( 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 .

O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O B-TISSUE O O B-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-GENEPROD 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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-TISSUE O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD 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-ORGANISM O O O O O O O O O O

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-GENEPROD O O B-ORGANISM O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O 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-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O 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-SUBCELLULAR O 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-TISSUE O B-GENEPROD O O B-ORGANISM O B-SMALL_MOLECULE 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 analysis of peptides corresponding to amino acids 380 - 399 of STAT2 . 293T cells 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 cells expressing flag - tagged STAT2 .

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


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 .

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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 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 .

O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

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 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 .

O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O

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 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 .

O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O

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 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 .

O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 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 .

O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O

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 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 .

O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE 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 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 ;

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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 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 .

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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 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 .

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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 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 .

O O B-CELL_LINE O B-CELL_LINE O O O O O B-GENEPROD O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

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 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 ;

O O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-SMALL_MOLECULE 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) 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 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 .

O O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O

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 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 .

O O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O

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 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 .

O O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O

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 cells expressing wild - type or T387A STAT2 treated with IFN - β for 1 h were analyzed . The position of ISGF3 is labeled .

O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O

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 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 .

O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE B-DISEASE O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O 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-DISEASE B-ORGANISM O O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O

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

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


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

O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O

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 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 .

O B-TISSUE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM O O O

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 + 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 ) .

O B-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

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 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 ) .

O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O B-TISSUE I-TISSUE O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O B-ORGANISM O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE 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 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 .

O B-GENEPROD O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-EXP_ASSAY B-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY 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 ) 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 .

B-DISEASE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O 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-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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 + 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 .

O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

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 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 .

O B-EXP_ASSAY O O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

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 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 ) .

O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 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 .

O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

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 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 .

B-CELL_TYPE O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O

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.


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 .

O B-CELL_TYPE O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

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.


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 .

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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 and 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 . Cytoplasmic and nuclear extracts were separated and analyzed by WB with indicated antibodies .

O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O

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 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 ) .

B-CELL_TYPE O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O

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).


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 ) .

O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O

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).


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 .

O B-CELL_TYPE O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O

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.


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 .

O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O

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.


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 .

O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O

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.


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 ) .

O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O

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).


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 ) .

O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD 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).


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 .

O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O

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.


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 .

O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

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.


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 .

O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE 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-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O

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.


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 .

O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O

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.


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 .

O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

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.


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 .

O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 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 =

O O B-TISSUE I-TISSUE O O O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O

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 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 .

O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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

B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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

B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) 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 =

O O O O O O O O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-ORGANISM O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D) 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 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 ) .

B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O O O O O

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 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 .

B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) 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 .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E) 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 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 .

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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 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 .

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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 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

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-TISSUE I-TISSUE O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O 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-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O B-EXP_ASSAY O O O O O O O O O O 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) 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 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 =

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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 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

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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 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

O O O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O 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) 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 ) 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 )

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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 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

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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 the plane of the 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 the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue - level organization during stage

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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 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 : :

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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 ) 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 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 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 =

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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 . Cell outlines are marked with Basigin : : YFP . This egg chamber has no mitotic cells and AR of 1 . 58 . Scale bars = 5

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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 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 .

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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 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 )

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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 ) . 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 .

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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 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 .

O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O 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-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.