Northern blot analysis of Tf2 retrotransposons , U6 snRNA , and act1 + mRNA ( loading control ) in wt , cay1Δ , and prp1 ‐ 1 cells . prp1 ‐ 1 cells were grown at 36 ° C for 4 h before harvesting . Black arrowhead indicates unprocessed Tf2 transcripts . The asterisk indicates unspliced U6 . Numbers at the bottom are the ratios between Tf2 and act1 + signals ( Tf2 / Act1 ) and expressed as fold increase over wt .

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

Northern blot analysis of Tf2 retrotransposons, U6 snRNA, and act1+ mRNA (loading control) in wt, cay1Δ, and prp1‐1 cells. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Black arrowhead indicates unprocessed Tf2 transcripts. The asterisk indicates unspliced U6. Numbers at the bottom are the ratios between Tf2 and act1+ signals (Tf2/Act1) and expressed as fold increase over wt.


Northern blot analysis as in ( A ) in mpn1 + and mpn1 ∆ strains carrying a stably integrated extra copy of the snu6 + gene or empty vector control plasmids ( ev ) .

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

Northern blot analysis as in (A) in mpn1+ and mpn1∆ strains carrying a stably integrated extra copy of the snu6+ gene or empty vector control plasmids (ev).


OTULIN DNA sequence chromatograms showing the homozygous single base substitution ( c . 841G > A , p . Gly281Arg , arrowhead ) . Data are representative to two independent experiments .

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

OTULIN DNA sequence chromatograms showing the homozygous single base substitution (c.841G>A, p.Gly281Arg, arrowhead). Data are representative to two independent experiments.


Affinity measurements by fluorescence polarisation ( FP ) with recombinant catalytically inactive OTULINC129A or OTULINC129A / G281R and FlAsH - labelled M1 - linked diUb . Data represent mean ± SD of one experiment performed in technical triplicate . Data are representative of three independent experiments . a . u . , arbitrary units . K D , dissociation constant .

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

Affinity measurements by fluorescence polarisation (FP) with recombinant catalytically inactive OTULINC129A or OTULINC129A/G281R and FlAsH-labelled M1-linked diUb. Data represent mean ± SD of one experiment performed in technical triplicate. Data are representative of three independent experiments. a.u., arbitrary units. KD, dissociation constant.


M1 - linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver - stained SDS - PAGE gels . Data are representative of three independent experiments .

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

M1-linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver-stained SDS-PAGE gels. Data are representative of three independent experiments.


First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R ( 1 . 0 mg / mL ) measured by nano differential scanning fluorimetry ( nanoDSF ) . Apparent melting temperatures ( Tm ) are indicated ( dashed lines ) . Data are representative to two 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

First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R (1.0 mg/mL) measured by nano differential scanning fluorimetry (nanoDSF). Apparent melting temperatures (Tm) are indicated (dashed lines). Data are representative to two independent experiments.


Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts . Data are representative of 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

Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts. Data are representative of three independent experiments.


Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 ( 10 μM ) for 24 h . Data are representative of two independent experiments .

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

Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 (10 μM) for 24 h. Data are representative of two independent experiments.


Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts . Data are representative of three independent experiments .

Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts. Data are representative of three independent experiments.


Immunoblot ( left ) and densitometry ( right ) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of 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 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

Immunoblot (left) and densitometry (right) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.


Immunoblot analysis of IκBα phosphorylation and degradation as well as p65 / RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of 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

Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments. Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.


Densitometry analysis of p65 / RelA phosphorylation as presented in ( A ) from three independent experiments .

Densitometry analysis of p65/RelA phosphorylation as presented in (A) from three independent experiments.


ELISA analysis of IL - 8 secretion in response to TNF stimulation ( 10 ng / mL ) in healthy control or OTULING281R primary fibroblasts . Bars represent mean ± SEM ( n = 4 ) .

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

ELISA analysis of IL-8 secretion in response to TNF stimulation (10 ng/mL) in healthy control or OTULING281R primary fibroblasts. Bars represent mean ± SEM (n=4).


Immunoblot analysis of the native TNF - RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts . Data are representative of 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

Immunoblot analysis of the native TNF-RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts. Data are representative of three independent experiments.


Immunoblot analysis of whole cell lysate from untreated human THP - 1 monocytes with stable expression of a non - targeting control shRNA or and shRNA targeting OTULIN . Data are representative of two independent experiments .

Immunoblot analysis of whole cell lysate from untreated human THP-1 monocytes with stable expression of a non-targeting control shRNA or and shRNA targeting OTULIN. Data are representative of two independent experiments.


Immunoblot ( left ) and densitometry ( right ) analysis of IκBα levels in shControl and shOTULIN THP - 1 cells treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of 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 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

Immunoblot (left) and densitometry (right) analysis of IκBα levels in shControl and shOTULIN THP-1 cells treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.


ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP - 1 cells . Data represents mean ± SEM ( n = 4 ) and were analysed using the two - way ANOVA test of statistical significance with Sidak ' s correction for multiple comparisons .

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

ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP-1 cells. Data represents mean ± SEM (n=4) and were analysed using the two-way ANOVA test of statistical significance with Sidak's correction for multiple comparisons.


Immunoblot analysis of IκBα phosphorylation and degradation as well as p65 / RelA phosphorylation in shControl and shOTULIN THP - 1 cells in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of 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

Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in shControl and shOTULIN THP-1 cells in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.


Densitometry analysis of p65 / RelA phosphorylation as presented in ( D ) from three independent experiments .

Densitometry analysis of p65/RelA phosphorylation as presented in (D) from three independent experiments.


Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , as indicated was analysed using MTT reduction assays .

O O O O 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

Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), as indicated was analysed using MTT reduction assays.


Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , Q - VD - OPh ( 10 μM ) , and Nec - 1 ( 10 μM ) as indicated was analysed using MTT reduction assays .

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

Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.


Viability of shControl and shOTULIN THP - 1 cells ( C - D ) after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) ,

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

Viability of shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL),


shControl and shOTULIN THP - 1 cells ( C - D ) after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , Q - VD - OPh ( 10 μM ) , and Nec - 1 ( 10 μM ) as indicated was analysed using MTT reduction assays .

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

shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.


Increased apoptotic cell death in the skin of ORAS patient III . 2 . Serial sections of normal skin ( top panels ) and a skin biopsy from patient III . 2 taken at an inflammatory flare ( bottom panels ) were immunostained for cleaved caspase - 3 ( centre panels ) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) assay ( right panels ) . Arrowheads indicate cleaved caspase - 3 - positive mesenchymal cells . None of these markers were present in the healthy control skin . H & E , haematoxylin and eosin . Boxes in H & E panels indicate the areas magnified in the cleaved caspase - 3 and TUNEL panels . Scale bars , 100 μm . Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III . 2 .

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

Increased apoptotic cell death in the skin of ORAS patient III.2. Serial sections of normal skin (top panels) and a skin biopsy from patient III.2 taken at an inflammatory flare (bottom panels) were immunostained for cleaved caspase-3 (centre panels) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (right panels). Arrowheads indicate cleaved caspase-3-positive mesenchymal cells. None
of these markers were present in the healthy control skin. H&E, haematoxylin and eosin. Boxes in H&E panels indicate the areas magnified in the cleaved caspase-3 and TUNEL panels. Scale bars, 100 μm.
Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III.2.


Analysis of the blood cell chimerism in patient III . 2 after HSCT . Short terminal repeat ( STR ) analysis was used to determine the percentage of donor cells in peripheral blood . Arrow indicates point of relapse .

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

Analysis of the blood cell chimerism in patient III.2 after HSCT. Short terminal repeat (STR) analysis was used to determine the percentage of donor cells in peripheral blood. Arrow indicates point of relapse.


Photograph of patient III . 2 at age ~ 10 years at an episode of inflammation caused by delayed etanercept administration . Arrows indicate the erythematous subcutaneous nodules ( panniculitis ) .

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

Photograph of patient III.2 at age ~10 years at an episode of inflammation caused by delayed etanercept administration. Arrows indicate the erythematous subcutaneous nodules (panniculitis).


A , B Representative time - lapse images ( A ) and kymograph ( B ) of mouse zygotes expressing H2B - RFP ( magenta ) and labelled with SiR - Tubulin ( green ) . Shown is an example of marked chromosomal displacement to the cortex . C Changes in chromosome - to - cell centre distances ( d ) over time relative to anaphase - onset ( n = 50 blastomeres ) . Data information : In ( A , time is shown as h post - metaphase and scale bar = 20 µm . In ( C , data are shown as mean ± SEM .

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

A, B Representative time-lapse images (A) and kymograph (B) of mouse zygotes expressing H2B-RFP (magenta) and labelled with SiR-Tubulin (green). Shown is an example of marked chromosomal displacement to the cortex. C Changes in chromosome-to-cell centre distances (d) over time relative to anaphase-onset (n = 50 blastomeres). Data information: In (A, time is shown as h post-metaphase and scale bar = 20 µm. In (C, data are shown as mean ± SEM.


B , C Representative time - lapse images of embryos in which DMSO ( B ) or nocodazole ( C ) was added after anaphase completion . D Changes in nucleus - to - cell centre distances ( d ) over time relative to time of drug addition following DMSO - ( n = 22 blastomeres ) or nocodazole - treatment ( n = 26 blastomeres ) at anaphase completion . Data information : In ( B , C , time is shown as h post - drug addition and scale bar = 20 µm . In ( D , data are shown as mean ± SEM . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

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

B, C Representative time-lapse images of embryos in which DMSO (B) or nocodazole (C) was added after anaphase completion. D Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition following DMSO- (n = 22 blastomeres) or nocodazole-treatment (n = 26 blastomeres) at anaphase completion. Data information: In (B,C, time is shown as h post-drug addition and scale bar = 20 µm. In (D, data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


E - G Representative time - lapse images of embryos in which DMSO ( E ) , Nocodazole ( F ) or Cytochalasin D ( G ) was added when nuclei had travelled to the cortex . H Changes in nucleus - to - cell centre distances ( d ) over time relative to time of drug addition DMSO - ( n = 20 blastomeres ) , nocodazole - ( n = 12 blastomeres ) or cytochalasin D - treatment ( n = 14 blastomeres ) after nuclei reached the cortex . Data information : In E - G ) , time is shown as h post - drug addition and scale bar = 20 µm . In H ) , data are shown as mean ± SEM . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G Representative time-lapse images of embryos in which DMSO (E), Nocodazole (F) or Cytochalasin D (G) was added when nuclei had travelled to the cortex. H Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition DMSO- (n = 20 blastomeres), nocodazole- (n = 12 blastomeres) or cytochalasin D-treatment (n = 14 blastomeres) after nuclei reached the cortex. Data information: In E - G), time is shown as h post-drug addition and scale bar = 20 µm. In H), data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


A , B Representative time - lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase ( A , n = 15 embryos ) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin ( B , n = 22 embryos ) . C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not . Each data point represents the mean maximal d value of the two blastomeres from the same embryo ( n = 45 embryos ) . D Graph of time taken to develop to the blastocyst stage in relation to maximal d values ( n = 27 embryos ) . E Graph comparing the distribution of times taken for nuclei to re - centre in embryos that developed to the blastocyst stage or not ( n = 45 embryos ) . F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re - centre ( n = 27 embryos ) . Data information : In ( A , B ) , time is shown as h post - drug washout and scale bar = 20 µm . In ( C - F ) , data are shown as mean ± SEM . In ( C , E ) , statistical significance was assessed by Welch ' s t - test , ns : not significant . In ( D , F ) , the strength of correlation ( r ) and statistical significance were assessed using the Pearson correlation test , ns : not significant .

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

A, B Representative time-lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase (A, n = 15 embryos) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin (B, n = 22 embryos). C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not. Each data point represents the mean maximal d value of the two blastomeres from the same embryo (n = 45 embryos). D Graph of time taken to develop to the blastocyst stage in relation to maximal d values (n = 27 embryos). E Graph comparing the distribution of times taken for nuclei to re-centre in embryos that developed to the blastocyst stage or not (n = 45 embryos). F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re-centre (n = 27 embryos). Data information: In (A, B), time is shown as h post-drug washout and scale bar = 20 µm. In (C - F), data are shown as mean ± SEM. In (C, E), statistical significance was assessed by Welch's t-test, ns: not significant. In (D, F), the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test, ns: not significant.


A Representative time - lapse image of DNA and SiR - tubulin in DMSO - and BFA - treated embryos during the first mitotic division and subsequent interphase . Time is shown as h relative to the first mitotic anaphase . B Changes in nucleus - to - cell centre distances ( d ) over time in BFA - treated embryos , relative to maximal nuclear displacement to the cortex ( n = 14 blastomeres ) . Data information : In ( A , scale bar = 20 µm .

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

A Representative time-lapse image of DNA and SiR-tubulin in DMSO- and BFA-treated embryos during the first mitotic division and subsequent interphase. Time is shown as h relative to the first mitotic anaphase. B Changes in nucleus-to-cell centre distances (d) over time in BFA-treated embryos, relative to maximal nuclear displacement to the cortex (n = 14 blastomeres). Data information: In (A, scale bar = 20 µm.


D Representative time - lapse images of the second mitotic division in DMSO - and BFA - treated embryos . Dashed white lines highlight position of nuclei shortly before NEBD . Dashed yellow lines outline daughter - cells . Time is shown as h relative to the second mitotic NEBD . E Dot plot of the duration between first mitotic division anaphase - onset and second mitotic division NEBD for DMSO - ( n = 47 blastomeres ) and BFA - treated ( n = 47 blastomeres ) embryos . F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO - ( n = 44 blastomeres ) and BFA - treated ( n = 43 blastomeres ) embryos . G Plot of the spindle - to - cell centre distances against pre - NEBD nucleus - to - cell centre distance ( n = 34 blastomeres ) . Line indicates mathematically regressed fit . Data information : In D ) , scale bar = 20 µm . In E - G , data are shown as mean ± SEM . In ( E , F , statistical significance was assessed by Mann - Whitney test , * * * * P < 0 . 0001 , ns : not significant . In ( G , the strength of correlation ( r ) and statistical significance were assessed using the Pearson correlation test . Two - tailed p values are reported ( * * P < 0 . 01 ) .

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

D Representative time-lapse images of the second mitotic division in DMSO- and BFA-treated embryos. Dashed white lines highlight position of nuclei shortly before NEBD. Dashed yellow lines outline daughter-cells. Time is shown as h relative to the second mitotic NEBD. E Dot plot of the duration between first mitotic division anaphase-onset and second mitotic division NEBD for DMSO- (n = 47 blastomeres) and BFA-treated (n = 47 blastomeres) embryos. F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO- (n = 44 blastomeres) and BFA-treated (n = 43 blastomeres) embryos. G Plot of the spindle-to-cell centre distances against pre-NEBD nucleus-to-cell centre distance (n = 34 blastomeres). Line indicates mathematically regressed fit. Data information: In D), scale bar = 20 µm. In E - G, data are shown as mean ± SEM. In (E, F, statistical significance was assessed by Mann-Whitney test, ****P < 0.0001, ns: not significant. In (G, the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test. Two-tailed p values are reported (**P < 0.01).


A Representative time - lapse images of 2 - cell embryos cultured with SiR - DNA to label nuclei ( white circles ) and microinjected with oil droplets ( yellow circles ) . Time is shown as h : min relative to maximal nuclear displacement . B Plot of the distances between oil droplets and nuclei to the cell - centre over time ( n = 14 oil - nucleus pairs ) . Data information : Scale bar = 20 µm in A , In ( B ) , data are shown as mean ± SEM .

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A Representative time-lapse images of 2-cell embryos cultured with SiR-DNA to label nuclei (white circles) and microinjected with oil droplets (yellow circles). Time is shown as h:min relative to maximal nuclear displacement. B Plot of the distances between oil droplets and nuclei to the cell-centre over time (n = 14 oil-nucleus pairs). Data information: Scale bar = 20 µm in A, In (B), data are shown as mean ± SEM.


E Top panel : an example of fluorescent bead distribution in a 2 - cell embryo . Lower panel : an example of region imaged for bead tracking at high temporal resolution . F , G Trajectories of bead movement over a 20 s - duration in oocytes ( F , n = 217 beads from 23 oocytes ) and 2 - cell embryos ( n = 277 beads from 26 embryos ) . Each coloured line represents a single bead track . Data information : Scale bar = 20 µm in E , except 2 µm in the lower panel of E .

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E Top panel: an example of fluorescent bead distribution in a 2-cell embryo. Lower panel: an example of region imaged for bead tracking at high temporal resolution. F, G Trajectories of bead movement over a 20 s-duration in oocytes (F, n = 217 beads from 23 oocytes) and 2-cell embryos (n = 277 beads from 26 embryos). Each coloured line represents a single bead track. Data information: Scale bar = 20 µm in E, except 2 µm in the lower panel of E.


B Shown is a representative 2 - cell embryo immunostained for β - tubulin ( green ) and DNA ( magenta ) at anaphase completion ( n = 8 embryos ) . C Dot plot of mean β - tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus ( cortex - facing aspect , dashed white triangle in B ) or behind the nucleus ( cleavage plane - facing aspect , dashed yellow triangle in B ) ( n = 14 embryos ) . Data information : In ( B scale bar = 20 µm . In ( C , data are shown as mean ± SEM . In ( C , statistical significance was assessed by Welch ' s t - test , * * * * P < 0 . 0001 , ns = not significant .

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B Shown is a representative 2-cell embryo immunostained for β-tubulin (green) and DNA (magenta) at anaphase completion (n = 8 embryos). C Dot plot of mean β-tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus (cortex-facing aspect, dashed white triangle in B) or behind the nucleus (cleavage plane-facing aspect, dashed yellow triangle in B) (n = 14 embryos). Data information: In (B scale bar = 20 µm. In (C, data are shown as mean ± SEM. In (C, statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.


D Representative time - lapse images of embryos expressing H2B - RFP and labelled with SiR - tubulin following the addition of dynarrestin at anaphase completion ( n = 14 embryos ) . Data information : In ( D , time is shown as h post - drug addition . In D , scale bar = 20 µm .

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D Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of dynarrestin at anaphase completion (n = 14 embryos). Data information: In (D, time is shown as h post-drug addition. In D, scale bar = 20 µm.


E Dot plot of maximal d values between DMSO - and dynarrestin - treated embryos ( n = 28 blastomeres per group ) . Data information : In E , data are shown as mean ± SEM . In E ) , statistical significance was assessed by Welch ' s t - test , * * * * P < 0 . 0001 , ns = not significant .

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E Dot plot of maximal d values between DMSO- and dynarrestin-treated embryos (n = 28 blastomeres per group). Data information: In E, data are shown as mean ± SEM. In E), statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.


G Representative time - lapse images of embryos expressing H2B - RFP and labelled with SiR - tubulin following the addition of taxol at maximal nuclear displacement . Data information : In G , scale bar = 20 µm .

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G Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of taxol at maximal nuclear displacement. Data information: In G, scale bar = 20 µm.


H Changes in nucleus - to - cell centre distances ( d ) over time relative to the time of drug addition in DMSO - ( n = 19 ) and taxol - ( n = 19 ) treated 2 - cell blastomeres ( mean ± SEM ) . Data information : In H ) , data are shown as mean ± SEM . In ( H ) , statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unremarked datapoints = not significant ) .

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H Changes in nucleus-to-cell centre distances (d) over time relative to the time of drug addition in DMSO- (n = 19) and taxol- (n = 19) treated 2-cell blastomeres (mean ± SEM). Data information: In H), data are shown as mean ± SEM. In (H), statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, unremarked datapoints = not significant).


I Representative images of embryos immunostained for β - tubulin and Hoechst following the completion of live imaging ( shown in E ) in DMSO - and taxol - treated embryos ( n = 9 embryos per group ) . Time is shown as h post - drug addition . Scale bar = 20 µm . Data information : In I ) , scale bar = 20 µm .

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I Representative images of embryos immunostained for β-tubulin and Hoechst following the completion of live imaging (shown in E) in DMSO- and taxol-treated embryos (n = 9 embryos per group). Time is shown as h post-drug addition. Scale bar = 20 µm. Data information: In I), scale bar = 20 µm.


B Pim1 , 2 , or 3 and GFP - EDC3 expression vectors were transfected into HEK - 293T . GFP - Trap Nano - body beads ( GFP - Trap ) were used to immunoprecipitate ( IP ) GFP - EDC3 , and the associated proteins were analyzed by Western blotting with the indicated Abs .

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B Pim1, 2, or 3 and GFP-EDC3 expression vectors were transfected into HEK-293T. GFP-Trap Nano-body beads (GFP-Trap) were used to immunoprecipitate (IP) GFP-EDC3, and the associated proteins were analyzed by Western blotting with the indicated Abs.


B HEK - 293T cells were transfected with Pim1 and each EDC3 domain ( Fig . 2A ) fused to the C - terminus of the GFP - tagged vector . The individual fragments were immunoprecipitated with GFP - Trap beads and analyzed by Western blotting with the indicated Abs . EDC3 wild type ( W ) , F1 , F2 , F3 , F4 were EDC3 fragments vector fused to GFP . The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control ( pcDNA3 ) .

B HEK-293T cells were transfected with Pim1 and each EDC3 domain (Fig. 2A) fused to the C-terminus of the GFP-tagged vector. The individual fragments were immunoprecipitated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs. EDC3 wild type (W), F1, F2, F3, F4 were EDC3 fragments vector fused to GFP. The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control (pcDNA3).


F PC3 - LN4 CRISPR - Ctr and Pim1 KO cells were treated with PIMi ( PIM447 , 3μM ) , AKTi ( GSK690693 , 5μM ) , or the combination for 24 h . Extracts were subjected to Western blotting with the indicated Abs .

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F PC3-LN4 CRISPR-Ctr and Pim1 KO cells were treated with PIMi (PIM447, 3μM), AKTi (GSK690693, 5μM), or the combination for 24 h. Extracts were subjected to Western blotting with the indicated Abs.


Quantification of EDC3 / DCP1a foci number from Control ( Ctrl ) , PIMi , AKTi and the AKTi + PIMi combination in ( A ) . Data information : The quantification data of ( B are presented as mean + / - SEM ( n = 30 cells / treatment group ) . p values < 0 . 05 , calculated using unpaired student t - test , are considered significant . p values > 0 . 05 not significant ( n . s . ) . The analyses done included multiple independent fields .

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Quantification of EDC3/DCP1a foci number from Control (Ctrl), PIMi, AKTi and the AKTi + PIMi combination in (A). Data information: The quantification data of (B are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.


D Quantification of DCP1a foci number for Control ( Ctrl ) , PIMi , AKTi , and AKTi + PIMi combination in ( C ) . Data information : The quantification data of D ) are presented as mean + / - SEM ( n = 30 cells / treatment group ) . p values < 0 . 05 , calculated using unpaired student t - test , are considered significant . p values > 0 . 05 not significant ( n . s . ) . The analyses done included multiple independent fields .

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D Quantification of DCP1a foci number for Control (Ctrl), PIMi, AKTi, and AKTi + PIMi combination in (C). Data information: The quantification data of D) are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.


A , B Western blot of whole - cell ( WCL ) and P - body ( PB ) purified from PC3 - LN4 cells stably expressing GFP - DCP1a used as a P - body tag . The P - bodies ( PB ) were isolated with GFP - Trap beads and analyzed by Western blotting with the indicated Abs .

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A, B Western blot of whole-cell (WCL) and P-body (PB) purified from PC3-LN4 cells stably expressing GFP-DCP1a used as a P-body tag. The P-bodies (PB) were isolated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs.


A PC3 - LN4 Naïve and CRISPR / Cas9 genome - edited EDC3 S161A ( SA ) , S161D ( Fredriksson et al . ) , and EDC3 knock - out ( KO ) cell lines were plated ( 4000 cells / well in 24 well plates ) and imaged every 12 h using phase contrast in the IncuCyte ZOOM platform . The graph shown was generated with IncuCyte Basic Software following 5 days of growth . The experiments were done in triplicates . Values are mean + / - S . E . M . p values < 0 . 05 , calculated using unpaired student t - test . Data information : All invasion and migration assays were performed in triplicates and 6 different fields at 10x magnification were counted in 3 separate inserts . The data are presented as the mean + / - S . D . p values < 0 . 05 , calculated using unpaired student ' s t - test .

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A PC3-LN4 Naïve and CRISPR/Cas9 genome-edited EDC3 S161A (SA), S161D (Fredriksson et al.), and EDC3 knock-out (KO) cell lines were plated (4000 cells/well in 24 well plates) and imaged every 12 h using phase contrast in the IncuCyte ZOOM platform. The graph shown was generated with IncuCyte Basic Software following 5 days of growth. The experiments were done in triplicates. Values are mean +/- S.E.M. p values<0.05, calculated using unpaired student t-test. Data information: All invasion and migration assays were performed in triplicates and 6 different fields at 10x magnification were counted in 3 separate inserts. The data are presented as the mean +/- S.D. p values<0.05, calculated using unpaired student's t-test.


H Relative mRNA expression of ITGA6 , ITGB1 , and KLF4 in PC3 - LN4 EDC3 KO - C60 cells , containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs Data information : Real - time PCR

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H Relative mRNA expression of ITGA6, ITGB1, and KLF4 in PC3-LN4 EDC3 KO-C60 cells, containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs Data information: Real-time PCR data


J Western blot analysis of proteins extracted from PC3 - LN4 cells treated with DMSO or the combination of Pim447 and AZD5363 for 96 h using the specified antibodies .

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J Western blot analysis of proteins extracted from PC3-LN4 cells treated with DMSO or the combination of Pim447 and AZD5363 for 96 h using the specified antibodies.


A Two independently derived CRISPR / Cas9 PC3 - LN4 EDC3 S161A mutants ( EDC3 SA # 1 , # 2 ) and wild type cells were injected subcutaneously in the flank ( 5X106 ) of SCID mice ( n = 8 / group ) , and tumor growth was monitored until the control group reached 1000 mm3 . The subcutaneous tumor volume measurement ( mm3 ) was done as outlined in Materials and Methods . Data are presented as mean ± SEM , ( n = 8 / group ) .

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A Two independently derived CRISPR/Cas9 PC3-LN4 EDC3 S161A mutants (EDC3 SA#1, #2) and wild type cells were injected subcutaneously in the flank (5X106) of SCID mice (n=8/group), and tumor growth was monitored until the control group reached 1000 mm3. The subcutaneous tumor volume measurement (mm3) was done as outlined in Materials and Methods. Data are presented as mean ± SEM, (n = 8/group).


B Phosphorylated EDC3 levels were examined in normal immortalized breast cell lines , MCF10A and MCF12A , human breast cancer cell lines HCC38 , HCC1954 , MDA - MB - 231 , MDA - MD - 468 , BT - 549 , MRKNU1 , and mouse 4T1 by Western blotting with the indicated Abs .

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B Phosphorylated EDC3 levels were examined in normal immortalized breast cell lines, MCF10A and MCF12A, human breast cancer cell lines HCC38, HCC1954, MDA-MB-231, MDA-MD-468, BT-549, MRKNU1, and mouse 4T1 by Western blotting with the indicated Abs.


C Dual immunohistochemistry staining of human breast tumor with p - EDC3 Ab ( Fredriksson et al . ) and total EDC3 ( brown ) . Scale bar : 50µm . D Dual immunohistochemistry staining of human normal resected breast tissue sections with p - EDC3 Ab ( Fredriksson et al . ) and total EDC3 ( brown ) . Scale bar : 50µm .

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C Dual immunohistochemistry staining of human breast tumor with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm. D Dual immunohistochemistry staining of human normal resected breast tissue sections with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm.


A Montage of class averages and 3D volumes of humanTFG ( amino acids 1 ‐ 193 ; EMDataBank accession code EMD ‐ 6076 ) . Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference ‐ free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left . Scale bars , 50 Å .

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A Montage of class averages and 3D volumes of humanTFG (amino acids 1‐193; EMDataBank accession code EMD‐6076). Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference‐free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left. Scale bars, 50 Å.


B Montage of class averages and 3D volumes of C . elegans TFG ( amino acids 1 ‐ 195 ; EMDataBank accession code EMD ‐ 6075 ) , generated as described in ( A ) . Scale bars , 50 Å .

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B Montage of class averages and 3D volumes of C. elegans TFG (amino acids 1‐195; EMDataBank accession code EMD‐6075), generated as described in (A). Scale bars, 50 Å.


C Superposition of 3D volumes of full ‐ length human TFG and truncated TFG ( amino acids 1 ‐ 193 ) . The truncated form is depicted in yellow , while the full ‐ length form is depicted as gray mesh . The top view ( top ) exhibits limited differences in the structures , while the side view ( bottom ) shows extra density in the full ‐ length TFG isoform . Scale bar , 25 Å .

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C Superposition of 3D volumes of full‐length human TFG and truncated TFG (amino acids 1‐193). The truncated form is depicted in yellow, while the full‐length form is depicted as gray mesh. The top view (top) exhibits limited differences in the structures, while the side view (bottom) shows extra density in the full‐length TFG isoform. Scale bar, 25 Å.


A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations . Error bars represent mean ± SEM ; n = 10 replicates .

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A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations. Error bars represent mean ± SEM; n = 10 replicates.


B Confocal microscopy images of recombinant , BODIPY ‐ labeled TFG in the presence of varying potassium acetate concentrations . Scale bar , 2 μm .

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B Confocal microscopy images of recombinant, BODIPY‐labeled TFG in the presence of varying potassium acetate concentrations. Scale bar, 2 μm.


C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain ‐ EM . Large arrowheads highlight TFG polymers ( only found in the presence of elevated potassium acetate ) . Small arrowheads highlight individual 11 nm cup ‐ like TFG octamers . Scale bar , 30 nm .

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C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain‐EM. Large arrowheads highlight TFG polymers (only found in the presence of elevated potassium acetate). Small arrowheads highlight individual 11 nm cup‐like TFG octamers. Scale bar, 30 nm.


D Circular dichroism spectroscopy was used to characterize the carboxyl ‐ terminus of C . elegans TFG ( amino acids 195 ‐ 486 ) . Samples were analyzed at different concentrations , and the data were normalized relative to one another . CD spectra were collected at 25 ° C in 25 mM sodium phosphate ( pH 7 . 2 ) using a 1 mm path length quartz cell . The spectra are characteristic of an intrinsically disordered protein .

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D Circular dichroism spectroscopy was used to characterize the carboxyl‐terminus of C. elegans TFG (amino acids 195‐486). Samples were analyzed at different concentrations, and the data were normalized relative to one another. CD spectra were collected at 25°C in 25 mM sodium phosphate (pH 7.2) using a 1 mm path length quartz cell. The spectra are characteristic of an intrinsically disordered protein.


A The distributions of endogenous TFG and ERGIC ‐ 53 were examined in hTERT ‐ RPE ‐ 1 cells stably expressing low levels of mApple ‐ Sec16B using SR ‐ SIM ( more than 15 cells analyzed on 3 separate occasions ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

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A The distributions of endogenous TFG and ERGIC‐53 were examined in hTERT‐RPE‐1 cells stably expressing low levels of mApple‐Sec16B using SR‐SIM (more than 15 cells analyzed on 3 separate occasions). Scale bar, 5 μm. Inset scale bar, 1 μm.


B Cells stably expressing low levels of GFP ‐ Sec16B and mRuby ‐ Sec23A ( Sec23A ) were fixed and imaged using SR ‐ SIM ( n = 12 ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

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B Cells stably expressing low levels of GFP‐Sec16B and mRuby‐Sec23A (Sec23A) were fixed and imaged using SR‐SIM (n = 12). Scale bar, 5 μm. Inset scale bar, 1 μm.


C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby ‐ Sec23A ( Sec23A ) using SR ‐ SIM ( n = 10 ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby‐Sec23A (Sec23A) using SR‐SIM (n = 10). Scale bar, 5 μm. Inset scale bar, 1 μm.


D The distributions of endogenous TFG and Sec31A were examined using g ‐ STED ( n = 8 ) . Scale bar , 1 μm .

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D The distributions of endogenous TFG and Sec31A were examined using g‐STED (n = 8). Scale bar, 1 μm.


E The distributions of endogenous TFG and Sec31A were examined using immunogold EM . Large arrowheads highlight 15 nm gold particles bound to α ‐ TFG antibodies . The small arrowhead highlights 5 nm gold particles bound to α ‐ Sec31A antibodies . At least 15 different cells were examined , and representative images are shown . Scale bar , 100 nm .

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E The distributions of endogenous TFG and Sec31A were examined using immunogold EM. Large arrowheads highlight 15 nm gold particles bound to α‐TFG antibodies. The small arrowhead highlights 5 nm gold particles bound to α‐Sec31A antibodies. At least 15 different cells were examined, and representative images are shown. Scale bar, 100 nm.


A Cells depleted of endogenous TFG were transfected with a variety of TFG transgenes ( including full ‐ length , untagged TFG , a truncation mutant encoding amino acids 1 ‐ 300 of untagged TFG , and a truncation mutant encoding amino acids 1 ‐ 350 of untagged TFG ) and stained using α ‐ TFG and α ‐ Sec31A antibodies . Scale bar , 5 μm . Inset scale bar , 1 μm . Images shown are representative of at least 15 individual cells analyzed for each condition .

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A Cells depleted of endogenous TFG were transfected with a variety of TFG transgenes (including full‐length, untagged TFG, a truncation mutant encoding amino acids 1‐300 of untagged TFG, and a truncation mutant encoding amino acids 1‐350 of untagged TFG) and stained using α‐TFG and α‐Sec31A antibodies. Scale bar, 5 μm. Inset scale bar, 1 μm. Images shown are representative of at least 15 individual cells analyzed for each condition.


B A recombinant polyhistidine ‐ and SUMO ‐ tagged TFG fragment ( Sumo ‐ TFGc , amino acids 194 ‐ 400 ; total molecular mass of 42 kDa ) was expressed and purified from Escherichia coli extracts using nickel affinity resin . Coomassie ‐ stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column ( top ) or a 10 - 30 % glycerol gradient ( bottom ) are shown . To determine the native molecular weight of the protein , the following equation was used : M = 6πηNas / ( 1 − υρ ) , where M is the native molecular weight , η is the viscosity of the medium , N is Avogadro ' s number , a is the Stokes radius , s is the sedimentation value , υ is the partial specific volume , and ρ is the density of the medium ( Siegel Monty , ) . These data suggest that this fragment of TFG is capable of forming dimers in solution .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A recombinant polyhistidine‐ and SUMO‐tagged TFG fragment (Sumo‐TFGc, amino acids 194‐400; total molecular mass of 42 kDa) was expressed and purified from Escherichia coli extracts using nickel affinity resin. Coomassie‐stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column (top) or a 10-30% glycerol gradient (bottom) are shown. To determine the native molecular weight of the protein, the following equation was used: M = 6πηNas/(1 − υρ), where M is the native molecular weight, η is the viscosity of the medium, N is Avogadro's number, a is the Stokes radius, s is the sedimentation value, υ is the partial specific volume, and ρ is the density of the medium (Siegel Monty, ). These data suggest that this fragment of TFG is capable of forming dimers in solution.


C , D Control cells ( C ) or cells depleted of endogenous TFG ( D ) were transfected with a construct expressing TFG with a carboxyl ‐ terminal GFP tag and stained using α ‐ Sec31A antibodies . Scale bar , 5 μm . Images shown are representative of at least 20 individual cells analyzed for each condition .

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C, D Control cells (C) or cells depleted of endogenous TFG (D) were transfected with a construct expressing TFG with a carboxyl‐terminal GFP tag and stained using α‐Sec31A antibodies. Scale bar, 5 μm. Images shown are representative of at least 20 individual cells analyzed for each condition.


A HumanRPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h . Cells were fixed and stained using α ‐ Sec16A and α ‐ Sec31A antibodies and imaged using confocal microscopy . Images shown are projections of 3D data sets ( 4 μm in z ) . Both individual and merged images with Sec16A in green and Sec31A in red are shown ( at least 15 cells for each condition ) . Scale bar , 5 μm . Higher magnification views of the indicated regions ( boxed ) are also shown in the lower or upper right portion of each image . Inset scale bar , 1 μm . B Quantification of the number of Sec31A ‐ labeled structures in a 4 μm2 region . For both conditions ( control or TFG siRNA ‐ treated ) , at least 50 distinct regions away from the peri ‐ nuclear Golgi were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . * * P 0 . 01 compared with control , calculated using a paired t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HumanRPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α‐Sec16A and α‐Sec31A antibodies and imaged using confocal microscopy. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and Sec31A in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Inset scale bar, 1 μm.B Quantification of the number of Sec31A‐labeled structures in a 4 μm2 region. For both conditions (control or TFG siRNA‐treated), at least 50 distinct regions away from the peri‐nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P 0.01 compared with control, calculated using a paired t‐test.


C Quantification of the number of Sec16A ‐ labeled structures in a 4 μm2 region . For both conditions ( control or TFG siRNA ‐ treated ) , at least 50 distinct regions away from the peri ‐ nuclear Golgi were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . No statistically significant difference was observed , following analysis using a paired 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

C Quantification of the number of Sec16A‐labeled structures in a 4 μm2 region. For both conditions (control or TFG siRNA‐treated), at least 50 distinct regions away from the peri‐nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. No statistically significant difference was observed, following analysis using a paired t‐test.


D Cells stably expressing low levels of GFP ‐ ERGIC ‐ 53 and mRuby ‐ Sec23A were fixed and imaged using SR ‐ SIM ( top ) , or depleted of endogenous TFG prior to fixation and SR ‐ SIM imaging ( bottom ) . Images shown are representative of at least 15 individual cells analyzed for each condition . Scale bar , 5 μm . Inset scale bar , 1 μm .

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D Cells stably expressing low levels of GFP‐ERGIC‐53 and mRuby‐Sec23A were fixed and imaged using SR‐SIM (top), or depleted of endogenous TFG prior to fixation and SR‐SIM imaging (bottom). Images shown are representative of at least 15 individual cells analyzed for each condition. Scale bar, 5 μm. Inset scale bar, 1 μm.


E HumanRPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h . Cells were fixed and stained using α ‐ Sec16A and α ‐ ERGIC ‐ 53 antibodies and imaged using SR ‐ SIM . Images shown are projections of 3D data sets ( 4 μm in z ) . Both individual and merged images with Sec16A in green and ERGIC ‐ 53 in red are shown ( at least 15 cells for each condition ) . Scale bar , 5 μm . Higher magnification views of the indicated regions ( boxed ) are also shown in the lower or upper right portion of each image . Arrowheads highlight Sec16A ‐ labeled structures that do not exhibit juxtaposed ERGIC ‐ 53 staining . Inset scale bar , 1 μm . F Based on structured illumination microscopy , quantification of the number of Sec16A ‐ labeled structures juxtaposed ( within 500 nm ) to ERGIC ‐ 53 ‐ labeled structures is shown . For both conditions ( control or TFG siRNA ‐ treated ) , at least 1 , 000 unique Sec16A ‐ labeled structures were examined . Error bars represent mean ± SEM ; at least 15 different cells per condition . * * P 0 . 01 compared with control , calculated using a paired t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HumanRPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α‐Sec16A and α‐ERGIC‐53 antibodies and imaged using SR‐SIM. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and ERGIC‐53 in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Arrowheads highlight Sec16A‐labeled structures that do not exhibit juxtaposed ERGIC‐53 staining. Inset scale bar, 1 μm.F Based on structured illumination microscopy, quantification of the number of Sec16A‐labeled structures juxtaposed (within 500 nm) to ERGIC‐53‐labeled structures is shown. For both conditions (control or TFG siRNA‐treated), at least 1,000 unique Sec16A‐labeled structures were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P 0.01 compared with control, calculated using a paired t‐test.


A HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple ( ManII ‐ mApple ) were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed and stained using TFG antibodies and imaged using confocal microscopy . Alternatively , cells were either treated with DMSO ( top row ) or brefeldin A ( BFA ) for 1 h ( middle row ) , followed by fixation , or washed into fresh media following DMSO or BFA treatment for 30 min ( bottom row ) , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with TFG in green and ManII in red are shown . Scale bar , 5 μm . Images shown are representative of at least 10 individual cells analyzed for each condition . B Human RPE ‐ 1 cells were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy . Alternatively , cells were either treated with DMSO ( top row ) or brefeldin A ( BFA ) for 1 h ( middle row ) , followed by fixation , or washed into fresh media following DMSO or BFA treatment for 30 min ( bottom row ) , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with TFG in green and GM130 in red are shown . Scale bar , 5 μm . Images shown are representative of at least 10 individual cells analyzed for each condition .

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

A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple (ManII‐mApple) were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and ManII in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.B Human RPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and GM130 in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.


C Higher magnification views of the indicated regions in ( B , boxed ) are shown ( GM130 distribution only ) . Scale bar , 2 μm .

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C Higher magnification views of the indicated regions in (B, boxed) are shown (GM130 distribution only). Scale bar, 2 μm.


A HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . Cells were subsequently treated with brefeldin A ( BFA ) for 1 h , followed by a wash into fresh media and further incubation for 2 h in the absence of BFA , prior to fixation . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with Sec31A in green and ManII in red are shown ( representative of at least 15 cells analyzed ) . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with brefeldin A (BFA) for 1 h, followed by a wash into fresh media and further incubation for 2 h in the absence of BFA, prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A in green and ManII in red are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.


B HumanRPE ‐ 1 cells stably expressing low levels of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . Cells were subsequently treated with BFA for 1 h , followed by a wash into fresh media and further incubation for 1 h in the absence of BFA , prior to fixation and staining using α ‐ Sec16A and α ‐ Sec31A antibodies . Images shown are projections of 3D data sets ( 4 μm in z ) . Merged images with Sec31A ( green ) , ManII ( red ) , and Sec16A ( blue ) are shown ( representative of at least 15 cells analyzed ) . Scale bar , 5 μm . C Higher magnification views of the indicated regions in ( B , boxed ) are shown . Arrows highlight COPII ‐ positive transport carriers that contain the cargo ManII , which are not juxtaposed to Sec16A ‐ labeled sites on the ER . Additionally , arrowheads point out distinct foci in which COPII continues to associate with Sec16A ‐ labeled sites , indicating that COPII vesicle formation continues in the absence of TFG . Scale bar , 1 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with BFA for 1 h, followed by a wash into fresh media and further incubation for 1 h in the absence of BFA, prior to fixation and staining using α‐Sec16A and α‐Sec31A antibodies. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A (green), ManII (red), and Sec16A (blue) are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.C Higher magnification views of the indicated regions in (B, boxed) are shown. Arrows highlight COPII‐positive transport carriers that contain the cargo ManII, which are not juxtaposed to Sec16A‐labeled sites on the ER. Additionally, arrowheads point out distinct foci in which COPII continues to associate with Sec16A‐labeled sites, indicating that COPII vesicle formation continues in the absence of TFG. Scale bar, 1 μm.


Jurkat T cells were treated with either DMSO ( vehicle ) or with 100μM of Dieldrin for the indicated times . Abundance of mRNA for the indicated genes or HERVs was assessed by RT - qPCR .

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

Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of Dieldrin for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.


Jurkat T cells were treated with either DMSO ( vehicle ) or with 100μM of PMA for the indicated times . Abundance of mRNA for the indicated genes or HERVs was assessed by RT - qPCR .

Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of PMA for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.


Jurkat T cells were treated with either DMSO ( vehicle ) or with 100μM of either Dieldrin or PMA for the indicated times . Abundance of mRNA for the indicated genes by RT - qPCR .

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

Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of either Dieldrin or PMA for the indicated times. Abundance of mRNA for the indicated genes by RT-qPCR.


Jurkat cells were exposed to either DMSO , PMA in DMSO , or dieldrin in DMSO for 30 min in triplicates . RNA - seq was then performed on cDNA libraries prepared with poly ( A ) selection : principal component analysis on the 485 genes the most affected by DMSO , PMA , or Dieldrin .

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

Jurkat cells were exposed to either DMSO, PMA in DMSO, or dieldrin in DMSO for 30 min in triplicates. RNA-seq was then performed on cDNA libraries prepared with poly(A) selection: principal component analysis on the 485 genes the most affected by DMSO, PMA, or Dieldrin.


Jurkat cells were exposed to either DMSO , or dieldrin in DMSO for 30 min . RNA - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of the Jurkat cells to dieldrin . Top tracks show coverage from sequencing of poly ( A ) - enriched libraries for comparison . All coverage tracks are at the same scale and all the sequencing result files are of comparable size . Blue and red reads ( rds ) are in opposite orientation . CAGE peaks use the same color code . Reads mapping inside a series of 4686 HERVs located away ( 30Kb ) from protein - coding genes were quantified both in the poly ( A ) - enriched and the ribo - depleted data . Read counts from each replicate were then summed and plotted .

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

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of the Jurkat cells to dieldrin. Top tracks show coverage from sequencing of poly(A)-enriched libraries for comparison. All coverage tracks are at the same scale and all the sequencing result files are of comparable size. Blue and red reads (rds) are in opposite orientation. CAGE peaks use the same color code. Reads mapping inside a series of 4686 HERVs located away (30Kb) from protein-coding genes were quantified both in the poly(A)-enriched and the ribo-depleted data. Read counts from each replicate were then summed and plotted.


Jurkat cells were exposed to either DMSO , or dieldrin in DMSO for 30 min . RNA - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Distribution of regions annotated " LTR " in RepeatMasker relative to sites of divergent transcription in Jurkat cells treated with either DMSO or dieldrin in DMSO .

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 B-CONTROLLED_VAR O O O

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Distribution of regions annotated "LTR" in RepeatMasker relative to sites of divergent transcription in Jurkat cells treated with either DMSO or dieldrin in DMSO.


Jurkat cells were exposed to either DMSO , or dieldrin in DMSO for 30 min . RNA - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Jurkat T cells were treated with either DMSO ( vehicle ) or 100μM dieldrin for the indicated times . Abundance of IL2 mRNA was assessed by RT - qPCR . Data shown are means + / - SEM from four independent experiments . Significance ( p - value ) was estimated using the two - sided student t test appropriate for small sample numbers . * * * : p - value < 0 . 001 .

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

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Jurkat T cells were treated with either DMSO (vehicle) or 100μM dieldrin for the indicated times. Abundance of IL2 mRNA was assessed by RT-qPCR. Data shown are means +/- SEM from four independent experiments. Significance (p-value) was estimated using the two-sided student t test appropriate for small sample numbers. ***: p-value<0.001.


Jurkat cells were exposed to either DMSO , or dieldrin in DMSO for 30 min . RNA - seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs . Screen captures from IGV showing increased divergent transcription inside a HERV sequence upon exposure of the Jurkat cells to dieldrin in the intergenic region between MUC21 and MUC22 . Color code as in ( A ) .

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

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing increased divergent transcription inside a HERV sequence upon exposure of the Jurkat cells to dieldrin in the intergenic region between MUC21 and MUC22. Color code as in (A).


A human phospho - kinase antibody array was probed with extracts from Jurkat cells treated with either DMSO , dieldrin , or PMA for 30 min as indicated . The array processed as a western and revealed by ECL . Graphic shows selected antibodies . Values shown are means + / - SEM from two measures .

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

A human phospho-kinase antibody array was probed with extracts from Jurkat cells treated with either DMSO, dieldrin, or PMA for 30 min as indicated. The array processed as a western and revealed by ECL. Graphic shows selected antibodies. Values shown are means +/- SEM from two measures.


Jurkat cells were treated with either DMSO ( vehicle ) , dieldrin , or PMA for the indicated times . Cell extracts were resolved by PAGE and western blotting was carried out with the indicated antibodies . Nearest size marker is indicated to the right ( kDa ) .

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

Jurkat cells were treated with either DMSO (vehicle), dieldrin, or PMA for the indicated times. Cell extracts were resolved by PAGE and western blotting was carried out with the indicated antibodies. Nearest size marker is indicated to the right (kDa).


Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min . Abundance of mRNA for the indicated genes or HERVs was assessed by RT - qPCR .

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

Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.


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


Jurkat cells treated as in ( C ) were subject to fractionation as indicated in the Material and Method section to distinguish a fraction with mostly RNAPII S2p ( F1 ) , one with mostly histone H3 ( F3 ) , and one with both RNAPII S2p and histone H3 ( P ) . Fractions were resolved by PAGE , and western blots were carried out with the indicated antibodies .

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

Jurkat cells treated as in (C) were subject to fractionation as indicated in the Material and Method section to distinguish a fraction with mostly RNAPII S2p (F1), one with mostly histone H3 (F3), and one with both RNAPII S2p and histone H3 (P). Fractions were resolved by PAGE, and western blots were carried out with the indicated antibodies.


( A ) Proteins detected in Vps34 - TAP immunoprecipitations by MS analysis are listed . The number of identified peptides of each protein and the peptide coverage are also 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

(A) Proteins detected in Vps34-TAP immunoprecipitations by MS analysis are listed. The number of identified peptides of each protein and the peptide coverage are also shown.


( B ) Cells expressing TAP - tagged Atg38 and the indicated HA - tagged proteins were grown in YPD . The extracts were prepared and were immunoprecipitated by IgG - Dynabeads as described in Materials and methods . The whole - cell extract ( left , " input " ) and the precipitated proteins ( right ) were analyzed by immunoblotting with the indicated antibodies .

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(B) Cells expressing TAP-tagged Atg38 and the indicated HA-tagged proteins were grown in YPD. The extracts were prepared and were immunoprecipitated by IgG-Dynabeads as described in Materials and methods. The whole-cell extract (left, "input") and the precipitated proteins (right) were analyzed by immunoblotting with the indicated antibodies.


( C ) Relative abundances of proteins identified by LC - MS / MS of Atg14 - TAP and Atg38 - TAP purifications from YPD - grown cells . Relative abundances are defined as emPAIprey / emPAIbait and are shown as mean ± SD of two independent experiments .

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(C) Relative abundances of proteins identified by LC-MS/MS of Atg14-TAP and Atg38-TAP purifications from YPD-grown cells. Relative abundances are defined as emPAIprey/emPAIbait and are shown as mean ± SD of two independent experiments.


( D ) Cell extracts were prepared from wild - type and atg14Δ cells expressing Atg38 - TAP as well as HA - tagged proteins of complex I components . The IgG Dynabead - precipitated proteins ( bottom ) , together with the whole - cell extracts ( top ) , were immunoblotted with the indicated antibodies .

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(D) Cell extracts were prepared from wild-type and atg14Δ cells expressing Atg38-TAP as well as HA-tagged proteins of complex I components. The IgG Dynabead-precipitated proteins (bottom), together with the whole-cell extracts (top), were immunoblotted with the indicated antibodies.


( E ) ATG38 - 2GFP ATG17 - 2mCherry cells in wild - type and atg14Δ were cultured at 30 ° C with or without rapamycin . After 180 min , cells were analyzed by fluorescence microscopy . Bar , 5 µm .

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(E) ATG38-2GFP ATG17-2mCherry cells in wild-type and atg14Δ were cultured at 30°C with or without rapamycin. After 180 min, cells were analyzed by fluorescence microscopy. Bar, 5 µm.


( A ) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD ( - N ) medium for 3 h at 30 ° C . Lysates from each group of cells were tested for ALP activity . ALP activity is shown as mean ± SD of three independent experiments with the activity of wild - type cells normalized to 1 .

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(A) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD(-N) medium for 3 h at 30°C. Lysates from each group of cells were tested for ALP activity. ALP activity is shown as mean ± SD of three independent experiments with the activity of wild-type cells normalized to 1.


( B ) Cells with the indicated genotypes were grown in YPD and treated with rapamycin for 3 h at 30 ° C . Total protein was separated by SDS - PAGE and detected by immunoblotting with anti - API .

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(B) Cells with the indicated genotypes were grown in YPD and treated with rapamycin for 3 h at 30°C. Total protein was separated by SDS-PAGE and detected by immunoblotting with anti-API.


( C ) Cells were grown in YPD medium for 1 h at 30 ° C and separated to the intracellular ( I ; cells ) and extracellular ( E ; medium ) fractions by centrifugation . The fractions were treated as detailed in Materials and methods and aliquots of each were separated by SDS - PAGE . The positions of the precursor ( p2 - CPY ) and mature ( m - CPY ) forms of CPY are indicated .

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(C) Cells were grown in YPD medium for 1 h at 30°C and separated to the intracellular (I; cells) and extracellular (E; medium) fractions by centrifugation. The fractions were treated as detailed in Materials and methods and aliquots of each were separated by SDS-PAGE. The positions of the precursor (p2-CPY) and mature (m-CPY) forms of CPY are indicated.


( A ) Wild - type and atg38Δ cells expressing the indicated GFP fusion proteins and Ape1 - mRFP were cultured at 30 ° C in the presence of rapamycin . After 60 min , cells were analyzed by fluorescence microscopy . Of the total pool of Ape1 - mRFP signal , the percentage of Ape1 - mRFP colocalizing with GFP was calculated . This percentage is shown as mean ± SD of two independent experiments , with the number of cells counted indicated at the bottom of the graph . ( B ) Examples of wild - type and atg38Δ cells expressing either Atg14 - 3 × GFP or Vps34 - 3 × GFP in A . Bar , 5 µm .

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(A) Wild-type and atg38Δ cells expressing the indicated GFP fusion proteins and Ape1-mRFP were cultured at 30°C in the presence of rapamycin. After 60 min, cells were analyzed by fluorescence microscopy. Of the total pool of Ape1-mRFP signal, the percentage of Ape1-mRFP colocalizing with GFP was calculated. This percentage is shown as mean ± SD of two independent experiments, with the number of cells counted indicated at the bottom of the graph. (B) Examples of wild-type and atg38Δ cells expressing either Atg14-3 × GFP or Vps34-3 × GFP in A. Bar, 5 µm.


( C ) Cell extracts were prepared from the cells in A , then analyzed by immunoblotting with anti - GFP and anti - Pgk1 antibodies .

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(C) Cell extracts were prepared from the cells in A, then analyzed by immunoblotting with anti-GFP and anti-Pgk1 antibodies.


( A ) Cell extracts were prepared from wild - type and atg38Δ strains expressing TAP - tagged Atg38 and the indicated HA proteins . The extracts were immunoprecipitated by IgG - Dynabeads . The whole - cell extracts ( top , " input " ) and the precipitates ( bottom ) were analyzed by immunoblotting with the indicated antibodies . Relative binding efficiency was determined as described in Materials and methods . To compare results from multiple experiments , the value for the reference sample ( wild - type cells ) was normalized to 1 . 0 . Relative binding efficiency is shown as mean ± SD of three independent experiments .

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(A) Cell extracts were prepared from wild-type and atg38Δ strains expressing TAP-tagged Atg38 and the indicated HA proteins. The extracts were immunoprecipitated by IgG-Dynabeads. The whole-cell extracts (top, "input") and the precipitates (bottom) were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods. To compare results from multiple experiments, the value for the reference sample (wild-type cells) was normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.


( B ) Relative abundances of proteins identified in TAP purifications from the indicated strains were analyzed as described in Fig . 1 C and are shown as mean ± SD of two independent experiments .

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(B) Relative abundances of proteins identified in TAP purifications from the indicated strains were analyzed as described in Fig. 1 C and are shown as mean ± SD of two independent experiments.


( C ) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD ( - N ) medium for 3 h at 30 ° C . Cell lysates from each group of cells were tested for ALP activity . ALP activity is shown as mean ± SD of three independent experiments with the activity of wild - type cells normalized to 1 .

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(C) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD(-N) medium for 3 h at 30°C. Cell lysates from each group of cells were tested for ALP activity. ALP activity is shown as mean ± SD of three independent experiments with the activity of wild-type cells normalized to 1.


( D ) Cells expressing Vps34 - 6HA , Vps30 - 9myc , Atg38 - 3FLAG , and Atg14 - TAP were lysed in a buffer containing 150 mM NaCl . The Atg14 - TAP immunoprecipitate with IgG - Dynabeads from this lysate was split into eight fractions and washed with lysis buffer containing the indicated concentrations of NaCl . The resulting precipitates were analyzed by immunoblotting with the indicated antibodies . Relative binding efficiency was determined as described in Materials and methods , although a 150 - mM NaCl sample was used as a control and normalized to 1 . 0 . Relative binding efficiency is shown as mean ± SD of three independent experiments .

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(D) Cells expressing Vps34-6HA, Vps30-9myc, Atg38-3FLAG, and Atg14-TAP were lysed in a buffer containing 150 mM NaCl. The Atg14-TAP immunoprecipitate with IgG-Dynabeads from this lysate was split into eight fractions and washed with lysis buffer containing the indicated concentrations of NaCl. The resulting precipitates were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods, although a 150-mM NaCl sample was used as a control and normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.


( A ) AH109 cells containing the indicated GAL4 activation domain fusions ( bait ) and GAL4 DNA - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . The interaction of fusion proteins was examined by plating cells on SD - Leu - Trp - His ( − LWH , middle ) and SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

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(A) AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( B ) Interactions between the indicated fusion proteins were analyzed as in A . AH109 cells containing the indicated GAL4 activation domain fusions ( bait ) and GAL4 DNA - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . The interaction of fusion proteins was examined by plating cells on SD - Leu - Trp - His ( − LWH , middle ) and SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

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(B) Interactions between the indicated fusion proteins were analyzed as in A. AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( C ) The TAP - tagged Vps34 protein complex was purified from the vps30Δ atg14Δ vps38Δ cells . The purified proteins were separated on SDS - PAGE and visualized by Coomassie staining . Recombinant GST - Atg38 ( WT ) , GST - Atg381 - 120 ( N ) , and GST - Atg38121 - 226 ( C ) were incubated with Vps15 - Vps34 - TAP complex bound to IgG - Dynabeads . The eluted proteins were analyzed by immunoblotting .

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(C) The TAP-tagged Vps34 protein complex was purified from the vps30Δ atg14Δ vps38Δ cells. The purified proteins were separated on SDS-PAGE and visualized by Coomassie staining. Recombinant GST-Atg38 (WT), GST-Atg381-120 (N), and GST-Atg38121-226 (C) were incubated with Vps15-Vps34-TAP complex bound to IgG-Dynabeads. The eluted proteins were analyzed by immunoblotting.


( A ) Interactions between the indicated fusion proteins were analyzed as in Fig . 4 A . AH109cells containing the indicated GAL4 activation domain fusions ( bait ) and GAL4 DNA - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . The interaction of fusion proteins was examined by plating cells on SD - Leu - Trp - His ( − LWH , middle ) and SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

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(A) Interactions between the indicated fusion proteins were analyzed as in Fig. 4 A. AH109cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( B ) Analysis of Atg38 by gel filtration . Recombinant Atg38 protein was fractionated over a gel filtration column . Each fraction was subjected to SDS - PAGE and visualized by Coomassie staining .

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(B) Analysis of Atg38 by gel filtration. Recombinant Atg38 protein was fractionated over a gel filtration column. Each fraction was subjected to SDS-PAGE and visualized by Coomassie staining.


( A ) Cell extracts were prepared from atg38Δ strains expressing Atg14 - TAP and either Vps34 - 6HA or Vps15 - 6HA with Atg381 - 120 fused with GFP and either GBP or GBP3A . The cell extracts were immunoprecipitated with IgG - Dynabeads . The precipitated proteins , together with the whole - cell extracts , were immunoblotted with the indicated antibodies ( left ) . Relative binding efficiency was determined as described in Fig . 3 A and was shown as mean ± SD of three independent experiments .

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(A) Cell extracts were prepared from atg38Δ strains expressing Atg14-TAP and either Vps34-6HA or Vps15-6HA with Atg381-120 fused with GFP and either GBP or GBP3A. The cell extracts were immunoprecipitated with IgG-Dynabeads. The precipitated proteins, together with the whole-cell extracts, were immunoblotted with the indicated antibodies (left). Relative binding efficiency was determined as described in Fig. 3 A and was shown as mean ± SD of three independent experiments.


Assay principle : RNAi was performed with luciferase - expressing cells that were challenged with or without CTLs and bi - specific antibody . Before readout , cell supernatant was removed and the remaining intact cells were lysed to measure the residual - cell - associated luciferase . To identify immune - checkpoint regulators , the difference between normalized luciferase measurements for conditions with CTLs and without CTLs was calculated . siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase levels under conditions with CTLs ; hence , the difference between luciferase measurements will be > 0 .

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Assay principle: RNAi was performed with luciferase-expressing cells that were challenged with or without CTLs and bi-specific antibody. Before readout, cell supernatant was removed and the remaining intact cells were lysed to measure the residual-cell-associated luciferase. To identify immune-checkpoint regulators, the difference between normalized luciferase measurements for conditions with CTLs and without CTLs was calculated. siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase levels under conditions with CTLs; hence, the difference between luciferase measurements will be > 0.


Luc - CTL assay performed at different T cell to MCF7 cell ratio with PBMC - derived CD8 + T cells and anti - EpCAM x CD3 bi - specific antibody ( ○ ) . Anti - CD19 × CD3 bi - specific Ab ( ▪ ) was used as a specificity control since CD19 is a B - lymphocyte - specific antigen and therefore this bsAb fails to cross - link tumor to T cells . Lower luciferase intensity indicates higher lysis . Error bars denote ± SEM .

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Luc-CTL assay performed at different T cell to MCF7 cell ratio with PBMC-derived CD8+ T cells and anti-EpCAM x CD3 bi-specific antibody (○). Anti-CD19 × CD3 bi-specific Ab (▪) was used as a specificity control since CD19 is a B-lymphocyte-specific antigen and therefore this bsAb fails to cross-link tumor to T cells. Lower luciferase intensity indicates higher lysis. Error bars denote ± SEM.