( a ) Histogram showing relative phospholipid in CHO treated with 0 . 3 % ethanol for 30 min before harvesting . Total PLD activity is measured as phosphatidylethanol ( PEtOH ) under following conditions : normal medium ( Nm ) , serum - free medium ( SFM ) , serum - free medium with 100 nM wortmannin ( SFM + W ) , nutrient deprivation ( 0 . 5 h St ; 1 . 5 h St ; 3 . 0 h St ) , 30 min of nutrient deprivation in presence of 100nM wortmannin ( 0 . 5h St + W ) . Other phospholipids were used as controls : phosphatidylserine ( PS ) , phosphatidylethanolamine ( PE ) , phosphatidylcholine ( PC ) . Values denote means ± s . e . m . ( n = 9 , except for SFM + W 0 . 5 h St + W , where n = 3 5 , respectively ) . * * P0 . 01 ; * , P0 . 05 .

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(a) Histogram showing relative phospholipid levels in CHO cells treated with 0.3% ethanol for 30 min before cell harvesting. Total PLD activity is measured as phosphatidylethanol (PEtOH) levels under the following conditions: normal medium (Nm), serum-free medium (SFM), serum-free medium with 100 nM wortmannin (SFM+W), nutrient deprivation (0.5 h St; 1.5 h St; 3.0 h St), 30 min of nutrient deprivation in presence of 100nM wortmannin (0.5h St+W). Other phospholipids were used as controls: phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC). Values denote means±s.e.m. (n=9, except for SFM+W and 0.5 h St+W, where n=3 and 5, respectively). **P0.01; *, P0.05.


( b ) Western blot of HA - PLD1 endogenous LC3 in post - supernatant ( PNS , upper panel ) , as well as in / soluble ( S ) particulate ( P ) fractions prepared from PNS after high - speed centrifugation ( middle panel ) . GAPDH was used as loading control in PNS fractions . HeLa were transfected with expression vectors encoding HA - PLD1 for 24 h subjected to 2 h of nutrient deprivation in presence or absence of PI3K inhibitor wortmannin ( 100 nM ) . Lower panel : quantitative of HA - PLD1 immunoreactivity expressed as P / S . Conditions were as follows : normal medium ( Nm ) ; normal medium with wortmannin ( Nm + W ) ; starvation condition ( St ) ; starvation condition + wortmannin ( St + W ) . Values denote means ± s . e . m . ( n = 3 ) .

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

(b) Western blot analysis of HA-PLD1 and endogenous LC3 levels in post-nuclear supernatant (PNS, upper panel), as well as in cytosolic/soluble (S) and particulate (P) fractions prepared from PNS after high-speed centrifugation (middle panel). GAPDH was used as loading control in PNS fractions. HeLa cells were transfected with expression vectors encoding HA-PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin (100 nM). Lower panel: quantitative analysis of HA-PLD1 immunoreactivity expressed as P/S ratio. Conditions were as follows: normal medium (Nm); normal medium with wortmannin (Nm+W); starvation condition (St); starvation condition+wortmannin (St+W). Values denote means±s.e.m. (n=3).


( c ) Confocal of co - localization between HA - PLD1 Lamp1 in HeLa subjected to nutrient deprivation for 2 h in presence ( ST + W ) or absence ( St ) of wortmannin 100 nM . Insets show high magnification of selected areas . Scale bar , 5 μm .

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(c) Confocal analysis of the co-localization between HA-PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence (ST+W) or absence (St) of wortmannin 100 nM. Insets show high magnification of the selected areas. Scale bar, 5 μm.


( a ) Western blot showing effects of siRNA - mediated Vps34 ( siVps34 ) downregulation on expression of HA - PLD1 Vps34 compared with control ( siCtrl ) . were incubated in normal medium ( Nm ) or subjected to 2 h of nutrient deprivation in presence of 50 nM bafilomycin ( St + B ) . HeLa were transfected for 48 h with siRNA directed to Vps34 ( siVps34 ) or a control ( siCtrl ) then transfected for 24 h with a plasmid encoding HA - PLD1 .

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(a) Western blot analysis showing the effects of siRNA-mediated Vps34 (siVps34) downregulation on the expression levels of HA-PLD1 and Vps34 compared with control cells (siCtrl). Cells were incubated in normal medium (Nm) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin (St+B). HeLa cells were transfected for 48 h with siRNA directed to Vps34 (siVps34) or a control sequence (siCtrl) and then transfected for 24 h with a plasmid encoding HA-PLD1.


( b ) Confocal imaging showing effects of Vps34 ( siVps34 ) downregulation on HA - PLD1 ( green ) , endogenous LC3 ( blue ) Lamp1 ( red ) on 2 h of nutrient deprivation compared with control treatment ( siCtrl ) . Scale bar , 5 μm . ( c ) Confocal showing subcellular localization of HA - PLD1 mutated in its PX domain ( F120A / R179Q ) , so as to abolish phosphoinositide binding ( HA - PXmut ) . HeLa were transiently transfected with a plasmid encoding HA - PXmut fixed after 2 h of nutrient deprivation . HA - PXmut ( green ) , endogenous LC3 ( blue ) Lamp1 ( red ) . Scale bar , 10 μm . In b c , insets show high magnification of selected areas . triple insets on right indicate high magnification of selected areas for two channels in various combinations .

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(b) Confocal imaging showing the effects of Vps34 (siVps34) downregulation on HA-PLD1 (green), endogenous LC3 (blue) and Lamp1 (red) on 2 h of nutrient deprivation compared with control treatment (siCtrl). Scale bar, 5 μm. (c) Confocal analysis showing the subcellular localization of HA-PLD1 mutated in its PX domain (F120A/R179Q), so as to abolish phosphoinositide binding (HA-PXmut). HeLa cells were transiently transfected with a plasmid encoding HA-PXmut and fixed after 2 h of nutrient deprivation. HA-PXmut (green), endogenous LC3 (blue) and Lamp1 (red). Scale bar, 10 μm. In b and c, insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations.


( a ) Confocal showing effects of PLD inhibition ( FIPI , 750 nM ) on of endogenous LC3 . CHO were treated with PLD inhibitor FIPI or DMSO for 3 h in presence of serum ( normal medium , Nm ) or in absence of nutrients ( starvation , St ) . Insets denote high magnification of selected areas . Scale bar , 10 μm . ( b ) of average number , surface of LC3 - positive compartments per after FIPI treatment . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 180 - 285 for Nm ; n = 449 - 519 for St ) . * * P0 . 01 .

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

(a) Confocal analysis showing the effects of PLD inhibition (FIPI, 750 nM) on the levels of endogenous LC3. CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum (normal medium, Nm) or in the absence of nutrients (starvation, St). Insets denote high magnification of the selected areas. Scale bar, 10 μm. (b) Quantification of the average number, size and surface area of LC3-positive compartments per cell after FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=180-285 cells for Nm; n=449-519 cells for St). **P0.01.


( c ) Western blot showing of LC3 - I LC3 - II in control - FIPI - treated CHO grown in normal medium ( Nm ) , after nutrient starvation ( St ) or after nutrient starvation in presence of 50 nM bafilomycin ( St + B ) . Treatments were for 3 h . Right panel : of signal intensities showing LC3 - II in extracts from nutrient - starved bafilomycin - treated CHO following FIPI treatment . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 4 ) .

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

(c) Western blot analysis showing levels of LC3-I and LC3-II in control- and FIPI-treated CHO cells grown in normal medium (Nm), after nutrient starvation (St) or after nutrient starvation in the presence of 50 nM bafilomycin (St+B). Treatments were for 3 h. Right panel: quantification of protein signal intensities showing LC3-II protein levels in extracts from nutrient-starved and bafilomycin-treated CHO cells following FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=4).


( d ) Western blot of effects of PLD inhibition on of total Tau , phosphorylated Tau ( p - Tau ) detergent - insoluble Tau . Organotypic brain slices from hTau mice were treated with either DMSO ( D ) or 750nM FIPI ( F ) harvested after 5 days of treatment . Left panel : western blot showing immunoreactivities for total human Tau ( CP27 ) , hyperphosphorylated tau ( p - Tau , ser202 / ser205 , CP13 ) , p62 tubulin from supernatant fraction aggregated tau ( CP27 ) p62 from sarkosyl - insoluble fraction . Two representative pairs are displayed . Right panel , of total ( Tau , p - Tau , p62 tubulin ) sarkosyl - insoluble of tau ( insoluble Tau ) p62 ( insoluble p62 ) . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 7 ) . * * P0 . 01 ; * , P0 . 05 . For statistical of FIPI - treated samples , tau p62 were compared with tubulin .

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

(d) Western blot analysis of the effects of PLD inhibition on the levels of total Tau, phosphorylated Tau (p-Tau) and detergent-insoluble Tau. Organotypic brain slices from hTau mice were treated with either DMSO (D) or 750nM FIPI (F) and harvested after 5 days of treatment. Left panel: western blot analysis showing immunoreactivities for total human Tau (CP27), hyperphosphorylated tau (p-Tau, ser202/ser205, CP13), p62 and tubulin from the supernatant fraction and aggregated tau (CP27) and p62 from the sarkosyl-insoluble fraction. Two representative pairs are displayed. Right panel, quantification of total proteins (Tau, p-Tau, p62 and tubulin) and sarkosyl-insoluble aggregates of tau (insoluble Tau) and p62 (insoluble p62). Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=7). **P0.01; *, P0.05. For the statistical analysis of FIPI-treated samples, tau and p62 protein levels were compared with tubulin.


a ) Western blot showing reduced of LC3 - II in KO MEFs . ( b ) of signal intensities from western blots showing LC3 - II in MEFs extracts after normalization to control GAPDH . In a b , MEFs were grown in normal medium ( Nm ) , treated for 2 h with 50 nM bafilomycin in normal medium ( B ) , or grown in nutrient - deprived medium in absence ( St ) or presence of 50 nM bafilomycin ( St + B ) . Values denote means ± s . e . m . ( n = 4 ) .

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a) Western blot analysis showing reduced levels of LC3-II in KO MEFs. (b) Quantification of protein signal intensities from western blots showing LC3-II levels in MEFs extracts after normalization to the control protein GAPDH. In a and b, MEFs were grown in normal medium (Nm), treated for 2 h with 50 nM bafilomycin in normal medium (B), or grown in nutrient-deprived medium in the absence (St) or presence of 50 nM bafilomycin (St+B). Values denote means±s.e.m. (n=4).


( c ) Immunostaining of endogenous LC3 in Pld1 WT KO MEFs showing a reduced number of LC3 - positive compartment in KO MEFs . DAPI is shown in blue . were deprived of nutrients for 2 h in absence ( St , upper panels ) or presence of bafilomycin ( St + B , lower panels ) . Scale bar , 5 μm . ( d ) of average ( top panel ) number of LC3 - positive compartments per ( bottom panel ) in Pld1 WT KO MEFs starved for 2 h . Values denote means ± s . e . m . ( n = 6 , number of = 26 - 45 for conditions ) . * * P0 . 01 ; * P0 . 05 .

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(c) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3-positive compartment in KO MEFs. The DAPI staining is shown in blue. Cells were deprived of nutrients for 2 h in the absence (St, upper panels) or presence of bafilomycin (St+B, lower panels). Scale bar, 5 μm. (d) Quantification of the average size (top panel) and number of LC3-positive compartments per cell (bottom panel) in Pld1 WT and KO MEFs starved for 2 h. Values denote means±s.e.m. (n=6, number of cells=26-45 for all the conditions). **P0.01; *P0.05.


( a ) Immunofluorescent detection of endogenous LC3 ( red ) in liver sections from fed or starved Pld1 WT KO mice . Sections were counterstained with DAPI . Scale bar , 5 μm . ( b ) Quantitative of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT KO mice . Histograms show average number , surface of LC3 - positive compartments . Values denote means ± s . e . m . ( ( n = 25 - 28 ) , 4 mice per genotype ) . * * P0 . 01 .

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(a) Immunofluorescent detection of endogenous LC3 (red) in liver sections from fed or starved Pld1 WT and KO mice. Sections were counterstained with DAPI. Scale bar, 5 μm. (b) Quantitative analysis of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT and KO mice. Histograms show the average number, size and surface area of LC3-positive compartments. Values denote means±s.e.m. ((n=25-28), 4 mice per genotype). **P0.01.


( c ) Electron microscopic showing a reduction in number of autophagosome / amphisome - like structures ( AP ) in PLD1 - deficient mice after food restriction . Representative electron from analysed in a showing APs ( arrows ) autophagolysosomes / lysosomes ( AL , arrowheads ) in liver sections from starved WT KO mice . Insets show high magnification of selected areas . Scale bar , 1 μm . ( d ) Morphometric of number ( per 100 μm2 ) relative surface ( % ) of APs ALs . Values denote means ± s . e . m . ( n = 36 electron micrograms , from a total of 3 , 600 μm2 per genotype ) . * * P0 . 01 . ( c ) Morphometric of diameter of APs ALs in liver of PLD1 - deficient mice after food restriction . Values denote means ± s . e . m . ( n = 60 ) . * * P0 . 01 .

O O 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 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

(c) Electron microscopic analysis showing a reduction in the size and number of autophagosome/amphisome-like structures (AP) in PLD1-deficient mice after food restriction. Representative electron micrographs from animals analysed in a showing APs (arrows) and autophagolysosomes/lysosomes (AL, arrowheads) in liver sections from starved WT and KO mice. Insets show high magnification of the selected areas. Scale bar, 1 μm. (d) Morphometric analysis of the number (per 100 μm2 cytoplasmic area) and relative surface area (% cytoplasmic area) of APs and ALs. Values denote means±s.e.m. (n=36 electron micrograms, from a total of 3,600 μm2 cytoplasmic area per genotype). **P0.01. (c) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1-deficient mice after food restriction. Values denote means±s.e.m. (n=60). **P0.01.


A . MS / MS of peptides corresponding to 380 - 399 of STAT2 . 293T expressing flag - tagged STAT2 were used .

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A. MS/MS analysis of peptides corresponding to amino acids 380-399 of STAT2. 293T cells expressing flag-tagged STAT2 were used.


B . Abundance of T387 phosphorylated STAT2 in 293T expressing flag - tagged STAT2 .

B. Abundance of T387 phosphorylated STAT2 in 293T cells expressing flag-tagged STAT2.


A . U6A expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) . were harvested after 4 h total RNAs were analyzed by real - time PCR . Values are means ± SD from 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

A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.


B . U6A expressing wild - type , T387A , or T387D STAT2 were treated with IFN - β ( 100 IU / ml ) . were harvested after 4 h total RNAs were analyzed by real - time PCR . Values are means ± SD from three independent experiments .

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B. U6A cells expressing wild-type, T387A, or T387D STAT2 were treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.


C , D . U6A expressing wild - type or T387A STAT2 were treated with IFN - β for 0 , 4 , 8 , or 24 h . Total RNAs were analyzed by using an Illumina HumanHT - 12 v4 Expression BeadChip array . average signal for each probe was used to determine expression . Genes with average signals below 25 detection P values greater than 0 . 01 in untreated or treated were excluded from . Inductions of less than 2 - fold were not scored . Venn diagram shows numbers of genes induced by 1 . 2 fold or more in U6A expressing T387A STAT2 , relative to untreated control . numbers in table are fold changes calculated from ratios T387A STAT2 treated / untreated wild - type STAT2 treated / untreated .

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C, D. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β for 0, 4, 8, or 24 h. Total RNAs were analyzed by using an Illumina HumanHT-12 v4 Expression BeadChip array. The average signal for each probe was used to determine expression levels. Genes with average signals below 25 and detection P values greater than 0.01 in the untreated or treated cells were excluded from the analysis. Inductions of less than 2-fold were not scored. The Venn diagram shows the numbers of genes induced by 1.2 fold or more in U6A cells expressing T387A STAT2, relative to untreated control cells. The numbers in the table are fold changes calculated from the ratios T387A STAT2 treated / untreated and wild-type STAT2 treated / untreated.


A . HME expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 / well . were exposed to vesicular stomatitis ( VSV ) for 2 h , with or without pre - treatment with IFN - β ( 100 IU / ml ) . After 20 h , amounts of VSV - M STAT2 were analyzed by Western method .

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A. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. The cells were exposed to vesicular stomatitis virus (VSV) for 2 h, with or without pre-treatment with IFN-β (100 IU/ml). After 20 h, the amounts of the VSV-M and STAT2 proteins were analyzed by the Western method.


B , C . HME expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 / well . On second day , were treated with IFN - β ( 10 IU / ml ) for 2 h . were then exposed to different multiplicities of infection of VSV for 2 h , followed by fresh media containing 10 IU / ml of IFN - β . were analyzed by with crystal violet ( B ) after 48 h .

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B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by staining with crystal violet (B) after 48 h.


B , C . HME expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 / well . On second day , were treated with IFN - β ( 10 IU / ml ) for 2 h . were then exposed to different multiplicities of infection of VSV for 2 h , followed by fresh media containing 10 IU / ml of IFN - β . were analyzed by MTT ( C ) after 48 h .

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B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by the MTT assay (C) after 48 h.


D . U6A expressing wild - type , T387A , or T387D STAT2 were placed into 96 well plates ( 2000 / well ) were treated with IFN - β ( 1000 IU / ml ) for 96 h . survival was analyzed by MTT . Each experiment was carried out two independent times , with results similar to representative examples that are shown .

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D. U6A cells expressing wild-type, T387A, or T387D STAT2 were placed into 96 well plates (2000 cells/well) and the cells were treated with IFN-β (1000 IU/ml) for 96 h. Cell survival was analyzed by the MTT assay. Each experiment was carried out two independent times, with results similar to the representative examples that are shown.


A . An immuno - precipitation was performed using whole lysates from expressing Flag - tagged wild - type STAT2 , samples were analyzed by Western method . numbers in chart are proportions of T387 phosphorylated STAT2 in each type , calculated in comparison with 293T .

O O O O O O O O O O O O O O O O O O O O O O O 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. An immuno-precipitation was performed using whole cell lysates from cells expressing Flag-tagged wild-type STAT2, and samples were analyzed by the Western method. The numbers in the chart are the proportions of T387 phosphorylated STAT2 in each cell type, calculated in comparison with 293T cells.


B . HME expressing wild - type STAT2 were treated with IFN - β ( 100 IU / ml ) . Whole lysates harvested at times indicated were analyzed by Western method ;

O O O O O O O O O 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. HME cells expressing wild-type STAT2 were treated with IFN-β (100 IU/ml). Whole cell lysates harvested at the times indicated were analyzed by the Western method;


C . HME expressing wild - type STAT2 were seeded in complete medium . On second day , were re - fed with medium lacking supplements . On third day , were treated with each supplement for 4 h , total lysates were analyzed by Western method . 1 . EGF , 2 . Insulin , 3 . Hydrocortisone , 4 . GA - 1000 , 5 . BPE .

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C. HME cells expressing wild-type STAT2 were seeded in complete medium. On the second day, the cells were re-fed with medium lacking supplements. On the third day, the cells were treated with each supplement for 4 h, and total lysates were analyzed by the Western method. 1. EGF, 2. Insulin, 3. Hydrocortisone, 4. GA-1000, 5. BPE.


D . HME were pretreated with hydrocortisone ( 0 . 04mg / ml 0 . 4mg / ml ) for 3 h then treated with IFN - β ( 100 IU / ml ) . were harvested after 4 h total RNAs were analyzed by real - time PCR . Values are means ± SD from three independent experiments .

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D. HME cells were pretreated with hydrocortisone (0.04mg/ml and 0.4mg/ml) for 3 h and then treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.


F . HCC827 LnCAP expressing wild - type STAT2 were treated with CDK inhibitors ( 1 µM ) for 4 h whole - lysates were analyzed by Western method . 1 . Flavopiridol , 2 . AT7519 , 3 . Dinaciclib .

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F. HCC827 and LnCAP cells expressing wild-type STAT2 were treated with CDK inhibitors (1 µM) for 4 h and whole-cell lysates were analyzed by the Western method. 1. Flavopiridol, 2. AT7519, 3. Dinaciclib.


A . U6A expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 30 min or were untreated . were washed with PBS media was replaced with fresh media containing staurosporine ( 500 nM ) . Whole - lysates were harvested analyzed by Western method ;

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A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 30 min or were untreated. The cells were washed with PBS and the media was replaced with fresh media containing staurosporine (500 nM). Whole-cell lysates were harvested and analyzed by the Western method;


B . U6A expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - lysates were used for immunoprecipitations of IRF9 Flag - STAT2 .

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B. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitations of IRF9 and Flag-STAT2.


C . U2A expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - lysates were used for immunoprecipitation of Flag - STAT2 .

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C. U2A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitation of Flag-STAT2.


D . U3A expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - lysates were used for immunoprecipitation of Flag - tagged STAT2 .

D. U3A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitation of Flag-tagged STAT2.


E . EMSAs using an ISRE probe . Extracts from U6A expressing wild - type or T387A STAT2 treated with IFN - β for 1 h were analyzed . position of ISGF3 is labeled .

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E. EMSAs using an ISRE probe. Extracts from U6A cells expressing wild-type or T387A STAT2 treated with IFN-β for 1 h were analyzed. The position of ISGF3 is labeled.


Activated GFAP + astrocytes in matter MS lesions with inflammatory infiltrates demyelination show an upregulation of OTUB1 ( arrows ) . Photomicrographs are obtained from case 9 ( A ) case 5 ( B ) listed in Table EV1 . Data are representative for 10 MS analyzed . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) mouse anti - GFAP ( FITC ) ; original magnification × 400 ; scale bars correspond to 50 µm .

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Activated GFAP+ astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 (arrows). Photomicrographs are obtained from case 9 (A) and case 5 (B) listed in Table EV1. Data are representative for all 10 MS patients analyzed. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC); original magnification ×400; scale bars correspond to 50 µm.


WB of OTUB1 expression in cultured astrocytes from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice .

WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.


Direct ex vivo WB of OTUB1 expression in astrocytes isolated from adult OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice .

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Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.


Normal CNS architecture in OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice . Myelination is normal in brain spinal cord of an OTUB1fl / fl a GFAP - Cre OTUB1fl / fl mouse . CV - LFB ; original magnification × 50 ; scale bars correspond to 100 µm . are representative of three mice per group .

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Normal CNS architecture in OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Myelination is normal in the brain and spinal cord of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse. CV-LFB staining; original magnification ×50; scale bars correspond to 100 µm. All photographs are representative of three mice per group.


Absolute numbers of CD45 + in spinal cord of OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice ( n = 5 for both groups , mean + ) . Absolute numbers of different subpopulations of leukocytes infiltrating spinal cord of OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were analyzed by flow cytometry ( n = 5 for both groups , mean + ) .

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Absolute numbers of CD45+ cells in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice (n = 5 for both groups, mean + SEM). Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry (n = 5 for both groups, mean + SEM).


In an OTUB1fl / fl a GFAP - Cre OTUB1fl / fl mouse , OTUB1 is strongly expressed by NeuN + neurons in brain spinal cord . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) mouse anti - NeuN ( FITC ) . Single NogoA + oligodendrocytes in brain spinal cord ( arrows ) of an OTUB1fl / fl a GFAP - Cre OTUB1fl / fl mouse express OTUB1 . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) mouse anti - NogoA ( Alexa Fluor 488 ) . GFAP + astrocytes are of normal distribution in brain spinal cord of a non - immunized OTUB1fl / fl mouse a GFAP - Cre OTUB1fl / fl mouse . Note that GFAP + astrocytes of an OTUB1fl / fl mouse do not express OTUB1 . GFAP + astrocytes of a GFAP - Cre OTUB1fl / fl mouse are also OTUB1 - negative represent a negative control for OTUB1 . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) mouse anti - GFAP ( FITC ) .

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

In an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse, OTUB1 is strongly expressed by NeuN+ neurons in the brain and spinal cord. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NeuN (FITC). Single NogoA+ oligodendrocytes in the brain and spinal cord (arrows) of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse express OTUB1. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488). GFAP+ astrocytes are of normal morphology and distribution in the brain and spinal cord of a non-immunized OTUB1fl/fl mouse and a GFAP-Cre OTUB1fl/fl mouse. Note that GFAP+ astrocytes of an OTUB1fl/fl mouse do not express OTUB1. GFAP+ astrocytes of a GFAP-Cre OTUB1fl/fl mouse are also OTUB1-negative and represent a negative control for OTUB1 staining. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC).


Activated GFAP + astrocytes in spinal cord upregulate OTUB1 in EAE at maximal disease activity ( day 15 p . i . ) in an OTUB1fl / fl mouse ( arrowheads ) , but not in a GFAP - Cre OTUB1fl / fl mouse . Note OTUB1 - expressing neuron in an OTUB1fl / fl mouse which is surrounded by GFAP - expressing processes of an activated astrocyte ( asterisk ) . At day 22 p . i . , astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl / fl mouse ( asterisk ) . Astrocytes in a GFAP - Cre OTUB1fl / fl mouse are OTUB1 - negative while neurons express OTUB1 ( asterisk ) . At day 22 p . i . , demyelination in a GFAP - Cre OTUB1fl / fl mouse is much more severe extended as compared to an OTUB1fl / fl mouse . Inflammation persists in GFAP - Cre OTUB1fl / fl mouse , whereas it has resolved in an OTUB1fl / fl mouse . are representative of three mice per group ; original magnification × 400 ; scale bars correspond to 50 µm . ( A , C ) Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) mouse anti - GFAP ( FITC ) . ( B , D ) CV - LFB .

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

Activated GFAP+ astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity (day 15 p.i.) in an OTUB1fl/fl mouse (arrowheads), but not in a GFAP-Cre OTUB1fl/fl mouse. Note the OTUB1-expressing neuron in an OTUB1fl/fl mouse which is surrounded by GFAP-expressing processes of an activated astrocyte (asterisk). At day 22 p.i., astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl/fl mouse (asterisk). Astrocytes in a GFAP-Cre OTUB1fl/fl mouse are OTUB1-negative while neurons express OTUB1 (asterisk). At day 22 p.i., demyelination in a GFAP-Cre OTUB1fl/fl mouse is much more severe and extended as compared to an OTUB1fl/fl mouse. Inflammation persists in the GFAP-Cre OTUB1fl/fl mouse, whereas it has resolved in an OTUB1fl/fl mouse. All photographs are representative of three mice per group; original magnification ×400; scale bars correspond to 50 µm. (A, C) Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). (B, D) CV-LFB staining.


EAE was induced in GFAP - Cre OTUB1fl / fl mice ( n = 29 ) OTUB1fl / fl control ( n = 29 ) by MOG35 - 55 peptide immunization with pertussis toxin . Graph represents data pooled from 4 experiments with 7 - 8 mice per group shows mean clinical scores + . Statistical performed using Mann - Whitney U ; * p < 0 . 05 . EAE was induced in GFAP - Cre OTUB1fl / fl mice ( n = 12 ) OTUB1fl / fl control ( n = 12 ) by MOG35 - 55 peptide immunization without pertussis toxin . Graph represents mean clinical scores + . Statistical performed using Mann - Whitney U ; * p < 0 . 05 .

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EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control littermates (n = 29) by MOG35-55 peptide immunization with pertussis toxin. Graph represents data pooled from 4 experiments with 7-8 mice per group and shows the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05. EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 12) and OTUB1fl/fl control littermates (n = 12) by MOG35-55 peptide immunization without pertussis toxin. Graph represents the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05.


Percentages of CD4 + CD8 + T infiltrating spinal cord of OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were analyzed by flow cytometry at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . Representative dot plots are shown .

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Percentages of CD4+ and CD8+ T cells infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. Representative dot plots are shown.


Absolute number of infiltrating leukocytes in spinal cord of OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . was calculated on flow cytometry results . Data show mean + .

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Absolute number of infiltrating leukocytes in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. was calculated based on flow cytometry results. Data show mean + SEM.


Infiltrating CD4 + T were further analyzed by flow cytometry for their ability to produce GM - CSF , IFN - γ , IL - 17 at day 15 22 p . i . , respectively . Representative dot plots are shown . Absolute number of infiltrating GM - CSF - , IFN - γ - , IL - 17 - producing CD4 + T at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . was calculated on flow cytometry results ( mean + ) .

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Infiltrating CD4+ T cells were further analyzed by flow cytometry for their ability to produce GM-CSF, IFN-γ, and IL-17 at day 15 and 22 p.i., respectively. Representative dot plots are shown. Absolute number of infiltrating GM-CSF-, IFN-γ-, and IL-17-producing CD4+ T cells at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. was calculated based on flow cytometry results (mean + SEM).


Relative expression of GM - CSF , IFN - γ , IL - 17 , TNF , IL - 6 , CXCL1 , CXCL10 , CXCL11 , CCL2 NOS2 mRNA in spinal cord of OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice at day 15 22 p . i . was determined by quantitative real - time PCR . Data are presented as relative increase over unimmunized control mice ( n = 3 for groups ) . Data show mean + .

Relative expression of GM-CSF, IFN-γ, IL-17, TNF, IL-6, CXCL1, CXCL10, CXCL11, CCL2 and NOS2 mRNA in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 and 22 p.i. was determined by quantitative real-time PCR. Data are presented as the relative increase over unimmunized control mice (n = 3 for all groups). Data show mean + SEM.


Astrocytes were isolated from unimmunized diseased OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice at day 15 p . i . , respectively . Relative expression of CXCL10 , CXCL11 , CCL2 , NOS2 mRNA was determined by quantitative real - time PCR . Data are presented as relative increase of genes at day 15 p . i . over gene expression of astrocytes from unimmunized control mice ( n = 3 for groups ) . Data show mean + .

Astrocytes were isolated from unimmunized and diseased OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 p.i., respectively. Relative expression of CXCL10, CXCL11, CCL2, and NOS2 mRNA was determined by quantitative real-time PCR. Data are presented as relative increase of genes at day 15 p.i. over gene expression of astrocytes from unimmunized control mice (n = 3 for all groups). Data show mean + SEM.


astrocytes isolated from OTUB1fl / fl ( n = 3 ) GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were left untreated or stimulated with IFN - γ ( 10 ng / ml ) , IL - 17 ( 50 ng / ml ) , TNF ( 10 ng / ml ) , respectively , for 16 h . mRNA of CXCL10 , CXCL11 , CCL2 , NOS2 were detected by quantitative real - time PCR .

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Primary astrocytes isolated from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were left untreated or stimulated with IFN-γ (10 ng/ml), IL-17 (50 ng/ml), and TNF (10 ng/ml), respectively, for 16 h. mRNA levels of CXCL10, CXCL11, CCL2, and NOS2 were detected by quantitative real-time PCR.


astrocytes from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were treated with IFN - γ ( 10 ng / ml ) for indicated times analyzed by western blot with indicated antibodies .

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-γ (10 ng/ml) for indicated times and analyzed by western blot with indicated antibodies.


astrocytes from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were treated with IFN - γ ( 10 ng / ml ) for indicated times . extracts were separated analyzed by WB with indicated antibodies .

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Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-γ (10 ng/ml) for indicated times. Cytoplasmic and nuclear extracts were separated and analyzed by WB with indicated antibodies.


Astrocytes were selectively isolated by MACS from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice at days 0 15 p . i . , respectively . Relative expression of STAT1 mRNA was determined by quantitative real - time PCR . Data show relative increase of STAT1 mRNA over that of unimmunized control mice ( n = 3 for groups ) ( mean + ) .

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Astrocytes were selectively isolated by MACS from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at days 0 and 15 p.i., respectively. Relative expression of STAT1 mRNA was determined by quantitative real-time PCR. Data show the relative increase of STAT1 mRNA over that of unimmunized control mice (n = 3 for all groups) (mean + SEM).


astrocytes from OTUB1fl / fl ( n = 3 ) GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were stimulated with IFN - γ ( 10 ng / ml ) for indicated times . mRNA was isolated analyzed by quantitative real - time PCR for SOCS1 . Data show relative increase over untreated controls ( mean + ) .

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Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were stimulated with IFN-γ (10 ng/ml) for indicated times. mRNA was isolated and analyzed by quantitative real-time PCR for SOCS1. Data show the relative increase over untreated controls (mean + SEM).


astrocytes from wild type mice were stimulated with IFN - γ ( 10 ng / ml ) for indicated times . were immunoprecipitated from whole lysates with anti - OTUB1 antibody analyzed by WB with antibodies against JAK1 , JAK2 , SOCS1 , OTUB1 .

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Primary astrocytes from wild type mice were stimulated with IFN-γ (10 ng/ml) for indicated times. Proteins were immunoprecipitated from whole cell lysates with anti-OTUB1 antibody and analyzed by WB with antibodies against JAK1, JAK2, SOCS1, and OTUB1.


astrocytes from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were left untreated or treated with IFN - γ ( 10 ng / ml ) for 30 min . were immunoprecipitated with anti - SOCS1 antibody analyzed by WB for SOCS1 , OTUB1 , K48 Ubiquitin .

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Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were left untreated or treated with IFN-γ (10 ng/ml) for 30 min. Proteins were immunoprecipitated with anti-SOCS1 antibody and analyzed by WB for SOCS1, OTUB1, and K48 Ubiquitin.


astrocytes from GFAP - Cre OTUB1fl / fl mice were cotransfected with SOCS1 - MYC , K48 ubiquitin - HA , OTUB1 - GFP / OTUB1 - ∆ N - GFP / OTUB1 - C91S - GFP plasmids . Twenty - four hours after transfection , were left untreated or stimulated with IFN - γ ( 10 ng / ml ) for 30 min before lysation . were immunoprecipitated with anti - MYC antibody analyzed by WB for MYC , GFP , HA .

Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC, K48 ubiquitin-HA, and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were left untreated or stimulated with IFN-γ (10 ng/ml) for 30 min before lysation. Proteins were immunoprecipitated with anti-MYC antibody and analyzed by WB for MYC, GFP, and HA.


astrocytes from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids . Twenty - four hours after transfection , were treated with cycloheximide ( CHX , 100 μg / ml ) for indicated times . SOCS1 were analyzed by WB ( upper panel ) . lower panel shows relative of SOCS1 normalized to Tubulin ( n = 3 for both groups ) .

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Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Twenty-four hours after transfection, cells were treated with cycloheximide (CHX, 100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups).


astrocytes from GFAP - Cre OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids or cotransfected with SOCS1 - MYC + OTUB1 - GFP plasmids . Twenty - four hours after transfection , were treated with CHX ( 100 μg / ml ) for indicated times . SOCS1 were analyzed by WB ( upper panel ) . lower panel shows relative of SOCS1 normalized to Tubulin ( n = 3 for both groups ) ( mean + ) .

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

Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids or cotransfected with SOCS1-MYC + OTUB1-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups) (mean + SEM).


astrocytes from GFAP - Cre OTUB1fl / fl mice were cotransfected with SOCS1 - MYC OTUB1 - GFP / OTUB1 - ∆ N - GFP / OTUB1 - C91S - GFP plasmids . Twenty - four hours after transfection , were treated with CHX ( 100 μg / ml ) for indicated times . SOCS1 were analyzed by WB ( left panel ) . right panel shows relative of SOCS1 normalized to Tubulin ( n = 4 for both groups ) . Data represent mean + .

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Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (left panel). The right panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 4 for both groups). Data represent mean + SEM.


astrocytes from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were stimulated with IFN - γ ( 10 ng / ml ) for four hours . Thereafter , were treated with CHX ( 100 μg / ml ) for indicated times . Whole lysates were analyzed by WB with indicated antibodies .

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Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were stimulated with IFN-γ (10 ng/ml) for four hours. Thereafter, cells were treated with CHX (100 μg/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.


astrocytes from OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids . Five hours later , astrocytes were subsequently transfected with different siRNAs or left untreated . Twenty - four hours after siRNA transfection , was isolated analyzed by WB for SOCS1 expression .

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Primary astrocytes from OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Five hours later, astrocytes were subsequently transfected with different siRNAs or left untreated. Twenty-four hours after siRNA transfection, protein was isolated and analyzed by WB for SOCS1 expression.


astrocytes from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were transfected with nonsense siRNA SOCS1 siRNA - 2 . Twenty - four hours after transfection , were stimulated with IFN - γ ( 10 μg / ml ) for 16 hours before mRNA isolation . Quantitative real - time PCR was performed to detect mRNA of CXCL10 , CCL2 , NOS2 ( n = 4 for groups ) . Data show mean + .

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Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with nonsense siRNA and SOCS1 siRNA-2. Twenty-four hours after transfection, cells were stimulated with IFN-γ (10 μg/ml) for 16 hours before mRNA isolation. Quantitative real-time PCR was performed to detect mRNA levels of CXCL10, CCL2, and NOS2 (n = 4 for all groups). Data show mean + SEM.


astrocytes from OTUB1fl / fl GFAP - Cre OTUB1fl / fl mice were treated with IFN - β ( 10 ng / ml ) for indicated times . Whole lysates were analyzed by WB with indicated antibodies .

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-β (10 ng/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.


astrocytes from OTUB1fl / fl ( n = 3 ) GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were treated with IFN - β ( 10 ng / ml ) for 16 hours . Quantitative real - time PCR was performed to detect OTUB1 ( B ) , CXCL10 ( C ) , CXCL11 ( D ) , CCL2 ( E ) , NOS2 ( F ) mRNA . Data show mean + .

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Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were treated with IFN-β (10 ng/ml) for 16 hours. Quantitative real-time PCR was performed to detect OTUB1 (B), CXCL10 (C), CXCL11 (D), CCL2 (E), and NOS2 (F) mRNA levels. Data show mean + SEM.


B ) Egg chambers flatten along coverslip , allowing for follicle morphologies to be compared in two dimensions . In ( B ) , three live w1118 ( control ) egg chambers expressing marker Basigin : : YFP are shown in multiple planes of focus , with a z - reconstruction at bottom . Because egg chambers flatten at coverslip , basal follicle morphologies can be revealed in a single focal plane . To account for differences in follicle height between three egg chambers , which increase in maturity from left to right , separate focal planes are shown for apical morphologies . A diagram illustrating position of three focal planes with respect to flattened egg chamber is shown in ( B " ) . Scale bars =

B) Egg chambers flatten along the coverslip, allowing for follicle cell morphologies to be compared in two dimensions. In (B), three live w1118 (control) egg chambers expressing the membrane marker Basigin::YFP are shown in multiple planes of focus, with a z-reconstruction at the bottom. Because the egg chambers flatten at the coverslip, the basal follicle cell morphologies can be revealed in a single focal plane. To account for differences in follicle cell height between the three egg chambers, which increase in maturity from left to right, separate focal planes are shown for the apical morphologies. A diagram illustrating the position of the three focal planes with respect to the flattened egg chamber is shown in (B"). Scale bars =


C - E ) Follicle cross - sectional decreases ( C ) shape becomes more regular ( D ) hexagonal ( E ) as early - stage egg chambers mature . These measurements were derived from at least three egg chambers from at least three flies per AR range . Bars in ( C ) ( D ) represent mean standard deviation . Statistical significance for side distributions was calculated using a chi - squared . Number of used for morphological : 1 . 0 - 1 . 2 apical n = 23 , basal n = 27 ; 1 . 2 - 1 . 4 apical n = 114 , basal n = 61 ; 1 . 4 - 1 . 6 , apical n = 160 , basal n = 149 . Vertex : 1 . 0 - 1 . 2 apical n = 38 , basal n = 33 ; 1 . 2 - 1 . 4 apical n = 73 , basal n = 70 ; 1 . 4 - 1 . 6 , apical n = 284 , basal n = 259 .

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C-E) Follicle cell cross-sectional area decreases (C) and cell shape becomes more regular (D) and hexagonal (E) as early-stage egg chambers mature. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars in (C) and (D) represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for morphological analysis: 1.0-1.2 apical n=23, basal n=27; 1.2-1.4 apical n=114, basal n=61; 1.4-1.6, apical n=160, basal n=149. Vertex counts: 1.0-1.2 apical n =38, basal n=33; 1.2-1.4 apical n=73, basal n=70; 1.4-1.6, apical n=284, basal n=259.


Egg chamber elongation is unaffected by reduced proliferation . String - shRNA expressed for 24 hours alters epithelial shape number ( A Chambers shown in ( A ) have approximately equal widths , with cross - sectional corresponding to developmental stage 6 . Scale bars = 20 μm . Number of chambers analyzed : w1118 1 . 0 - 1 . 2 n = 24 , 1 . 2 - 1 . 4 n = 14 , 1 . 4 - 1 . 6 n = 18 String - shRNA 1 . 0 - 1 . 2 n = 11 , 1 . 2 - 1 . 4 n = 10 , 1 . 4 - 1 . 6 n = 15 . Bars represent mean standard

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Egg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell number (A Chambers shown in (A) have approximately equal widths, with the cross-sectional area corresponding to developmental stage 6. Scale bars = 20 μm. Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviation


Egg chamber elongation is unaffected by reduced proliferation . String - shRNA expressed for 24 hours alters epithelial shape numbe but not relationship between egg chamber aspect cross - sectional , which is quantified in ( B ) Number of chambers analyzed : w1118 1 . 0 - 1 . 2 n = 24 , 1 . 2 - 1 . 4 n = 14 , 1 . 4 - 1 . 6 n = 18 String - shRNA 1 . 0 - 1 . 2 n = 11 , 1 . 2 - 1 . 4 n = 10 , 1 . 4 - 1 . 6 n = 15 . Bars represent mean standard

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Egg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell numbe but not the relationship between egg chamber aspect ratio and cross-sectional area, which is quantified in (B) Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviation


C , D ) proliferation is necessary for normal tissue Follicle shape regularity ( C ) polygonality ( D ) measurements in control String - shRNA egg chambers of different aspect ratios . These measurements were derived from at least three egg chambers from at least three flies per AR range . Bars represent mean standard deviation . Statistical significance for side distributions was calculated using a chi - squared . Number of used for : w1118 1 . 0 - 1 . 2 n = 23 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 ; String - shRNA 1 . 0 - 1 . 2 , n = 6 , 1 . 2 - 1 . 4 n = 19 , 1 . 4 - 1 . 6 n =

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C, D) Cell proliferation is necessary for normal tissue morphology Follicle cell shape regularity (C) and polygonality (D) measurements in control and String-shRNA egg chambers of different aspect ratios. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for analysis: w1118 1.0-1.2 n= 23, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=6, 1.2-1.4 n=19, 1.4-1.6 n=17


Epithelial shape is affected by decreased proliferation . Representative of epithelial taken from egg chambers with aspect ratios of approximately 1 . 4 ( A ) .

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Epithelial cell size and shape is affected by decreased proliferation. Representative images of epithelial cells taken from egg chambers with aspect ratios of approximately 1.4 (A).


Epithelial shape is affected by decreased proliferation . outlines are marked with Basigin : : YFP . Scale bars = 5 μm . become significantly larger ( B ) shown on a log4 scale in ( B ) . Number of analyzed : w1118 1 . 0 - 1 . 2 n = 14 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 Statistical significance calculated using a Mann - Whitney . Data information : Bars represent mean standard deviation .

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Epithelial cell size and shape is affected by decreased proliferation. Cell outlines are marked with Basigin::YFP. Scale bars = 5 μm. Cells become significantly larger (B) Cell area shown on a log4 scale in (B). Number of cells analyzed: w1118 1.0-1.2 n= 14, 1.2-1.4 n=114, 1.4-1.6 n=160 Statistical significance calculated using a Mann-Whitney test. Data information: Bars represent mean and standard deviation.


Epithelial shape is affected by decreased proliferation . outlines are marked with Basigin : : YFP . Scale bars = 5 μm . become more aligned with tissue elongation axis when proliferation is stopped using String - shRNA ( C ) . Number of analyzed : w1118 1 . 0 - 1 . 2 n = 14 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 ; String - shRNA 1 . 0 - 1 . 2 , n = 10 , 1 . 2 - 1 . 4 n = 19 , 1 . 4 - 1 . 6 n = 17 . Statistical significance calculated using a Mann - Whitney . Data information : Bars represent mean standard deviation .

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Epithelial cell size and shape is affected by decreased proliferation. Cell outlines are marked with Basigin::YFP. Scale bars = 5 μm. Cells become more aligned with the tissue elongation axis when proliferation is stopped using String-shRNA (C). Number of cells analyzed: w1118 1.0-1.2 n= 14, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=10, 1.2-1.4 n=19, 1.4-1.6 n=17. Statistical significance calculated using a Mann-Whitney test. Data information: Bars represent mean and standard deviation.


D , E ) follicular epithelium ' thins out ' over egg chamber equator when proliferation is blocked . Cartoon demonstrates tissue shape observed in follicular epithelium in control ( w1118 ) String - shRNA treated egg chambers . of phenomenon shown in ( D ) reveals that are shorter over equator than at anterior pole in elongated egg chambers when proliferation is blocked . Statistical significance calculated using unpaired t - with Welch ' s correction . Number of analyzed : w1118 1 . 0 - 1 . 2 n = 21 , 1 . 2 - 1 . 4 n = 11 , 1 . 4 - 1 . 6 n = 10 ; String - shRNA 1 . 0 - 1 . 2 , n = 9 , 1 . 2 - 1 . 4 n = 11 , 1 . 4 - 1 . 6 n = 13 . Data information : Bars represent mean standard deviation .

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D, E) The follicular epithelium 'thins out' over the egg chamber equator when proliferation is blocked. Cartoon demonstrates the tissue cell shape observed in the follicular epithelium in control (w1118) and String-shRNA treated egg chambers. Quantification of the phenomenon shown in (D) reveals that cells are shorter over the equator than at the anterior pole in elongated egg chambers when proliferation is blocked. Statistical significance calculated using unpaired t-test with Welch's correction. Number of cells analyzed: w1118 1.0-1.2 n= 21, 1.2-1.4 n=11, 1.4-1.6 n=10; String-shRNA 1.0-1.2, n=9, 1.2-1.4 n=11, 1.4-1.6 n=13. Data information: Bars represent mean and standard deviation.


F ) Recoil is observed when cuts are made in apical cortex with a 2 - photon with egg chambers in saggital view . show cortex before after cutting . Scale bars = 10 μm . Yellow arrows indicate edges of ablated region . G ) Recoil is higher when cuts are made at apical cortex at equator of egg chambers , rather than at poles . of experiment shown in ( F ) . Statistical significance tested by Mann - Whitney . Number of cuts : equator n = 30 , pole n = 20 . Data information ablation experiments performed on tissue from sqhAX3 , sqh : : GFP flies .

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F) Recoil is observed when cuts are made in apical cortex with a 2-photon laser with egg chambers in saggital view. Images show cortex before and after cutting. Scale bars = 10 μm. Yellow arrows indicate edges of ablated region. G) Recoil is higher when cuts are made at the apical cortex at the equator of egg chambers, rather than at the poles. Quantification of experiment shown in (F). Statistical significance tested by Mann-Whitney test. Number of laser cuts: equator n=30, pole n=20. Data information Laser ablation experiments performed on tissue from sqhAX3, sqh::GFP flies.


H ) Recoil is observed when larger cuts are made in apical cortex with a UV . show cortex before after cutting . Red lines indicate lines over which recoil was calculated , parallel to axes of egg chamber . Scale bars = 5 μm . Yellow arrows indicate edges of ablated region . I ) Recoil tends to be higher towards AP ( elongation ) axis compared to UD ( rotation ) axis . Graph shows of recoil speed plotted on a log2 scale in AP versus UD axis . Red at x = 1 indicates equal recoil in both axes . Values above 1 indicate higher recoil speeds in AP axis lower than 1 indicate higher recoil in UD axis . AP / UD Median = 1 . 242 ; Mean = 2 . 1 . Wilcoxon Signed Rank to for significance of recoil AP / UD shows that median is significantly different from 1 to p = 0 . 0172 . Number of cuts = 20 . Bar represents median . ablation experiments performed on tissue from sqhAX3 , sqh : : GFP flies .

H) Recoil is observed when larger cuts are made in apical cortex with a UV laser. Images show cortex before and after cutting. Red lines indicate the lines over which recoil was calculated, parallel to the axes of the egg chamber. Scale bars = 5 μm. Yellow arrows indicate edges of ablated region. I) Recoil tends to be higher towards the AP (elongation) axis compared to the the UD (rotation) axis. Graph shows the ratio of recoil speed plotted on a log2 scale in the AP versus the UD axis. Red line at x=1 indicates equal recoil in both axes. Values above 1 indicate higher recoil speeds in the AP axis lower than 1 indicate higher recoil in the UD axis. AP/UD Ratio Median = 1.242 ; Mean = 2.1. Wilcoxon Signed Rank Test to test for significance of the recoil ratio AP/UD shows that the median is significantly different from 1 to p = 0.0172. Number of cuts = 20. Bar represents median.Laser ablation experiments performed on tissue from sqhAX3, sqh::GFP flies.


B ) i ) Planar division orientation is initially random in follicular epithelium , with a significant bias towards egg chamber elongation axis evident at stage 6 ( asterisk , p = 0 . 0031 , Wilcoxon Signed Rank , Theoretical Mean of 45 ; p = 0 . 0218 when comparison to a randomly generated list of same ) . Number of analyzed : stage 2 / 3 n = 16 , stage 4 n = 27 stage 5 n = 30 , stage 6 n = 40 . ii ) of angle of tissue expansion as calculated by vector addition of minor major axis of egg chambers . Number of egg chambers analyzed : stage 2 / 3 n = 10 , stage 4 n = 15 , stage 5 n = 15 , stage 6 n = 10 . iii ) of elongation angles ( as shown in E ) with respect to chamber elongation axis by stage from live egg chambers . Number of analyzed : stage 2 / 3 n = 18 , stage 4 n = 49 stage 5 n = 64 , stage 6 n = 97 . Bars represent mean standard

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B) i) Planar division orientation is initially random in the follicular epithelium, with a significant bias towards the egg chamber elongation axis evident at stage 6 (asterisk, p = 0.0031, Wilcoxon Signed Rank Test, Theoretical Mean of 45; p = 0.0218 when comparison to a randomly generated list of the same size). Number of cells analyzed: stage 2/3 n=16, stage 4 n=27 stage 5 n=30, stage 6 n=40. ii) Quantification of the angle of tissue expansion as calculated by vector addition of the minor and major axis of egg chambers. Number of egg chambers analyzed: stage 2/3 n=10, stage 4 n=15, stage 5 n=15, stage 6 n=10. iii) Quantification of cell elongation angles (as shown in E) with respect to the chamber elongation axis by stage from live egg chambers. Number of cells analyzed: stage 2/3 n=18, stage 4 n=49 stage 5 n=64, stage 6 n=97. Bars represent mean and standard deviation


C ) Planar division orientation at Stage 6 correlates with tissue expansion in Fat2 - shRNA Pak - shRNA egg chambers . Mann Whitney w1118 versus Pak shRNA p = 0 . 0094 . Neither Fat2 - shRNA nor Pak - shRNA distributions are stastically different from random ( Wilcoxon Signed Rank , Theoretical Mean of 45 ) . Number of analyzed : w1118 n = 97 , Fat2 - shRNA n = 44 , Pak - shRNA n = 39 . Egg chambers analyzed : w1118 n = 40 , Fat2 - shRNA n = 21 , Pak - shRNA n =

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C) Planar division orientation at Stage 6 correlates with tissue expansion in Fat2-shRNA and Pak-shRNA egg chambers. Mann Whitney test w1118 versus Pak shRNA p = 0.0094. Neither the Fat2-shRNA nor Pak-shRNA distributions are stastically different from random (Wilcoxon Signed Rank Test, Theoretical Mean of 45). Number of cells analyzed: w1118 n =97, Fat2-shRNA n=44, Pak-shRNA n=39. Egg chambers analyzed: w1118 n=40, Fat2-shRNA n=21, Pak-shRNA n=21


D ) Neighboring can form new attachments underneath ( basal to ) a mitotic that has moved apically . Scale bars = 5 μm . Thin yellow lines in XZ reconstructions indicate z - position of two XY focal

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D) Neighboring cells can form new attachments underneath (basal to) a mitotic cell that has moved apically. Scale bars = 5 μm. Thin yellow lines in the XZ reconstructions indicate the z-position of the two XY focal planes


E ) Workflow for determining follicle shape shapes . Segmentation of an egg chamber with an aspect of 1 . 5 reveals that follicle elongation is usually perpendicular to elongation axis of egg chamber . outlines were marked with Basigin : : YFP . Scale bars = 5

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E) Workflow for determining follicle shape shapes. Segmentation of an egg chamber with an aspect ratio of 1.5 reveals that follicle cell elongation is usually perpendicular to the elongation axis of the egg chamber. Cell outlines were marked with Basigin::YFP. Scale bars = 5 μm


F ) Live confocal imaging shows that long axis of a follicle at interphase does not predict orientation of division . Long axis orientations at timepoints shown were determined as in ( E ) . Scale bars = 5 μm .

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F) Live confocal imaging shows that the long axis of a follicle cell at interphase does not predict the orientation of division. Long axis orientations at the timepoints shown were determined as in (E). Scale bars = 5 μm.


G ) interphase long axis does not correlate with division angle in both follicular epithelium ( FE ) embyronic neuroectoderm ( ENE ) . n = 10 ( FE ) , 18 ( ENE )

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G) The interphase long axis does not correlate with the division angle in both the follicular epithelium (FE) and embyronic neuroectoderm (ENE). n=10 cells (FE), 18 cells (ENE)


B , C ) Pins Mud are symmetric around entire cortex in ( B ) mitotic follicle ( C ) mitotic in ENE . ( B " C " ) Confocal imaging of immunostained fixed tissue . Scale

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B, C) Pins and Mud are symmetric around the entire cell cortex in (B) mitotic follicle cells and (C) mitotic cells in the ENE. (B" and C") Confocal imaging of immunostained fixed tissue. Scale b


E ) Live imaging reveals a mitotic spindle rotating in plane of FE until anaphase at timepoint 0 . Timepoints shown in minutes relative anaphase . Scale bars = 5 μm .

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E) Live imaging reveals a mitotic spindle rotating in the plane of the FE until anaphase at timepoint 0. Timepoints shown in minutes relative anaphase. Scale bars = 5 μm.


Live confocal imaging of Myosin - II shown by Zipper : : YFP ( A at apical cortex of follicle during egg chamber elongation showing a transition from to tissue - level organization during stage

Live confocal imaging of Myosin-II shown by Zipper::YFP (A at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6


Live confocal imaging of Myosin - II shown b Spaghetti squash - mCherry ( B ) at apical cortex of follicle during egg chamber elongation showing a transition from to tissue - level organization during stage 6 . ( B ) localization Sqh - mCherry is shown relative to adherens junction marked by Shg : :

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Live confocal imaging of Myosin-II shown b Spaghetti squash-mCherry (B) at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6. (B) The localization Sqh-mCherry is shown relative to adherens junction marked by Shg::GFP


A ) Apical Myo - II localization as marked by Sqh - GFP is disrupted in round ( stage 2 / 3 ) elongated ( stage 6 ) egg chambers . Red arrows point to seperation of Myo - II at junctions , indicating that actomyosin is no longer correctly linked to adherens junctions . Live confocal imaging . Scale bars = 5 μm .

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A) Apical Myo-II localization as marked by Sqh-GFP is disrupted in round (stage 2/3) and elongated (stage 6) egg chambers. Red arrows point to the seperation of Myo-II at cell junctions, indicating that actomyosin is no longer correctly linked to adherens junctions. Live confocal imaging. Scale bars = 5 μm.


Tissue topology is affected by disruption of Canoe , but not Mud ( D ) Statistical significance for side distributions was calculated using a chi - squared . Number of analyzed : w1118 1 . 4 - 1 . 6 n = 284 ; Canoe - shRNA 1 . 4 - 1 . 6 n = 310 ; mud3 / mud4 1 . 4 - 1 . 6 n =

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Tissue topology is affected by disruption of Canoe, but not Mud (D) Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells analyzed: w1118 1.4-1.6 n=284; Canoe-shRNA 1.4-1.6 n=310; mud3/mud4 1.4-1.6 n=251


Tissue topology is affected by disruption of Canoe , but not Mud Representative image of a Canoe - shRNA follicle epithelium . outlines are marked with Basigin : : YFP . This egg chamber has no mitotic AR of 1 . 58 . Scale bars = 5

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

Tissue topology is affected by disruption of Canoe, but not Mud Representative image of a Canoe-shRNA follicle epithelium. Cell outlines are marked with Basigin::YFP. This egg chamber has no mitotic cells and AR of 1.58. Scale bars = 5 μm


F - G ) HeLa were treated as in B ) then fixed stained with ULK1 ( green ) ( F ) or ATG13 ( red ) antibodies ( G ) respectively . Hoechst was used to stain nuclei . Graphs ( right ) show average of both ULK1 ( F ) ( n = 3 independent experiments , n = 11 fields analysed ) or ATG13 ( n = 3 independent experiments , n ≥ 16 fields analysed ) ( G ) number per both ULK1 ( n = 3 independent experiments ) ( F ) or ATG13 ( n = 3 independent experiments ) ( G ) fold of increase of average respectively . arrows indicate most representative of analysed conditions . data are reported as mean value ± . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison . * * , P < 0 , 01 ; * * * * , P < 0 . 0001 or by unpaired Student ' s t ‐ when two groups were compared . * , P < 0 . 05 ; * * * * , P < 0 . 0001 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-G) HeLa cells were treated as in B) and then fixed and stained with ULK1 (green) (F) or ATG13 (red) antibodies (G) respectively. Hoechst was used to stain nuclei. Graphs (right) show the average of both ULK1 (F) (n=3 independent experiments, n=11 fields analysed) or ATG13 (n=3 independent experiments, n≥16 fields analysed) (G) puncta number per cell and both ULK1 (n=3 independent experiments) (F) or ATG13 (n=3 independent experiments) (G) fold of increase of the average size respectively. White arrows indicate most representative puncta of analysed conditions. All data are reported as the mean value ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. *, P<0.05; ****, P<0.0001. Scale bar 5 μm.


HeLa were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) grown in fed or starved conditions for 1h . were fixed co - labelled with ULK1 ( red ) antibody A ) anti - CALNEXIN ( CANX ) ( green ) antibody to highlight ER structures ; ( n = 3 independent experiments , n ≥ 16 fields analysed ) B ) GOLGIN97 ( green ) antibody to label GOLGI ; ( n = 3 independent experiments , n = 12 fields analysed ) C ) SEC31A ( green ) to point out COPII vesicles . ( n = 3 independent experiments , n ≥ 16 fields analysed )

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

HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed or starved conditions for 1h. Cells were fixed and co-labelled with ULK1 (red) antibody and A) anti-CALNEXIN (CANX) (green) antibody to highlight ER structures; (n=3 independent experiments, n≥16 fields analysed) B) GOLGIN97 (green) antibody to label GOLGI complex; (n=3 independent experiments, n=12 fields analysed) C) SEC31A (green) to point out COPII vesicles. (n=3 independent experiments, n≥16 fields analysed)


D ) ERGIC53 ( green ) to mark ERGIC compartment . ( n = 3 independent experiments , n ≥ 14 fields analysed ) . of ULK1 localization with each compartment is reported in each graph . Co - localization analyses were performed by ImageJ plugin Jacop . Values of Mander ' s coefficient for ULK1 are expressed in as mean ± . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison . * * , P < 0 , 01 ; * * * , P < 0 , 001 ; * * * * , P < 0 , 0001 ; ns , not significant . Scale bars 5 μm . arrowheads point at ULK1 associated to analysed markers ; N , nucleus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ERGIC53 (green) to mark ERGIC compartment. (n=3 independent experiments, n≥14 fields analysed). Analysis of ULK1 localization with each compartment is reported in each graph. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for ULK1 are expressed in percentage as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ***, P<0,001; ****, P<0,0001; ns, not significant. Scale bars 5 μm. White arrowheads point at ULK1 puncta associated to the analysed markers; N, nucleus.


F ) HeLa were co - transfected with control siRNA an empty vector ( EV ) , or 3 ' UTR TFG siRNA together with an EV or HA - TFG plasmids . grown for 1h in starvation medium then fixed , permeabilized , labelled with anti - ULK1 ( green ) anti - ERGIC53 ( red ) antibodies respectively . Hoechst was used to stain nuclei . Co - localization were performed as in D ) . Values of Mander ' s coefficient for ULK1 , expressed as , are mean ± . Statistical analyses were performed by one - way ANOVA followed by Dunnetts ' s multiple comparison . * , P < 0 , 05 . ( n = 3 ) ( n = 3 independent experiments , n ≥ 9 fields analysed ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) HeLa cells were co-transfected with control siRNA and an empty vector (EV), or 3'UTR TFG siRNA together with an EV or HA-TFG plasmids. Cells grown for 1h in starvation medium then fixed, permeabilized, and labelled with anti-ULK1 (green) and anti-ERGIC53 (red) antibodies respectively. Hoechst was used to stain nuclei. Co-localization analysis were performed as in D). Values of Mander's coefficient for ULK1, expressed as percentage, are mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Dunnetts's multiple comparison test. *, P<0,05. (n=3) (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.


A ) HeLa were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) grown in fed ( FED ) or starvation conditions ( STV ) in presence or absence of CLQ for 1h . were then fixed stained using LC3B - II ( red ) antibody . of LC3B number is reported ( bottom ) . data are expressed as mean value ± . Statistical were performed by two - way ANOVA followed by Tukey ' s multiple comparison . * * * * , P < 0 . 0001 . ( n = 4 independent experiments , n ≥ 25 fields analysed ) . 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 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

A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed (FED) or starvation conditions (STV) in the presence or absence of CLQ for 1h. Cells were then fixed and stained using LC3B-II (red) antibody. Analysis of LC3B puncta number is reported (bottom). All data are expressed as the mean value ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. ****, P<0.0001. (n=4 independent experiments, n≥25 fields analysed). Scale bar 5 μm.


B ) HeLa were transfected as in A ) cultured in starvation conditions ( STV ) in presence or absence of Bafilomycin A1 ( Baf A1 ) for 1h . were then fixed stained using LC3B - II ( green ) LAMP1 ( red ) antibodies . Hoechst was used to mark nuclei . Co - localization analyses were performed by ImageJ plugin Jacop . Values of Mander ' s coefficient for LC3B - II are expressed as mean ± . Statistical were performed by two - way ANOVA followed by Tukey ' s multiple comparison . * , P < 0 , 05 ; ( n = 3 independent experiments , n ≥ 13 fields analysed ) . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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

B) HeLa cells were transfected as in A) and cultured in starvation conditions (STV) in the presence or absence of Bafilomycin A1 (Baf A1) for 1h. Cells were then fixed and stained using LC3B-II (green) and LAMP1 (red) antibodies. Hoechst was used to mark nuclei. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for LC3B-II are expressed as mean ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; (n=3 independent experiments, n≥13 fields analysed). Scale bar 5 μm.


C ) HeLa were transfected as in A ) grown in fed ( FED ) or starvation conditions ( STV ) for 1h . were then fixed stained using ATG16L1 ( yellow ) antibody . ATG16L1 number ( upper right ) average of ATG16L1 as fold increase ( bottom right ) were reported . values are expressed as mean ± . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison . * * , P < 0 , 01 ; * * * * , P < 0 . 0001 or by unpaired Student ' s t ‐ when two groups were compared . ns , not significant . ( n = 3 independent experiments , n ≥ 9 fields analysed ) . 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 O O O O O O O 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) HeLa cells were transfected as in A) and grown in fed (FED) or starvation conditions (STV) for 1h. Cells were then fixed and stained using ATG16L1 (yellow) antibody. ATG16L1 puncta number (upper right) and the average size of ATG16L1 as fold increase (bottom right) were reported. All values are expressed as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. ns, not significant. (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.


D ) HEK293 stably expressing GFP - DFCP1 were transfected as in A ) cultured in starved conditions for 40 ' . were visualised with cellSens microscope for live - imaging . Pictures show taken at same number of frames for each video . Analyses of number of DFCP1 structures per ( upper right ) mean of omegasomes structures diameter ( μm ) ( bottom right ) were reported . N , nucleus . values are expressed as mean ± . Statistical analyses were performed by unpaired Student ' s t ‐ . * , P < 0 , 05 ; * * * , P < 0 . 001 . ( n = 3 independent experiments ) . 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 O O O O O O O O O O O O O O O O O O O O

D) HEK293 cells stably expressing GFP-DFCP1 were transfected as in A) and cultured in starved conditions for 40'. Cells were visualised with cellSens microscope for live-cell imaging. Pictures show the images taken at the same number of frames for each video. Analyses of the number of DFCP1 structures per cell (upper right) and the mean of omegasomes structures diameter (μm) (bottom right) were reported. N, nucleus. All values are expressed as mean ± SEM. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; ***, P<0.001. (n=3 independent experiments). Scale bar 5 μm.


A ) HeLa were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) ; after culturing in fed ( FED ) or starvation conditions ( STV ) for 1h , were fixed labelled with indicated antibodies . Co - localization was performed by using ImageJ Jacop plugin mean ± of Mander ' s coefficients of ULK1 , expressed as , is reported ( bottom ) . arrowheads point at co - localization events between ULK1 LC3B ; arrows track not co - localizing ; N , nucleus . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison . * , P < 0 , 05 ; * * , P < 0 . 01 ; * * * * , P < 0 . 0001 . ( n = 3 independent experiments , n ≥ 16 fields analysed ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR); after culturing cells in fed (FED) or starvation conditions (STV) for 1h, cells were fixed and labelled with the indicated antibodies. Co-localization analysis was performed by using ImageJ Jacop plugin and mean ± SEM of Mander's coefficients of ULK1, expressed as percentage, is reported (bottom). White arrowheads point at co-localization events between ULK1 and LC3B; white arrows track puncta not co-localizing; N, nucleus. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; **, P<0.01; ****, P<0.0001. (n=3 independent experiments, n≥16 fields analysed). Scale bar 5 μm.


HEK293 stably expressing either GFP - ATG13 ( B ) were transiently transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) , together with CFP - LC3B plasmid ( visualized in red ) cultured in starved conditions for 40 ' . Wide - field live - imaging of starved were taken by cellSens microscope . Representative of lifespan of ATG13 ( B ) are reported . of ATG13 ( B ) forming association with LC3B - II are also shown . appearance of LC3B on ATG13 ( B ) ( bottom right ) ( C ) were quantified reported . values are expressed in seconds ( sec ) as mean ± . Yellow arrowheads point at DFCP1 LC3B particles in first frame from their onset , arrowheads arrows show omegasomes autophagosomes as indicated . Statistical analyses were performed by unpaired Student ' s t ‐ . * , P < 0 , 05 ; * * , P < 0 . 01 . ( n = 3 independent experiments , n = 50 events / condition analysed ) . 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

HEK293 cells stably expressing either GFP-ATG13 (B) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of ATG13 (B) are reported. Images of ATG13 (B) forming association with LC3B-II are also shown. The appearance of LC3B on ATG13 (B) (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.


HEK293 stably expressing either GFP - DFCP1 ( C ) were transiently transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) , together with CFP - LC3B plasmid ( visualized in red ) cultured in starved conditions for 40 ' . Wide - field live - imaging of starved were taken by cellSens microscope . Representative of lifespan of both ATG13 ( C ) are reported . of DFCP1 ( C ) forming association with LC3B - II are also shown . appearance of LC3B on on DFCP1 ( bottom right ) ( C ) were quantified reported . values are expressed in seconds ( sec ) as mean ± . Yellow arrowheads point at DFCP1 LC3B particles in first frame from their onset , arrowheads arrows show omegasomes autophagosomes as indicated . Statistical analyses were performed by unpaired Student ' s t ‐ . * , P < 0 , 05 ; * * , P < 0 . 01 . ( n = 3 independent experiments , n = 50 events / condition analysed ) . 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

HEK293 cells stably expressing either GFP-DFCP1 (C) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of both ATG13 (C) are reported. Images of DFCP1 (C) forming association with LC3B-II are also shown. The appearance of LC3B on on DFCP1 (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.


D ) HeLa were transfected as in A ) . were imaged by . Representative electron ( x26500 ) are reported . average number of abnormal structures per is shown ( bottom graph ) . N , nucleus ; M , mitochondria ; ER , endoplasmic reticulum . average number of abnormal vacuoles per was evaluated ( upper right ) . Statistical was performed by unpaired Student ' s t ‐ . * * * , P < 0 , 001 . A minimum of 50 were observed . was performed ( n = 3 independent experiments , n ≥ 51 analysed ) . Data are presented as mean ± . Scale bar 500 nm as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected as in A). Cells were imaged by TEM. Representative electron micrographs (x26500) are reported. The average number of abnormal structures per cell is shown (bottom graph). N, nucleus; M, mitochondria; ER, endoplasmic reticulum. The average number of abnormal vacuoles per cell was evaluated (upper right). Statistical analysis was performed by unpaired Student's t‐test. ***, P<0,001. A minimum of 50 cells were observed. Cell count was performed (n=3 independent experiments, n≥51 cells analysed). Data are presented as mean ± SEM. Scale bar 500 nm as indicated.