Left panel : Quantification of non - esterified cholesterol content by means of filipin staining in hearts of C57BL / 6 male mice after 25 weeks on HFD , long - term treated with isotype control ( n = 17 ) or VEGF - B blocking antibody ( 2H10 , n = 17 ) initiated after 5 weeks on HFD , normalized to age - matched mice on chow diet ( n = 10 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Right panel : Body weights ( mean ± SEM ) of HFD ( n = 17 + 17 ) and chow fed littermate mice ( n = 10 ) . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * * < 0 . 001 ( compared to HFD control mice ) .

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Left panel: Quantification of non-esterified cholesterol content by means of filipin staining in hearts of C57BL/6 male mice after 25 weeks on HFD, long-term treated with isotype control (n=17) or VEGF-B blocking antibody (2H10, n=17) initiated after 5 weeks on HFD, normalized to age-matched mice on chow diet (n=10). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Right panel: Body weights (mean ± SEM) of HFD (n=17+17) and chow fed littermate mice (n=10). Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, *** <0.001 (compared to HFD control mice).


Quantification of non - esterified cholesterol content by means of filipin staining in hearts of mice on HFD for 17 weeks followed by treatment for 1 week with 2H10 antibody ( n = 5 ) compared to control antibody treated mice ( n = 5 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using t - test , p - value : * * < 0 . 01 .

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

Quantification of non-esterified cholesterol content by means of filipin staining in hearts of mice on HFD for 17 weeks followed by treatment for 1 week with 2H10 antibody (n=5) compared to control antibody treated mice (n=5). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values.Statistical evaluation using t-test, p-value: ** <0.01.


At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA ( 10 nM each ) , the intracellular localization of TfnR in HeLa cells was examined ( shown as green ) . Nuclear DNA was stained with Hoechst 33342 ( shown as blue ) . Control knockdown ( left panel ) , Rab8a knockdown ( center panel ) , and BAG6 knockdown ( right panel ) .

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At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA (10 nM each), the intracellular localization of TfnR in HeLa cells was examined (shown as green). Nuclear DNA was stained with Hoechst 33342 (shown as blue). Control knockdown (left panel), Rab8a knockdown (center panel), and BAG6 knockdown (right panel).


Intracellular localization of Ptc1 ( green ) in HeLa cells . Nuclei were stained with Hoechst 33342 ( shown as blue ) .

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Intracellular localization of Ptc1 (green) in HeLa cells. Nuclei were stained with Hoechst 33342 (shown as blue).


Knockdown of Rab8a ( with Rab8a siRNA # 1 , # 2 , and # 3 ) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells .

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Knockdown of Rab8a (with Rab8a siRNA#1, #2, and #3) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells.


Knockdown of BAG6 ( with BAG6 siRNA # 1 ) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells .

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

Knockdown of BAG6 (with BAG6 siRNA#1) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells.


BAG6 protein co - precipitated Rab8a , while neither Rab7 nor Rab11a were co - precipitated with BAG6 . Flag - tagged Rab8a , Rab7 , Rab11a , and luciferase - CL1 ( Lc - CL1 ; a positive control ) were expressed in HeLa cells and the cells were treated with 10 μM MG - 132 for 4 h . Flag - immunoprecipitates were blotted with anti - BAG6 and anti - Flag antibodies , respectively . Note that all cells used were treated with 10 μM MG - 132 for 4 h .

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BAG6 protein co-precipitated Rab8a, while neither Rab7 nor Rab11a were co-precipitated with BAG6. Flag-tagged Rab8a, Rab7, Rab11a, and luciferase-CL1 (Lc-CL1; a positive control) were expressed in HeLa cells and the cells were treated with 10 μM MG-132 for 4 h. Flag-immunoprecipitates were blotted with anti-BAG6 and anti-Flag antibodies, respectively. Note that all cells used were treated with 10 μM MG-132 for 4 h.


S - tagged BAG6 pull - down efficiently co - precipitated Rab8a ( T22N ) , a GDP - bound mutant , while BAG6 scarcely co - precipitated Rab8a ( Q67L ) , a constitutively active mutant . Co - precipitation of Rab8a WT with BAG6 was used as a standard . MG - 132 ( 10 μM ) was included in the cell culture for 4 h , as indicated . Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L , and that this was partly due to increased degradation , as will be shown later . S - BAG6 stands for N - terminally S - tagged BAG6

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S-tagged BAG6 pull-down efficiently co-precipitated Rab8a (T22N), a GDP-bound mutant, while BAG6 scarcely co-precipitated Rab8a (Q67L), a constitutively active mutant. Co-precipitation of Rab8a WT with BAG6 was used as a standard. MG-132 (10 μM) was included in the cell culture for 4 h, as indicated. Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L, and that this was partly due to increased degradation, as will be shown later. S-BAG6 stands for N-terminally S-tagged BAG6 protein


Anti - Flag signals in ( C ) were quantified , and relative signal intensities are presented . The value of the WT Rab8a signal with MG - 132 was defined as 1 . 0 . Note that all signal intensities of Flag - tag were normalized by that of the Rab8a input signals .

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Anti-Flag signals in (C) were quantified, and relative signal intensities are presented. The value of the WT Rab8a signal with MG-132 was defined as 1.0. Note that all signal intensities of Flag-tag were normalized by that of the Rab8a input signals.


A series of Flag - Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag - Rab8a . Note that all cells used were treated with 10 μM MG - 132 for 4 h .

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A series of Flag-Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag-Rab8a. Note that all cells used were treated with 10 μM MG-132 for 4 h.


Deficiency of Rabin8 , a GEF for Rab8a , enhanced the physical interaction between BAG6 and WT Rab8a proteins . Flag - tagged WT Rab8a was expressed in Rabin8 siRNA - treated cells , and Flag immunoprecipitates were probed with an anti - BAG6 antibody . Note that all cells used were treated with 10 μM MG - 132 for 4 h .

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

Deficiency of Rabin8, a GEF for Rab8a, enhanced the physical interaction between BAG6 and WT Rab8a proteins. Flag-tagged WT Rab8a was expressed in Rabin8 siRNA-treated cells, and Flag immunoprecipitates were probed with an anti-BAG6 antibody. Note that all cells used were treated with 10 μM MG-132 for 4 h.


C - terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition .

C-terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition.


The N - terminal GTPase domain of Rab8a was essential for BAG6 recognition . A series of Flag - tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S - tagged BAG6 and treated with ( + ) or without ( - ) protease inhibitors for 4 h . Flag - Rab8a substrates were immunoprecipitated and probed with an anti - BAG6 antibody .

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The N-terminal GTPase domain of Rab8a was essential for BAG6 recognition. A series of Flag-tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S-tagged BAG6 and treated with (+) or without (-) protease inhibitors for 4 h. Flag-Rab8a substrates were immunoprecipitated and probed with an anti-BAG6 antibody.


Hydrophobicity recognition domain of BAG6 ( N465 ) was critical for Rab8a recognition . A schematic representation of the BAG6 - truncated proteins used in this experiment is shown in the upper panel . Numbers denote the corresponding amino acids of mammalian BAG6 . Positions of UBL , BUILD , and DUF3538 domains , which are all linked to hydrophobicity recognition by BAG6 [ 37 ] , are indicated . WT , wild - type ; ∆ N465 , N - terminal 465 residues - deleted mutant ; and Ν465 , C - terminal 689 residues - deleted mutant .

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Hydrophobicity recognition domain of BAG6 (N465) was critical for Rab8a recognition. A schematic representation of the BAG6-truncated proteins used in this experiment is shown in the upper panel. Numbers denote the corresponding amino acids of mammalian BAG6. Positions of UBL, BUILD, and DUF3538 domains, which are all linked to hydrophobicity recognition by BAG6 [37], are indicated. WT, wild-type; ∆N465, N-terminal 465 residues-deleted mutant; and Ν465, C-terminal 689 residues-deleted mutant.


CHX chase experiments show that GDP - bound Rab8a ( T22N ) was a highly labile protein , while the GTP - bound active mutant ( Q67L ) was a stable protein ( E ) . Instability of the T22N mutant was not perturbed by the C204S mutation ( F ) . Actin was used as a loading control .

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CHX chase experiments show that GDP-bound Rab8a (T22N) was a highly labile protein, while the GTP-bound active mutant (Q67L) was a stable protein (E). Instability of the T22N mutant was not perturbed by the C204S mutation (F). Actin was used as a loading control.


N - terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor , while the ∆ N100 fragment was not .

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N-terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor, while the ∆N100 fragment was not.


Rab8a ( T22N ) was polyubiquitinated in the presence of the proteasome inhibitor . Asterisks indicate non - specific bands .

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Rab8a (T22N) was polyubiquitinated in the presence of the proteasome inhibitor. Asterisks indicate non-specific bands.


Kyte - Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N - 3IS mutant . The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant ( indicated within the red box ) . The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins .

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Kyte-Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N-3IS mutant. The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant (indicated within the red box). The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins.


Substitution of the hydrophobic residues of Switch I with hydrophilic residues ( T22N 3IS ) abolished its binding to BAG6 protein .

Substitution of the hydrophobic residues of Switch I with hydrophilic residues (T22N 3IS) abolished its binding to BAG6 protein.


Rab8a ( T22N - 3IS ) mutant protein was highly stable , while Rab8a ( T22N ) protein was quite unstable in HeLa cells . Actin was used as a loading control .

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Rab8a (T22N-3IS) mutant protein was highly stable, while Rab8a (T22N) protein was quite unstable in HeLa cells. Actin was used as a loading control.


Rab8a ( T22N - 3IS ) mutant was not subject to polyubiquitin co - precipitation , while Rab8a ( T22N ) protein was .

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

Rab8a (T22N-3IS) mutant was not subject to polyubiquitin co-precipitation, while Rab8a (T22N) protein was.


Rab8a ( T22N ) protein accumulated in BAG6 - knockdown cells . HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA . At 48 h after siRNA transfection , Flag tagged - Rab8a ( T22N ) was expressed in the cells . At 24 h after Rab8a ( T22N ) transfection , the cells were chased with 50 μg / mL CHX and harvested at the indicated time after CHX addition . Actin was used as a loading control .

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Rab8a (T22N) protein accumulated in BAG6-knockdown cells. HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA. At 48 h after siRNA transfection, Flag tagged-Rab8a (T22N) was expressed in the cells. At 24 h after Rab8a (T22N) transfection, the cells were chased with 50 μg/mL CHX and harvested at the indicated time after CHX addition. Actin was used as a loading control.


Anti - Flag blot signals in the control or BAG6 siRNA - treated cells were quantified , and relative signal intensities after CHX addition were calculated . The value of the Flag signal at 0 h was defined as 1 . 0 . Note that all signal intensities of the Flag - tag were normalized by that of actin , a loading control , in each sample .

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Anti-Flag blot signals in the control or BAG6 siRNA-treated cells were quantified, and relative signal intensities after CHX addition were calculated. The value of the Flag signal at 0 h was defined as 1.0. Note that all signal intensities of the Flag-tag were normalized by that of actin, a loading control, in each sample.


Polyubiquitin modification of Rab8a was abolished in BAG6 - knockdown cells . Flag - Rab8a ( T22N ) immunoprecipitates were blotted with an anti - polyubiquitin antibody ( FK2 , left panel ) . As a negative control , siRNA # 1scr was used . Anti - polyubiquitin signals co - precipitated with Rab8a ( T22N ) ( a representative example is shown in the left panel ) were quantified ( right panel ) . Note that the intensities of the co - precipitated polyubiquitin signal were normalized both by the input ubiquitin - signal and bait Flag - signal .

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Polyubiquitin modification of Rab8a was abolished in BAG6-knockdown cells. Flag-Rab8a (T22N) immunoprecipitates were blotted with an anti-polyubiquitin antibody (FK2, left panel). As a negative control, siRNA#1scr was used. Anti-polyubiquitin signals co-precipitated with Rab8a (T22N) (a representative example is shown in the left panel) were quantified (right panel). Note that the intensities of the co-precipitated polyubiquitin signal were normalized both by the input ubiquitin-signal and bait Flag-signal.


Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a . Right panel shows a merged image of TfnR staining ( shown as green ) , Rab8a ( T22N ) staining ( magenta ) , and Hoechst 33342 nuclear staining ( blue ) .

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Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a. Right panel shows a merged image of TfnR staining (shown as green), Rab8a (T22N) staining (magenta), and Hoechst 33342 nuclear staining (blue).


A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti - BAG6 antibody . Co - precipitation of Rab8a ( T22N ) with BAG6 was used as a positive control for this experiment .

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

A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti-BAG6 antibody. Co-precipitation of Rab8a (T22N) with BAG6 was used as a positive control for this experiment.


Quantification of BAG6 co - precipitation with Rab family proteins . The graph shows the quantities of BAG6 protein in the respective anti - Flag precipitates that were normalized by the amount of Flag - Rab bait proteins . The value of BAG6 co - precipitation with Rab8a ( T22N ) was defined as 1 . 0 . The quantities of the respective immunosignals were also normalized to the actin signal of each sample , and represent the median calculated from at least 3 independent biological replicates . The number of independent biological replicates was as follows : Rab1a , n = 4 ; Rab1b , n = 5 ; Rab2b , n = 3 ; Rab3a , n = 4 ; Rab4 , n = 4 ; Rab5a , n = 3 ; Rab5b , n = 4 ; Rab6a , n = 5 ; Rab6b , n = 4 ; Rab7a , n = 3 ; Rab8a , n = 14 ; Rab9b , n = 4 ; Rab10 , n = 6 ; Rab11a , n = 3 ; Rab11b , n = 3 ; Rab12 , n = 2 ; Rab13 , n = 6 ; Rab14 , n = 3 ; Rab15 , n = 5 ; Rab35 , n = 3 .

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Quantification of BAG6 co-precipitation with Rab family proteins. The graph shows the quantities of BAG6 protein in the respective anti-Flag precipitates that were normalized by the amount of Flag-Rab bait proteins. The value of BAG6 co-precipitation with Rab8a (T22N) was defined as 1.0. The quantities of the respective immunosignals were also normalized to the actin signal of each sample, and represent the median calculated from at least 3 independent biological replicates. The number of independent biological replicates was as follows: Rab1a, n = 4; Rab1b, n = 5; Rab2b, n = 3; Rab3a, n = 4; Rab4, n = 4; Rab5a, n = 3; Rab5b, n = 4; Rab6a, n = 5; Rab6b, n = 4; Rab7a, n = 3; Rab8a, n = 14; Rab9b, n = 4; Rab10, n = 6; Rab11a, n = 3; Rab11b, n = 3; Rab12, n = 2; Rab13, n = 6; Rab14, n = 3; Rab15, n = 5; Rab35, n = 3.


BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers . Representative images of the trans - Golgi membrane protein Stx6 ( green ) in BAG6 - suppressed CHO cells with a Chinese hamster - specific siRNA ( cBAG6 siRNA # 2 ) . Scale bar : 10 μm ( A ) . Fluorescent signals were detected using a laser scanning confocal microscopy system . Nuclei were stained by Hoechst ( blue ) .

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BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Representative images of the trans-Golgi membrane protein Stx6 (green) in BAG6-suppressed CHO cells with a Chinese hamster-specific siRNA (cBAG6 siRNA#2). Scale bar: 10 μm (A). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).


BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers . Images of the cis - Golgi membrane protein GS28 and the cis - Golgi matrix protein GM130 in BAG6 - suppressed CHO cells with another Chinese hamster - specific siRNA ( cBAG6 siRNA # 5 ) . GS28 ( green ) and GM130 ( red ) are indicated by arrowheads . Scale bar : 10 μm . ( B ) . Fluorescent signals were detected using a laser scanning confocal microscopy system . Nuclei were stained by Hoechst ( blue ) .

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BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Images of the cis-Golgi membrane protein GS28 and the cis-Golgi matrix protein GM130 in BAG6-suppressed CHO cells with another Chinese hamster-specific siRNA (cBAG6 siRNA#5). GS28 (green) and GM130 (red) are indicated by arrowheads. Scale bar: 10 μm. (B). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).


Glycosylation of the IL - 2Rα transmembrane protein was not reduced by BAG6 knockdown . Flag - tagged WT IL - 2Rα protein was expressed in HeLa cells with ( + ) or without ( - ) BAG6 siRNA , and was immunoprecipitated with an anti - Flag antibody . The precipitates were incubated with ( + ) or without ( - ) 10 unit of the deglycosylation enzyme PNGase F , and subjected to Western blot analysis with an anti - Flag antibody . Low - mobility ( indicated as glycosylated ) and high mobility ( indicated as non - glycosylated ) signals of WT IL - 2Rα are indicated .

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Glycosylation of the IL-2Rα transmembrane protein was not reduced by BAG6 knockdown. Flag-tagged WT IL-2Rα protein was expressed in HeLa cells with (+) or without (-) BAG6 siRNA, and was immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F, and subjected to Western blot analysis with an anti-Flag antibody. Low-mobility (indicated as glycosylated) and high mobility (indicated as non-glycosylated) signals of WT IL-2Rα are indicated.


Defects in the distribution of cell surface glycoproteins in BAG6 - suppressed cells . The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S . D . calculated from 10 independent biological replicates ( E ) .

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Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S.D. calculated from 10 independent biological replicates (E).


Defects in the distribution of cell surface glycoproteins in BAG6 - suppressed cells . Lectin GS - II - derived cell surface signals were counted using ImageJ software . SRP54 knockdown was used as a positive control for this experiment t - test ) . Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization . BAG6 siRNA down - regulated the cell surface expression of glycoproteins ( F ) .

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Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. Lectin GS-II-derived cell surface signals were counted using ImageJ software. SRP54 knockdown was used as a positive control for this experiment t-test). Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization. BAG6 siRNA down-regulated the cell surface expression of glycoproteins (F).


C Immunostaining for the naive pluripotency marker KLF17 and the shared pluripotency marker OCT4 . Scale bar : 50μm . Data information : Representative images of n = 3 biological replicates are shown .

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C Immunostaining for the naive pluripotency marker KLF17 and the shared pluripotency marker OCT4. Scale bar: 50μm. Data information: Representative images of n=3 biological replicates are shown.


D Gene - expression analysis by qPCR of KiPS primed - iPSCs , KiPS - chemical resetting at day 14 , and KiPS naive - iPSCs . Data information : Bars indicate the mean ± SEM of n = 3 biological replicates shown as dots . ( D ) Expression was normalised to the mean of HPD06 naive - iPSCs samples .

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D Gene-expression analysis by qPCR of KiPS primed-iPSCs, KiPS-chemical resetting at day 14, and KiPS naive-iPSCs. Data information: Bars indicate the mean ± SEM of n=3 biological replicates shown as dots. (D) Expression was normalised to the mean of HPD06 naive-iPSCs samples.


I Barplots showing the absolute expression ( CPM ) of twelve trophoblast genes found to be significantly UP - regulated in KiPS - chemical resetting vs KiPS primed - iPSCs comparison . Data information : Bars indicate the mean ± SEM of n = 3 biological replicates shown as dots . * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns no significance ( 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

I Barplots showing the absolute expression (CPM) of twelve trophoblast genes found to be significantly UP-regulated in KiPS-chemical resetting vs KiPS primed-iPSCs comparison. Data information: Bars indicate the mean ± SEM of n=3 biological replicates shown as dots. ***P < 0.001, ****P < 0.0001, ns no significance (Student's t-test).


B Gene expression analysis by RT - qPCR for naive ( KLF17 , TFCP2L1 , and NANOG ) and trophoblast ( GATA3 , GATA2 , KRT7 , ELF5 , TP63 , and TFAP2A ) marker genes in naive - iPSCs and TSC cells derived from two different naive - iPSC lines ( HPD06 and H9 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Gene expression analysis by RT-qPCR for naive (KLF17, TFCP2L1, and NANOG) and trophoblast (GATA3, GATA2, KRT7, ELF5, TP63, and TFAP2A) marker genes in naive-iPSCs and TSC cells derived from two different naive-iPSC lines (HPD06 and H9).


I Gene - expression analysis by qPCR of KiPS primed - iPSCs , KiPS in PXGL medium at day 14 , KiPS in TSC medium at day 14 , H9 naive - TSCs on ColIV , H9 naive - TSCs on MEF and HPD06 naive - iPSCs . Data information : ( I ) Dots indicate biological replicates and bars indicate the mean ± SEM of n = 3 biological replicates . Top : naive , primed , and general pluripotency markers , expression was normalised to HPD06 naive iPSCs ; bottom : trophoblast markers , expression was normalised to H9 naive - TSCs .

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I Gene-expression analysis by qPCR of KiPS primed-iPSCs, KiPS in PXGL medium at day 14, KiPS in TSC medium at day 14, H9 naive-TSCs on ColIV, H9 naive-TSCs on MEF and HPD06 naive-iPSCs. Data information: (I) Dots indicate biological replicates and bars indicate the mean ± SEM of n=3 biological replicates. Top: naive, primed, and general pluripotency markers, expression was normalised to HPD06 naive iPSCs; bottom: trophoblast markers, expression was normalised to H9 naive-TSCs.


J Immunostaining for the pluripotency markers OCT4 and KLF17 , and the TSC markers GATA3 and KRT7 of HPD06 naive - iPSC and ccTSC . 01 cultured on MEF and on collagen IV ( ColIV ) after 3 passages . Scale bars : 30 μm . Data information : Representative images of n = 2 biological replicates are shown .

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J Immunostaining for the pluripotency markers OCT4 and KLF17, and the TSC markers GATA3 and KRT7 of HPD06 naive-iPSC and ccTSC.01 cultured on MEF and on collagen IV (ColIV) after 3 passages. Scale bars: 30 μm. Data information: Representative images of n=2 biological replicates are shown.


B Immunostaining for the pluripotency markers OCT4 , and the TSC markers GATA3 and KRT7 of KiPS primed - iPSCs , HPD06 naive - iPSCs , ccTSC . 02 at passage 4 , ccTSC . 01 at passage 4 and HPD06 naive - TSCs . Scale bars : 30 μm . Data information : ( B ) Representative images of n = 3 biological replicates are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunostaining for the pluripotency markers OCT4, and the TSC markers GATA3 and KRT7 of KiPS primed-iPSCs, HPD06 naive-iPSCs, ccTSC.02 at passage 4, ccTSC.01 at passage 4 and HPD06 naive-TSCs. Scale bars: 30 μm. Data information: (B) Representative images of n=3 biological replicates are shown.


F Barplots showing the absolute expression as log ( CPM + 1 ) of ADAP2 , HAVCR1 , SLC28A3 , ARID5B , and GATA2 in the reported conditions . Data information : ( F ) Bars indicate the mean ± SEM of at least n = 5 biological replicates from at least 2 independent studies shown with different symbols . * * * P < 0 . 001 , ns no significance ( 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

F Barplots showing the absolute expression as log(CPM+1) of ADAP2, HAVCR1, SLC28A3, ARID5B, and GATA2 in the reported conditions. Data information: (F) Bars indicate the mean ± SEM of at least n=5 biological replicates from at least 2 independent studies shown with different symbols. ***P < 0.001, ns no significance (Student's t-test).


A Gene - expression analysis by qPCR of syncytiotrophoblast ( left ) and extravillous ( right ) markers . Data information : ( A ) Dots indicate biological replicates . Left : expression was normalised to HPD06 naive - STBs and ccTSC . 02 - STBs ; Right : expression was normalised to HPD06 naive - EVTs and ccTSC . 02 - EVTs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Gene-expression analysis by qPCR of syncytiotrophoblast (left) and extravillous (right) markers. Data information: (A) Dots indicate biological replicates. Left: expression was normalised to HPD06 naive-STBs and ccTSC.02-STBs; Right: expression was normalised to HPD06 naive-EVTs and ccTSC.02-EVTs.


B Top : Immunostaining for the TSC marker KRT7 and the STB marker SDC1 of ccTSC . 02 - STBs , HPD06 naive - STBs , ccTSC . 02 , and HPD06 naive - TSCs . Scale bars : 50 μm . Bottom : Immunostaining for the TSC marker KRT7 and the EVT marker HLA - G of ccTSC . 02 - EVTs , HPD06 naive - EVTs , ccTSC . 02 , and HPD06 naive - TSCs . Scale bars : 25 μm . Data information : ( B ) Representative images of n = 2 biological replicates are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Top: Immunostaining for the TSC marker KRT7 and the STB marker SDC1 of ccTSC.02-STBs, HPD06 naive-STBs, ccTSC.02, and HPD06 naive-TSCs. Scale bars: 50 μm. Bottom: Immunostaining for the TSC marker KRT7 and the EVT marker HLA-G of ccTSC.02-EVTs, HPD06 naive-EVTs, ccTSC.02, and HPD06 naive-TSCs. Scale bars: 25 μm. Data information: (B) Representative images of n=2 biological replicates are shown.


D Barplots showing the absolute expression as log ( CPM + 1 ) of general pluripotency ( OCT4 and NANOG ) , naive ( SUSD2 ) , and TSC - specific genes ( GATA2 , GATA3 , KRT7 , and ADAP2 ) in the reported conditions highlighted in different colours . Data information : ( D ) Dots indicate biological replicates and bars indicate the mean ± SEM of n = 3 biological replicates .

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

D Barplots showing the absolute expression as log(CPM+1) of general pluripotency (OCT4 and NANOG), naive (SUSD2), and TSC-specific genes (GATA2, GATA3, KRT7, and ADAP2) in the reported conditions highlighted in different colours. Data information: (D) Dots indicate biological replicates and bars indicate the mean ± SEM of n=3 biological replicates.


B Immunostaining for the TSC markers GATA3 and KRT7 , the naive pluripotency marker SUSD2 and general pluripotency markers OCT4 of KiPS primed - iPSCs , KiPS at day 3 of the conversion , KiPS at day 7 in TSC medium or PXGL , KiPS at day 14 in TSC medium or PXGL , HPD06 naive - TSCs and HPD06 naive - iPSCs . Scale bars : 25 μm . Data information : ( B ) Representative images of n = 2 biological replicates are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunostaining for the TSC markers GATA3 and KRT7, the naive pluripotency marker SUSD2 and general pluripotency markers OCT4 of KiPS primed-iPSCs, KiPS at day 3 of the conversion, KiPS at day 7 in TSC medium or PXGL, KiPS at day 14 in TSC medium or PXGL, HPD06 naive-TSCs and HPD06 naive-iPSCs. Scale bars: 25 μm. Data information: (B) Representative images of n=2 biological replicates are shown.


C Single - cell scatter plots showing the log ( CPM + 1 ) integrated intensity of TSC markers GATA3 and KRT7 , naive pluripotency marker SUSD2 , and general pluripotency marker OCT4 of KiPS primed - iPSCs , KiPS at day 3 of the conversion , KiPS at day 7 in TSC medium or PXGL , KiPS at day 14 in TSCs medium or PXGL , HPD06 naive - TSCs and HPD06 naive - iPSCs . Black lines represent the threshold used to define positivity to a given marker . Data information : ( C ) Fluorescence intensity was measured by immunofluorescence in 5 to 12 independent fields from 2 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 O O O O O 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 Single-cell scatter plots showing the log(CPM+1) integrated intensity of TSC markers GATA3 and KRT7, naive pluripotency marker SUSD2, and general pluripotency marker OCT4 of KiPS primed-iPSCs, KiPS at day 3 of the conversion, KiPS at day 7 in TSC medium or PXGL, KiPS at day 14 in TSCs medium or PXGL, HPD06 naive-TSCs and HPD06 naive-iPSCs. Black lines represent the threshold used to define positivity to a given marker. Data information: (C) Fluorescence intensity was measured by immunofluorescence in 5 to 12 independent fields from 2 independent experiments.


a , Immunoprecipitation of Flag - beclin 1 constructs with Nef - HA in HeLa cells 24 h post - transfection .

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a, Immunoprecipitation of Flag-beclin 1 constructs with Nef-HA in HeLa cells 24 h post-transfection.


b , GFP - LC3 - positive dots ( autophagosomes ) in MCF7 cells expressing GFP - LC3 and Flag - beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h .

O O O O O O O O O O O O O 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, GFP-LC3-positive dots (autophagosomes) in MCF7 cells expressing GFP-LC3 and Flag-beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h.


d , Biochemical assessment of autophagy ( p62 and LC3 immunoblots ) in peptide - treated HeLa cells ( 3 h ) .

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d, Biochemical assessment of autophagy (p62 and LC3 immunoblots) in peptide-treated HeLa cells (3 h).


e , f , Representative images ( e ) and quantification of GFP - LC3 - positive dots ( f ) in peptide - treated HeLa / GFP - LC3 cells ( 30 µM , 3 h ) . Scale bars , 20 µm .

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e, f, Representative images (e) and quantification of GFP-LC3-positive dots (f) in peptide-treated HeLa/GFP-LC3 cells (30 µM, 3 h). Scale bars, 20 µm.


g , GFP - LC3 - positive dots in siRNA - transfected peptide - treated HeLa / GFP - LC3 cells ( 30 µM , 3 h ) .

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g, GFP-LC3-positive dots in siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (30 µM, 3 h).


h , Model of Tat - beclin 1 peptide ( left ) based on corresponding elements of the beclin 1 evolutionarily conserved domain ( ECD ) structure ( centre ) . Essential phenylalanine side chains , magenta ; positions of solubility mutations , pink ; lipid interaction site , yellow . ECD surface representation ( right ) illustrates exposure of corresponding peptide ( cyan ) .

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h, Model of Tat-beclin 1 peptide (left) based on corresponding elements of the beclin 1 evolutionarily conserved domain (ECD) structure (centre). Essential phenylalanine side chains, magenta; positions of solubility mutations, pink; lipid interaction site, yellow. ECD surface representation (right) illustrates exposure of corresponding peptide (cyan).


i , p62 and LC3 immunoblots in peptide - treated HeLa cells ( 3 h ) . In b , f , g , bars represent mean ± s . e . m . of triplicate samples ( 50 - 100 cells per sample ) . Similar results were observed in three independent experiments . * P 0 . 05 , * * P 0 . 01 ; 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

i, p62 and LC3 immunoblots in peptide-treated HeLa cells (3 h). In b, f, g, bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. *P 0.05, **P 0.01; t-test.


a , HeLa cells were treated with biotin - conjugated peptides ( 30 μM , 3 h ) and proteins bound to peptides were analysed by immunoblot with anti - GAPR - 1 . b - T - B , biotin - Tat - beclin 1 ; b - T - S , biotin - Tat - scrambled .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HeLa cells were treated with biotin-conjugated peptides (30 μM, 3 h) and proteins bound to peptides were analysed by immunoblot with anti-GAPR-1. b-T-B, biotin-Tat-beclin 1; b-T-S, biotin-Tat-scrambled.


b , Immunoprecipitation of Flag - beclin 1 with GAPR - 1 - Myc in HeLa cells 24 h post - transfection .

b, Immunoprecipitation of Flag-beclin 1 with GAPR-1-Myc in HeLa cells 24 h post-transfection.


c , GFP - LC3 - positive dots in GAPR - 1 siRNA - transfected peptide - treated HeLa / GFP - LC3 cells ( 20 & amp ; amp ; mgr ; M , 3 h ) with or without 100 nM bafilomycin A1 . Bars represent mean ± s . e . m . of triplicate samples ( 50 - 100 cells per sample ) . Similar results were observed in three independent experiments . NC , non - silencing control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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

c, GFP-LC3-positive dots in GAPR-1 siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (20 &amp;mgr;M, 3 h) with or without 100 nM bafilomycin A1. Bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. NC, non-silencing control.


d , Localization of beclin 1 and GM130 ( a Golgi marker ) in HeLa cells stably transduced with empty vector ( left panel ) or GAPR - 1 - Myc ( middle panel ) or transfected with GAPR - 1 siRNA ( right panel ) and treated with peptide ( 20 μM , 1 h ) . Scale bar , 20 μm .

d, Localization of beclin 1 and GM130 (a Golgi marker) in HeLa cells stably transduced with empty vector (left panel) or GAPR-1-Myc (middle panel) or transfected with GAPR-1 siRNA (right panel) and treated with peptide (20 μM, 1 h). Scale bar, 20 μm.


e , Immunoblot of beclin 1 in post - nuclear supernatant ( PNS ) and Golgi - enriched fractions in HeLa cells stably transduced with empty vector or GAPR - 1 - Myc after peptide treatment ( 20 μM , 2 h ) .

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e, Immunoblot of beclin 1 in post-nuclear supernatant (PNS) and Golgi-enriched fractions in HeLa cells stably transduced with empty vector or GAPR-1-Myc after peptide treatment (20 μM, 2 h).


f , WIPI2 dots in cells in the experimental conditions shown in d . Bars represent mean ± s . e . m . for 100 - 150 cells . * P 0 . 05 , * * * P 0 . 001 ; t - test . † † † P 0 . 001 ; two - way ANOVA for comparison of magnitude of changes between groups .

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f, WIPI2 dots in cells in the experimental conditions shown in d. Bars represent mean ± s.e.m. for 100-150 cells. *P 0.05, ***P 0.001; t-test. †††P 0.001; two-way ANOVA for comparison of magnitude of changes between groups.


a , Percentage of cells with small htt103Q aggregates ( left ) and number of aggregates per cell ( right ) in HeLa cells expressing doxycycline ( Dox ) - repressible CFP - htt103Q after daily treatment with doxycycline or peptide ( 20 μM , 4 h per day ) for 2 days . Bars represent mean ± s . e . m . of triplicate samples ( 60 - 120 cells per sample ) . Similar results were observed in three 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

a, Percentage of cells with small htt103Q aggregates (left) and number of aggregates per cell (right) in HeLa cells expressing doxycycline (Dox)-repressible CFP-htt103Q after daily treatment with doxycycline or peptide (20 μM, 4 h per day) for 2 days. Bars represent mean ± s.e.m. of triplicate samples (60-120 cells per sample). Similar results were observed in three independent experiments.


b , Filter trap assays for htt103Q large and small aggregates in HeLa / htt103Q cells .

b, Filter trap assays for htt103Q large and small aggregates in HeLa/htt103Q cells.


c , Viral titres in HeLa cells infected with 0 . 1 plaque - forming units ( p . f . u . ) per cell of SINV , CHIKV or WNV ( strain TX02 ) and treated with peptide ( 10 μM , 4 - 8 h post - infection ) . Values represent geometric mean ± s . e . m . for triplicate samples of supernatants collected 18 h post - infection ( SINV ) or 24 h post - infection ( CHIKV and WNV ) . Similar results were observed in three 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 O O O O O O O O O O O O O O

c, Viral titres in HeLa cells infected with 0.1 plaque-forming units (p.f.u.) per cell of SINV, CHIKV or WNV (strain TX02) and treated with peptide (10 μM, 4-8 h post-infection). Values represent geometric mean ± s.e.m. for triplicate samples of supernatants collected 18 h post-infection (SINV) or 24 h post-infection (CHIKV and WNV). Similar results were observed in three independent experiments.


d , Bacteriacolony - forming units ( c . f . u . ) in primary BMDMs infected with L . monocytogenes ΔactA mutant strain DPL - 4029 for 30 min and treated with peptide ( 10 µM from 0 to 2 h post - infection ) . Bars represent mean ± s . e . m . of triplicate samples . Similar results were observed in three 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

d, Bacteriacolony-forming units (c.f.u.) in primary BMDMs infected with L. monocytogenes ΔactA mutant strain DPL-4029 for 30 min and treated with peptide (10 µM from 0 to 2 h post-infection). Bars represent mean ± s.e.m. of triplicate samples. Similar results were observed in three independent experiments.


e , HIV - 1 p24 antigen release in primary human MDMs infected with HIV - 1 24 h after initiation of daily peptide treatment . Values represent mean ± s . e . m . of triplicate samples . Similar results were observed in MDMs from three independent donors .

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

e, HIV-1 p24 antigen release in primary human MDMs infected with HIV-1 24 h after initiation of daily peptide treatment. Values represent mean ± s.e.m. of triplicate samples. Similar results were observed in MDMs from three independent donors.


f , HIV - 1p24 antigen release in MDMs transduced with nonspecific scrambled shRNA ( shNS ) or ATG5 shRNA ( shATG5 ) and treated daily with peptide ( 5 µM ) . Values represent mean ± s . e . m . of triplicate wells . Similar results were observed in MDMs from two independent donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HIV-1p24 antigen release in MDMs transduced with nonspecific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and treated daily with peptide (5 µM). Values represent mean ± s.e.m. of triplicate wells. Similar results were observed in MDMs from two independent donors.


g , LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV - 1 infection . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; 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

g, LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV-1 infection. *P  0.05; **P  0.01; ***P  0.001; t-test.


a , GFP - LC3 - positive dots in tissues of 6 - week - old GFP - LC3 mice treated with the indicated peptide ( 20 mg kg − 1 i . p . , 6 h ) . A minimum of ten fields was counted per tissue section . Bars represent mean ± s . e . m . for three mice . Similar results were observed in three 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

a, GFP-LC3-positive dots in tissues of 6-week-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). A minimum of ten fields was counted per tissue section. Bars represent mean ± s.e.m. for three mice. Similar results were observed in three independent experiments.


b , p62 immunoblot of brains of 5 - day - old GFP - LC3 mice treated with the indicated peptide ( 20 mg kg − 1 i . p . , 6 h ) .

O O O O O O O O O O O O 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, p62 immunoblot of brains of 5-day-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h).


c , Survival curves of 5 - day - old C57BL / 6J mice infected with CHIKV ( 106 p . f . u . s . c . ) and treated daily with peptide ( 15 mg kg − 1 i . p . beginning 1 day post - infection ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Survival curves of 5-day-old C57BL/6J mice infected with CHIKV (106 p.f.u. s.c.) and treated daily with peptide (15 mg kg−1 i.p. beginning 1 day post-infection).


d - f , Representative images of WNV envelope antigen and TdT - mediated dUTP nick end labelling ( TUNEL ) staining ( d ) ( T , Tat alone ) , quantification of cell death in brain ( e ) and survival curves ( f ) for 5 - day - oldC57BL / 6Jmice infected with WNV ( Egypt strain 101 , 1 p . f . u . intracerebral ( i . c . ) ) and treated daily with peptide ( D - amino acid forms , 20mgkg − 1 i . p . beginning 1day post - infection ) . Images in d are from cerebral cortex day 6 post - infection . Similar results were observed in all regions of the brain for three mice per group . Scale bar , 20μm . Bars in e represent mean ± s . e . m . TUNEL - positive cells per unit area of brain for three mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e) and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. g


d - f , Representative images of WNV envelope antigen and TdT - mediated dUTP nick end labelling ( TUNEL ) staining ( d ) ( T , Tat alone ) , quantification of cell death in brain ( e ) , and survival curves ( f ) for 5 - day - oldC57BL / 6Jmice infected with WNV ( Egypt strain 101 , 1 p . f . u . intracerebral ( i . c . ) ) and treated daily with peptide ( D - amino acid forms , 20mgkg − 1 i . p . beginning 1day post - infection ) . Images in d are from cerebral cortex day 6 post - infection . Similar results were observed in all regions of the brain for three mice per group . Scale bar , 20μm . Bars in e represent mean ± s . e . m . TUNEL - positive cells per unit area of brain for three mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e), and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. g


g , Geometric mean + s . e . m . viral titres of WNV - infected mousebrains day 6 post - infection . Values represent combined data for 12 - 20 mice per treatment group from 10 to 12 litters . Data in c and f represent combined survival probabilities for three and four independent litters , respectively , in each group . Similar results were observed in each independent experiment . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; NS , not significant ; t - test .

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g, Geometric mean + s.e.m. viral titres of WNV-infected mousebrains day 6 post-infection. Values represent combined data for 12-20 mice per treatment group from 10 to 12 litters. Data in c and f represent combined survival probabilities for three and four independent litters, respectively, in each group. Similar results were observed in each independent experiment. *P 0.05; **P 0.01; ***P 0.001; NS, not significant; t-test.


A . Schematic depiction of the Caenorhabditis elegans phasmid cilia ( left ) . Two sensory cilia are aligned into the phasmid channel ( the dashed box and enlarged in the middle ) . Each cilium contains a ciliary base , a middle segment ( m . s . ) , and a distal segment ( d . s . ) . Right shows an inverted fluorescence Hessian - SIM image of GFP - tagged dynein - 2 heavy chain CHE - 3 in cilia , in which individual puncta were resolved . Scale bar : 1 μm .

A. Schematic depiction of the Caenorhabditis elegans phasmid cilia (left). Two sensory cilia are aligned into the phasmid channel (the dashed box and enlarged in the middle). Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.). Right shows an inverted fluorescence Hessian-SIM image of GFP-tagged dynein-2 heavy chain CHE-3 in cilia, in which individual puncta were resolved. Scale bar: 1 μm.


B . Frame series of an anterograde moving IFT - particle from a 7xGFP : : CHE - 3 worm . Localization precisions are shown under each frame . Time in millisecond ( ms ) ; Scale bar , 100 nm .

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B. Frame series of an anterograde moving IFT-particle from a 7xGFP::CHE-3 worm. Localization precisions are shown under each frame. Time in millisecond (ms); Scale bar, 100 nm.


C . Localization precisions ( mean ± SD ) of tracks from multi - GFP - labeled worms . The number of GFP copies was estimated as the following : the dynein - 2 heavy chain of CHE - 3 forms a dimer , 14 = 2 x 7 ; IFT - particle subunit CHE - 2 is also a dimer , 14 = 2 x 7 ; IFT - particle subunit DYF - 11 can be a tetramer , 12 = 4 x 3 ; the genetic cross combined 7xGFP : : CHE - 3 with DYF - 11 : : 3XGFP , 26 = 2 x 7 + 4 x 3 .

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C. Localization precisions (mean ± SD) of tracks from multi-GFP-labeled worms. The number of GFP copies was estimated as the following: the dynein-2 heavy chain of CHE-3 forms a dimer, 14 = 2 x 7; IFT-particle subunit CHE-2 is also a dimer, 14 = 2 x 7; IFT-particle subunit DYF-11 can be a tetramer, 12 = 4 x 3; the genetic cross combined 7xGFP::CHE-3 with DYF-11::3XGFP, 26 = 2 x 7 + 4 x 3.


D . The representative diagram shows the displacement of the cargo within the middle ciliary segment . The y - axis grid lines are spaced 16 nm . Protein CHE - 3 tagged with 7x GFP was used for tracking experiments . Trace was analyzed using the SIC fitting

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

D. The representative diagram shows the displacement of the cargo within the middle ciliary segment. The y-axis grid lines are spaced 16 nm. Protein CHE-3 tagged with 7x GFP was used for tracking experiments. Trace was analyzed using the SIC fitting algorithm


E . Histogram of 161 displacements detected in 15 tracks extracted from 12 WT worms . The plus and minus displacements are defined as the upward " jumping " or downward " jumping " Protein markers used for tracking experiments are CHE - 3 tagged with 7x GFP , CHE - 2 tagged with 7x GFP , or CHE - 3 tagged with 7x GFP plus DYF - 11 tagged with 3x GFP .

E. Histogram of 161 displacements detected in 15 tracks extracted from 12 WT worms. The plus and minus displacements are defined as the upward "jumping" or downward "jumping" Protein markers used for tracking experiments are CHE-3 tagged with 7x GFP, CHE-2 tagged with 7x GFP, or CHE-3 tagged with 7x GFP plus DYF-11 tagged with 3x GFP.


A . Frame series shows the off - axis movement of an IFT - particle ( # 1 ) . A neighboring particle ( # 2 ) serves as a fiduciary marker .

O O O O O O O O O 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. Frame series shows the off-axis movement of an IFT-particle (#1). A neighboring particle (#2) serves as a fiduciary marker.


B . Representative two - dimensional IFT - tracks from WT , kinesin - II ( klp - 11 ) , and osm - 3 null animals . Red and violet circles indicate the start and end of the tracks , respectively . Proteins used for tracking experiments are CHE - 3 tagged with 7x GFP , or DYF - 11 tagged with 3x GFP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative two-dimensional IFT-tracks from WT, kinesin-II (klp-11), and osm-3 null animals. Red and violet circles indicate the start and end of the tracks, respectively. Proteins used for tracking experiments are CHE-3 tagged with 7x GFP, or DYF-11 tagged with 3x GFP.


C . Time - averaged mean - square displacement ( MSD ) versus lag time ( tlag ) of 10 - 15 tracks from WT and IFT - kinesin mutant animals . Proteins used for tracking experiments are CHE - 3 tagged with 7x GFP , CHE - 3 tagged with 7x GFP plus DYF - 11 tagged with 3x GFP , CHE - 2 tagged with 7x GFP , or DYF - 11 tagged with 3x GFP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time-averaged mean-square displacement (MSD) versus lag time (tlag) of 10-15 tracks from WT and IFT-kinesin mutant animals. Proteins used for tracking experiments are CHE-3 tagged with 7x GFP, CHE-3 tagged with 7x GFP plus DYF-11 tagged with 3x GFP, CHE-2 tagged with 7x GFP, or DYF-11 tagged with 3x GFP.


E . Average deviation angles in 10 - 15 tracks from WT and mutant animals . Comparisons were performed with the WT .

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E. Average deviation angles in 10-15 tracks from WT and mutant animals. Comparisons were performed with the WT.


F . Representative tracks of the recombinant C . elegans kinesin - II and OSM - 3 - kinesin moving along suspended MTs in the optical trap assay .

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F. Representative tracks of the recombinant C. elegans kinesin-II and OSM-3-kinesin moving along suspended MTs in the optical trap assay.


G . Representative TEM images of the middle ciliary segments from WT and IFT - kinesin mutant animals . Red arrowheads indicate ectopic singlet microtubules . Blue arrowheads indicate ectopic doublet microtubules . The schematics below depict the phenotype of each image .

O O O O O O O O O O O O O O O O O O O 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. Representative TEM images of the middle ciliary segments from WT and IFT-kinesin mutant animals. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image.


B . Representative two - dimensional IFT tracks from WT and OSM - 3 ( 1 or 3xGS ) animals . Red and violet circles indicate the start and end of the tracks , respectively .

B. Representative two-dimensional IFT tracks from WT and OSM-3(1 or 3xGS) animals. Red and violet circles indicate the start and end of the tracks, respectively.


C . Time - averaged mean - square displacement ( MSD ) versus lag time ( tlag ) of 10 - 15 tracks from WT and mutant worms .

C. Time-averaged mean-square displacement (MSD) versus lag time (tlag) of 10-15 tracks from WT and mutant worms.


A . Histogram of anterograde IFT speeds of 3xGFP : : CHE - 3 at the middle ( top ) and distal ( bottom ) ciliary segments ( top ) . Comparisons were performed between the WT and osm - 3 mutants . * * *

O O O O O O O O O O O O O O O O O O O O O O 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. Histogram of anterograde IFT speeds of 3xGFP::CHE-3 at the middle (top) and distal (bottom) ciliary segments (top). Comparisons were performed between the WT and osm-3 mutants. ***


B . Schematic depiction of the C . elegans amphid and phasmid cilia . Each cilium contains a ciliary base , a middle segment ( m . s . ) , and a distal segment ( d . s . ) . C . Amphid ( top ) and phasmid ( bottom ) cilia in the WT and osm - 3 GS knock - in animals . Cilia are marked with 7xGFP : : CHE - 3 . Arrowheads indicate the ciliary base . Arrows indicate the junctions between the middle and distal segments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Schematic depiction of the C. elegans amphid and phasmid cilia. Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.). C. Amphid (top) and phasmid (bottom) cilia in the WT and osm-3 GS knock-in animals. Cilia are marked with 7xGFP::CHE-3. Arrowheads indicate the ciliary base. Arrows indicate the junctions between the middle and distal segments.


D . Cilium length ( mean ± SD ) in WT and osm - 3 mutant animals . Comparisons were performed between the WT and osm - 3 mutants .

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D. Cilium length (mean ± SD) in WT and osm-3 mutant animals. Comparisons were performed between the WT and osm-3 mutants.


E . Quantifications of dye - filling defects from WT and osm - 3 mutant animals .

E. Quantifications of dye-filling defects from WT and osm-3 mutant animals.


F . Representative TEM images of the middle ciliary segments from osm - 3 ( 1xGS ) and osm - 3 ( 3xGS ) worms . Red arrowheads indicate ectopic singlet microtubules . Blue arrowheads indicate ectopic doublet microtubules . The schematics below depict the phenotype of each image .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative TEM images of the middle ciliary segments from osm-3(1xGS) and osm-3(3xGS) worms. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image.


( B ) Growth inhibition upon overexpression of HOS3 - NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1 . 10 - fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids , were spotted onto SC - Glu and SC - Gal medium ( to activate the GAL1 promoter ) and incubated at 25 ° C for 3 days . Note that HOS3 - NLS ( under the control of the native HOS3 promoter ) does not affect cell growth .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth inhibition upon overexpression of HOS3-NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1. 10-fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids, were spotted onto SC-Glu and SC-Gal medium (to activate the GAL1 promoter) and incubated at 25 °C for 3 days. Note that HOS3-NLS (under the control of the native HOS3 promoter) does not affect cell growth.


( C ) esa1 - ts and gcn5Δ esa1 - ts mutants have bud emergence defects . ( Top ) Cells of the indicated strains were arrested in G1 by treatment with ɑ - factor for 2 . 5 h at 25 ºC , shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC . Cells were fixed at the indicated times and the presence of buds was assessed by microscopy . Mean and SEM are derived from n = number of independent experiments . At least 200 cells were scored for each strain and time point . ( Bottom ) Bright field images of the indicated strains 60 min after the ɑ - factor washout . Arrowheads point to cell buds . Scale bar , 4 µm .

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(C) esa1-ts and gcn5Δ esa1-ts mutants have bud emergence defects. (Top) Cells of the indicated strains were arrested in G1 by treatment with ɑ-factor for 2.5 h at 25 ºC, shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC. Cells were fixed at the indicated times and the presence of buds was assessed by microscopy. Mean and SEM are derived from n = number of independent experiments. At least 200 cells were scored for each strain and time point. (Bottom) Bright field images of the indicated strains 60 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.


( D ) Inactivation of ESA1 and GCN5 delays DNA replication . Cells of the indicated genotypes were synchronised as in panel C , and DNA content was evaluated by flow cytometry . Numbers indicate time in minutes after the release . This experiment was repeated three times with similar results ; one experiment is shown .

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(D) Inactivation of ESA1 and GCN5 delays DNA replication. Cells of the indicated genotypes were synchronised as in panel C, and DNA content was evaluated by flow cytometry. Numbers indicate time in minutes after the release. This experiment was repeated three times with similar results; one experiment is shown.


( A ) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60 . ( Top ) Nup60 - GFP was immunoprecipitated from extracts of the indicated strains , and its acetylation state probed with anti - AcLys antibodies . ( Bottom ) Total extracts probed with anti - HA antibodies to verify KAT overexpression .

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

(A) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60. (Top) Nup60-GFP was immunoprecipitated from extracts of the indicated strains, and its acetylation state probed with anti-AcLys antibodies. (Bottom) Total extracts probed with anti-HA antibodies to verify KAT overexpression.


( B ) nup60 - KN partially rescues the budding defect of esa1 - ts cells . ( Left ) Budding of cells of the indicated strains were determined as in Figure 1C . At least 200 cells were scored for each strain and time point . Data from 3 independent experiments is represented as mean and SEM ( esa1 - ts , esa1 - ts nup60 - KN ) . ( Right ) Bright field images of the indicated strains 40 min after the ɑ - factor washout . Arrowheads point to cell buds . Scale bar , 4 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

(B) nup60-KN partially rescues the budding defect of esa1-ts cells. (Left) Budding of cells of the indicated strains were determined as in Figure 1C. At least 200 cells were scored for each strain and time point. Data from 3 independent experiments is represented as mean and SEM (esa1-ts, esa1-ts nup60-KN). (Right) Bright field images of the indicated strains 40 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.


( C ) mRNA levels of CLN2 , CDC21 , SVS1 , and RNR1 were determined for cells of the indicated strains after G1 arrest and release at restrictive temperature , with samples collected at indicated times . Data from 3 independent experiments is represented as mean and SEM .

(C) mRNA levels of CLN2, CDC21, SVS1, and RNR1 were determined for cells of the indicated strains after G1 arrest and release at restrictive temperature, with samples collected at indicated times. Data from 3 independent experiments is represented as mean and SEM.


( D ) nup60 - KN mutation partially rescues the delay in synthesis of the G1 / S cyclin Cln2 in esa1 - ts cells . Cells of the indicated strains were processed as in ( B ) and the amount of Cln2 - HA protein at the indicated times was assessed by western blot . % of budded cells is indicated below for each corresponding strain and time point . Note the slow , inefficient budding in esa1 - ts mutants . In WT , the reduction of Cln2 at 60 min and its increase at 90 min reflects the start of a second cycle , which is absent in esa1 - ts cells .

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(D) nup60-KN mutation partially rescues the delay in synthesis of the G1/S cyclin Cln2 in esa1-ts cells. Cells of the indicated strains were processed as in (B) and the amount of Cln2-HA protein at the indicated times was assessed by western blot. % of budded cells is indicated below for each corresponding strain and time point. Note the slow, inefficient budding in esa1-ts mutants. In WT, the reduction of Cln2 at 60 min and its increase at 90 min reflects the start of a second cycle, which is absent in esa1-ts cells.


( E ) nup60 - KN partially rescues the Whi5 export defect of esa1 - ts daughter cells . Composite of bright field and Whi5 - mCherry ( left ) and quantification of Whi5 nuclear export ( right ) in mother ( M ) and daughter ( D ) cells of the indicated strains . Whi5 export ( arrows ) is delayed in esa1 - ts mothers and daughters compared to WT ( p < 000 . 1 , Log - rank Mantel - Cox test ) ; esa1 - ts nup60 - KN advances Whi5 export relative to esa1 - ts in daughters ( p = 0 . 0105 ) , but not in mothers ( p > 0 . 05 ) . 3 z - confocal slices spaced 0 . 5 µm were acquired every 3 min ; maximum projections of selected timepoints are shown . Time is indicated in minutes ; t = 0 marks Whi5 nuclear import . Scale bar , 5 µm . n = number of cells , pooled from two independent experiments with similar results .

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

(E) nup60-KN partially rescues the Whi5 export defect of esa1-ts daughter cells. Composite of bright field and Whi5-mCherry (left) and quantification of Whi5 nuclear export (right) in mother (M) and daughter (D) cells of the indicated strains. Whi5 export (arrows) is delayed in esa1-ts mothers and daughters compared to WT (p < 000.1, Log-rank Mantel-Cox test); esa1-ts nup60-KN advances Whi5 export relative to esa1-ts in daughters (p=0.0105), but not in mothers (p > 0.05). 3 z-confocal slices spaced 0.5 µm were acquired every 3 min; maximum projections of selected timepoints are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. n = number of cells, pooled from two independent experiments with similar results.


( A ) Composite of bright field and Whi5 - tdTomato ( BF / Whi5 ) and CLN2 - PP7 mRNA labelled with PCP - NLS - GFP ( CLN2 mRNA ) in mother ( M ) and daughter ( D ) cells of the indicated strains . Arrows indicate nuclear foci . Brighter GFP nucleoplasmic areas may correspond to the nucleolus . Insets show enhanced - contrast images to visualise cytoplasmic mRNA particles ( arrowheads ) . Numbers indicate minutes relative to Whi5 nuclear import . Maximum projections of whole - cell Z - stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1 - ts images , where single Z - slices are shown for clarity . Scale bar : 4 µm ( inset : 1 µm ) .

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(A) Composite of bright field and Whi5-tdTomato (BF/Whi5) and CLN2-PP7 mRNA labelled with PCP-NLS-GFP (CLN2 mRNA) in mother (M) and daughter (D) cells of the indicated strains. Arrows indicate nuclear foci. Brighter GFP nucleoplasmic areas may correspond to the nucleolus. Insets show enhanced-contrast images to visualise cytoplasmic mRNA particles (arrowheads). Numbers indicate minutes relative to Whi5 nuclear import. Maximum projections of whole-cell Z-stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1-ts images, where single Z-slices are shown for clarity. Scale bar: 4 µm (inset: 1 µm).


( B ) Fraction of cells of the indicated strains with nuclear CLN2 - PP7 mRNA foci , aligned relative to Whi5 import . Symbols represent individual values ; lines were generated by smoothing of the nearest 3 neighbouring values ( 0th order polynomial ) . ( C ) Fraction of cells of the indicated strains with nuclear CLN2 - PP7 mRNA foci , aligned relative to Whi5 export .

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(B) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 import. Symbols represent individual values; lines were generated by smoothing of the nearest 3 neighbouring values (0th order polynomial). (C) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 export.


( D ) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export , normalised relative to the nuclear background . Data information boxes include 50 % of data points , lines represent the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; * * , p ≤ 0 . 01 ; n . s . , p > 0 . 05 , ordinary one - way ANOVA with Tukey multiple comparisons test . Adjusted p - values in ( D ) : WT vs . esa1 - ts p < 0 . 0001 , WT vs . esa1 - ts nup60 - KN p < 0 . 0001 , esa1 - ts vs . esa1 - ts nup60 - KN p = 0 . 9845 Foci were scored in individual Z - slices spanning the entire cell volume . n = number of cells , pooled from two independent experiments with similar results .

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(D) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export, normalised relative to the nuclear background. Data information boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (D): WT vs. esa1-ts p < 0.0001, WT vs. esa1-ts nup60-KN p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.9845 Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.


( E ) Number of cytoplasmic CLN2 - PP7 mRNA foci in early G1 ( 5 minutes after Whi5 import ) and at the G1 / S transition ( 5 min after Whi5 export ) . Data information : boxes include 50 % of data points , lines represent the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; * * , p ≤ 0 . 01 ; n . s . , p > 0 . 05 , ordinary one - way ANOVA with Tukey multiple comparisons test . Adjusted p - values in ( E ) : WT vs . esa1 - ts p < 0 . 0001 , esa1 - ts vs . esa1 - ts nup60 - KN p = 0 . 002 . Foci were scored in individual Z - slices spanning the entire cell volume . n = number of cells , pooled from two independent experiments with similar results .

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(E) Number of cytoplasmic CLN2-PP7 mRNA foci in early G1 (5 minutes after Whi5 import) and at the G1/S transition (5 min after Whi5 export). Data information: boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (E): WT vs. esa1-ts p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.002. Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.


( F ) The position of nuclear CLN2 - PP7 foci ( marked with arrowheads in the examples ) was scored relatively to the nuclear periphery ( visualised with PCP - NLS - GFP ) in the indicated strains . The fraction of non - perinuclear CLN2 - PP7 foci is significantly increased in esa1 - ts cells compared to the WT and rescued by nup60 - KN mutation ( two - sided Fisher ' s exact test p = 0 . 0209 and p = 0 . 0227 ) . Scale bar , 1 µm .

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(F) The position of nuclear CLN2-PP7 foci (marked with arrowheads in the examples) was scored relatively to the nuclear periphery (visualised with PCP-NLS-GFP) in the indicated strains. The fraction of non-perinuclear CLN2-PP7 foci is significantly increased in esa1-ts cells compared to the WT and rescued by nup60-KN mutation (two-sided Fisher's exact test p = 0.0209 and p = 0.0227). Scale bar, 1 µm.


( B ) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3 - NLS overexpression . 10 - fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC - Glu and SC - Gal medium and incubated at 25 ° C for 3 days .

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(B) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3-NLS overexpression. 10-fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC-Glu and SC-Gal medium and incubated at 25 °C for 3 days.


( C ) A high - copy plasmid containing SAC3 rescues the lethality of Hos3 - NLS overexpression . Strains carrying a high copy plasmid containing the SAC3 ORF ( nucleotides 467 - 3905 ) , or the empty vector , were

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(C) A high-copy plasmid containing SAC3 rescues the lethality of Hos3-NLS overexpression. Strains carrying a high copy plasmid containing the SAC3 ORF (nucleotides 467-3905), or the empty vector, were grown


( D ) Overexpression of Hos3 - NLS promotes nuclear accumulation of mRNA . ( Left ) Cultures of the indicated strains were treated with galactose overnight to induce HOS3 - NLS expression , cells were fixed , and FISH was performed using Cy3 - Oligo ( dT ) . ( Right ) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions . Data information : , arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 µm .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overexpression of Hos3-NLS promotes nuclear accumulation of mRNA. (Left) Cultures of the indicated strains were treated with galactose overnight to induce HOS3-NLS expression, cells were fixed, and FISH was performed using Cy3-Oligo(dT). (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions. Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.


( E ) Inactivation of Esa1 impairs export of poly ( A ) RNA . ( Left ) Cultures of the indicated strains were incubated at 37 ºC . ( Right ) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information : , arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 µm .

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

(E) Inactivation of Esa1 impairs export of poly(A) RNA. (Left) Cultures of the indicated strains were incubated at 37 ºC. (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.


( F ) nup60 - KN mutation partially rescues the mRNA export defects of esa1 - ts . Cells of the indicated strains were processed Data information : arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 µm .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) nup60-KN mutation partially rescues the mRNA export defects of esa1-ts. Cells of the indicated strains were processed Data information: arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.


( A ) Depletion of Hos3 or expression of acetyl - mimic Nup60 ( nup60 - KN ) increases the nuclear localisation of Sac3 in G1 . Cells of the indicated strains were imaged by time - lapse microscopy and the fluorescence levels of the indicated proteins were determined in G1 ( after cytokinesis ) . The NPC component Nup49 was used as a control for Nuclear Pore Complex protein levels . Fluorescence intensity was measured in sum projections of whole - cell Z - stacks , by segmentation of either the nuclear area in the mCherry channel , or of the whole cell in the brightfield channel . The ratio of Sac3 / Nup49 intensities was then normalised relative to the mean intensity of wild - type mothers . Data information arrowheads point to daughter cells boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . Scale bar , 2 µm . n = number of cells , pooled from three independent experiments with similar results .

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(A) Depletion of Hos3 or expression of acetyl-mimic Nup60 (nup60-KN) increases the nuclear localisation of Sac3 in G1. Cells of the indicated strains were imaged by time-lapse microscopy and the fluorescence levels of the indicated proteins were determined in G1 (after cytokinesis). The NPC component Nup49 was used as a control for Nuclear Pore Complex protein levels. Fluorescence intensity was measured in sum projections of whole-cell Z-stacks, by segmentation of either the nuclear area in the mCherry channel, or of the whole cell in the brightfield channel. The ratio of Sac3/Nup49 intensities was then normalised relative to the mean intensity of wild-type mothers. Data information arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.


( B ) Inactivation of Esa1 decreases Sac3 nuclear levels . Wild - type ( WT ) and esa1 - ts cells were arrested in mitosis by treatment with nocodazole at 25 ºC , shifted to 37 ºC , released from the mitosis block in fresh medium at 37 ºC , and imaged by time - lapse microscopy . Fluorescence levels were quantified in G1 Data information : arrowheads point to daughter cells boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . Scale bar , 2 µm . n = number of cells , pooled from three independent experiments with similar results .

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(B) Inactivation of Esa1 decreases Sac3 nuclear levels. Wild-type (WT) and esa1-ts cells were arrested in mitosis by treatment with nocodazole at 25 ºC, shifted to 37 ºC, released from the mitosis block in fresh medium at 37 ºC, and imaged by time-lapse microscopy. Fluorescence levels were quantified in G1 Data information: arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.


( C ) Rapamycin - dependent dimerisation abolishes Sac3 mother / daughter asymmetries . NUP60 - mCherry - FKBP SAC3 - GFP - FRB cells were incubated with rapamycin ( RAPA ) to trigger FRB - FKBP heterodimerization , or with DMSO as control . Fluorescence levels were quantified in G1 cells 15 to 30 minutes after addition of the drug . Data information arrowheads point to daughter cells , boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . Scale bar , 2 µm . n = number of cells , pooled from three independent experiments with similar results .

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(C) Rapamycin-dependent dimerisation abolishes Sac3 mother/daughter asymmetries. NUP60-mCherry-FKBP SAC3-GFP-FRB cells were incubated with rapamycin (RAPA) to trigger FRB-FKBP heterodimerization, or with DMSO as control. Fluorescence levels were quantified in G1 cells 15 to 30 minutes after addition of the drug. Data information arrowheads point to daughter cells, boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.


( D ) Sac3 anchoring to the nuclear basket advances Start in esa1 - ts daughter cells . Composite of bright field and Whi5 - mGFP ( top ) and quantification of Whi5 nuclear export timing ( bottom ) in wild - type ( WT ) and esa1 - ts mother ( M ) and daughter ( D ) cells treated with either rapamycin ( RAPA ) or DMSO and expressing Nup60 - FRB and Sac3 - mCherry - FKBP . Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter cells ( DMSO vs RAPA , p > 0 . 05 , Log - rank Mantel - Cox test ) , but it advances Whi5 export in esa1 - ts daughters ( p = 0 . 0001 ) . Whi5 export efficiency was slightly improved also in mother cells ( p = 0 . 0374 ) . 8 z - confocal slices spaced 0 . 4 µm were acquired every 3 min ; maximum projections are shown . Time is indicated in minutes ; t = 0 marks Whi5 nuclear import . Scale bar , 5 µm . Data information : arrowheads point to Whi5 export .

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

(D) Sac3 anchoring to the nuclear basket advances Start in esa1-ts daughter cells. Composite of bright field and Whi5-mGFP (top) and quantification of Whi5 nuclear export timing (bottom) in wild-type (WT) and esa1-ts mother (M) and daughter (D) cells treated with either rapamycin (RAPA) or DMSO and expressing Nup60-FRB and Sac3-mCherry-FKBP. Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter cells (DMSO vs RAPA, p > 0.05, Log-rank Mantel-Cox test), but it advances Whi5 export in esa1-ts daughters (p = 0.0001). Whi5 export efficiency was slightly improved also in mother cells (p = 0.0374). 8 z-confocal slices spaced 0.4 µm were acquired every 3 min; maximum projections are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. Data information: arrowheads point to Whi5 export.


( A ) Time Lapse microscopy of WT and nup60 - KN cells expressing GAL1pr : sfGFP and Nup60 - mCherry at the indicated times of galactose induction . Scale bar , 4 µm .

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(A) Time Lapse microscopy of WT and nup60-KN cells expressing GAL1pr:sfGFP and Nup60-mCherry at the indicated times of galactose induction. Scale bar, 4 µm.


( B ) Depletion of Hos3 , and expression of acetyl - mimic Nup60 ( nup60 - KN ) enhances GAL1 expression . WT , hos3Δ , nup60 - KN and nup60 - KN hos3Δ cells were shifted to galactose and imaged by time - lapse microscopy to monitor GAL1pr : sfGFP expression during 7 hours . Nuclear fluorescence was scored by segmentation of the nuclear area in the mCherry channel and mean fluorescence of nuclear GFP and Nup60 - mCherry was quantified from sum projections of whole - cell Z - stacks at the indicated times . At least 200 cells were scored for each strain and time point . Shaded areas indicate the SEM .

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(B) Depletion of Hos3, and expression of acetyl-mimic Nup60 (nup60-KN) enhances GAL1 expression. WT, hos3Δ, nup60-KN and nup60-KN hos3Δ cells were shifted to galactose and imaged by time-lapse microscopy to monitor GAL1pr:sfGFP expression during 7 hours. Nuclear fluorescence was scored by segmentation of the nuclear area in the mCherry channel and mean fluorescence of nuclear GFP and Nup60-mCherry was quantified from sum projections of whole-cell Z-stacks at the indicated times. At least 200 cells were scored for each strain and time point. Shaded areas indicate the SEM.


( C ) mRNA levels of GAL1 were determined for wild type ( WT ) and nup60 - KN cells at the indicated times after galactose addition . One of two independent experiments with similar results is shown ( mean and SEM from 3 technical replicates ) .

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(C) mRNA levels of GAL1 were determined for wild type (WT) and nup60-KN cells at the indicated times after galactose addition. One of two independent experiments with similar results is shown (mean and SEM from 3 technical replicates).


( D ) The GFP intensity of mother / daughter pairs for cells in B , at 5 - minute intervals after galactose addition ( left ) and 425 min after galactose addition ( right ) . Boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed paired t - test . n = number of cells . One of two independent experiments with similar results is shown .

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(D) The GFP intensity of mother/daughter pairs for cells in B, at 5-minute intervals after galactose addition (left) and 425 min after galactose addition (right). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.001; ns, p > 0.05, two-tailed paired t-test. n = number of cells. One of two independent experiments with similar results is shown.


( E ) Expression of GAL1pr : sfGFP induced with galactose ( left ) or ß - estradiol ( right ) , in the presence of the ß - estradiol - dependent transactivator Gal4 - ER - VP16 . Smooth lines show GFP fluorescence intensity ( left x axis ) ; lines with circles show the difference in GFP intensity between the indicated strains ( right x axis ) . The difference between wild type and hos3 ∆ , and between wild type and nup60 - KN , increases continuously over time in response to galactose , but not to ß - estradiol .

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(E) Expression of GAL1pr:sfGFP induced with galactose (left) or ß-estradiol (right), in the presence of the ß-estradiol-dependent transactivator Gal4-ER-VP16. Smooth lines show GFP fluorescence intensity (left x axis); lines with circles show the difference in GFP intensity between the indicated strains (right x axis). The difference between wild type and hos3∆, and between wild type and nup60-KN, increases continuously over time in response to galactose, but not to ß-estradiol.