosomes ) in the absence ( 0 . 1 % DMSO ) or presence of CK666 treatment ( 0 . 4 mM ) for 2 hr . The right panel shows magnified images of the indicated region of interest ( ROI ) . S The normalized intensity of 𝑟 - tubulin at centrosome in control ( 0 . 1 % DMSO , 2 hr ) or CK666 ( 0 . 4 mM , 2 hr ) - treated cells . Individual data points and the mean ± SD ( in red ) are shown ( n = 150 and 150 cells in DMSO and CK666 - treated group , respectively ; four independent experiments ) .

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

U2Os cells were incubated with antibodies against 𝑟-tubulin (green; a marker of 𝑟-TuRC) or Pericentrin (red; a marker of centrosomes) in the absence (0.1% DMSO) or presence of CK666 treatment (0.4 mM) for 2 hr. The right panel shows magnified images of the indicated region of interest (ROI). S The normalized intensity of 𝑟-tubulin at centrosome in control (0.1% DMSO, 2 hr) or CK666 (0.4 mM, 2 hr)-treated cells. Individual data points and the mean ± SD (in red) are shown (n = 150 and 150 cells in DMSO and CK666-treated group, respectively; four independent experiments).


U2Os cells transfected with GCP4 - GFP were treated with or without CK666 ( 0 . 4 mM , 2 hr ) and then observed for the indicated time after photobleaching . The mobile fraction percentage ( left ) and mobile recovery half - time ( right ) of GCP4 - GFP in FRAP experiments ( c ) . Data ( red ) are shown as

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

U2Os cells transfected with GCP4-GFP were treated with or without CK666 (0.4 mM, 2 hr) and then observed for the indicated time after photobleaching. The mobile fraction percentage (left) and mobile recovery half-time (right) of GCP4- GFP in FRAP experiments (c). Data (red) are shown as mean


U2Os cells expressing EB1 - mNeon were incubated with 0 . 1 % DMSO or 0 . 4 mM CK666 for 2 hr . Images were captured over 150 sec and were overlaid to show the overall tracks of the microtubules . The insets show enlarged images of the centrosomal regions . F , G . The number of microtubule tracks that emanate from centrosomes ( F ) and the microtubule elongation rate ( G ) in the absence and presence of CK666 treatment ( 0 . 4 mM ) . Individual data points and the mean ± SD ( in red ) are shown ( n = 78449 and 54283 microtubule tracks from 27 and 22 cells in the DMSO - and CK666 - treated groups , respectively ; three independent experiments ) .

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

U2Os cells expressing EB1-mNeon were incubated with 0.1% DMSO or 0.4 mM CK666 for 2 hr. Images were captured over 150 sec and were overlaid to show the overall tracks of the microtubules. The insets show enlarged images of the centrosomal regions. F, G. The number of microtubule tracks that emanate from centrosomes (F) and the microtubule elongation rate (G) in the absence and presence of CK666 treatment (0.4 mM). Individual data points and the mean ± SD (in red) are shown (n = 78449 and 54283 microtubule tracks from 27 and 22 cells in the DMSO- and CK666-treated groups, respectively; three independent experiments).


( A - C ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS ( Earle ' s Balanced Salt Solution ) for 30 min . Total membranes ( mem ) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells . Reactions contained the indicated concentrations of PI3K inhibitor ( PI3KI ) 3 - methyladenine ( 3 - MA ) ( B ) or FYVE protein ( C ) . A diagram of the experimental scheme is shown in ( A ) . RPN1 , Ribophorin

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

(A-C) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS (Earle's Balanced Salt Solution) for 30 min. Total membranes (mem) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells. Reactions contained the indicated concentrations of PI3K inhibitor (PI3KI) 3-methyladenine (3-MA) (B) or FYVE protein (C). A diagram of the experimental scheme is shown in (A). RPN1, Ribophorin 1


( D , E ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS in the absence or presence of 20 nM wortmannin ( Wtm ) or 10 mM 3 - methyladenine ( 3 - MA ) for 30 min . Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K × g , 25K × g and 100K × g pellet fractions followed by a lipidation assay as above ( E ) . A diagram is shown in ( D ) .

O O O O O 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 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 O O O O O 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) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS in the absence or presence of 20 nM wortmannin (Wtm) or 10 mM 3-methyladenine (3-MA) for 30 min. Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K ×g, 25K ×g and 100K ×g pellet fractions followed by a lipidation assay as above (E). A diagram is shown in (D).


( F , G ) Atg5 KO MEFs were starved for 30 min . Membranes in the 25K × g and 100K × g pellets from a differential centrifugation were collected as described above . A similar lipidation assay was performed in the presence of indicated concentrations ( Conc in G ) of 3 - MA , wortmannin ( F ) and FYVE protein as well as a PI3P binding - deficient FYVE mutant protein ( C / S ) ( G ) . Quantification of lipidation activity is shown as the ratio of LC3 - II to LC3 - I ( II / I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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) Atg5 KO MEFs were starved for 30 min. Membranes in the 25K ×g and 100K ×g pellets from a differential centrifugation were collected as described above. A similar lipidation assay was performed in the presence of indicated concentrations (Conc in G) of 3-MA, wortmannin (F) and FYVE protein as well as a PI3P binding-deficient FYVE mutant protein (C/S) (G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).


( B ) A small vesicle generation assay as above was performed with the indicated conditions . Slowly - sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction . 3 - MA , 5 mM ; Wortmannin , 20 nM ; S , starved Atg5 KO MEF cytosol ; N , untreated Atg5 KO MEF cytosol ; − / AP , in the absence of ATP regeneration and GTP and in the presence of apyrase ( AP ) .

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

(B) A small vesicle generation assay as above was performed with the indicated conditions. Slowly-sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction. 3-MA, 5 mM; Wortmannin, 20 nM; S, starved Atg5 KO MEF cytosol; N, untreated Atg5 KO MEF cytosol; −/AP, in the absence of ATP regeneration and GTP and in the presence of apyrase (AP).


( D ) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K , P and F1 - 4 fractions ( upper four panels : Small vesicle ) . SDS - PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction ( lower five panels : Fraction input ) . TFR , transferrin

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K, P and F1-4 fractions (upper four panels: Small vesicle). SDS-PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction (lower five panels: Fraction input). TFR, transferrin receptor


( E ) A small vesicle generation assay as shown in ( A ) was performed in the presence of indicated proteins or drugs . Slowly - sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST , H79G ( Sar1A ( H79G ) , 0 . 7 μM ) , wortmannin ( 20 nM ) , or BFA ( brefeldin A , 0 . 5 μg / ml ) .

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

(E) A small vesicle generation assay as shown in (A) was performed in the presence of indicated proteins or drugs. Slowly-sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST, H79G (Sar1A (H79G), 0.7 μM), wortmannin (20 nM), or BFA (brefeldin A, 0.5 μg/ml).


( F ) A small vesicle generation assay as shown in ( A ) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A ( H79G ) or 3 - MA followed by a lipidation reaction .

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

(F) A small vesicle generation assay as shown in (A) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A (H79G) or 3-MA followed by a lipidation reaction.