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

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 .

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

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 O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O 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 .

O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O 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 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 O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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 ) .

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

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

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

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


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

Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were 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 O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O 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 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 O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O 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 O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR 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 O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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 O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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

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

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

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

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 ) .

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


Epithelial 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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

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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 =

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

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

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


F ) Live confocal imaging shows that 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 .

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


G ) 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 .

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


Tissue topology is affected by disruption of Canoe , but not Mud ( D ) Statistical significance for 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 O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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


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

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

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


D ) HEK293 cells stably expressing GFP - DFCP1 were transfected as in A ) and cultured in starved conditions for 40 ' . Cells were visualised with cellSens microscope for live - cell imaging . Pictures show the images taken at the same number of frames for each video . Analyses of the number of DFCP1 structures per cell ( upper right ) and the mean of omegasomes structures diameter ( μm ) ( bottom right ) were reported . N , nucleus . All values are expressed as mean ± SEM . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * * , P < 0 . 001 . ( n = 3 independent experiments ) . Scale bar 5 μm .

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

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


A ) HeLa cells were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) ; after culturing cells in fed ( FED ) or starvation conditions ( STV ) for 1h , cells were fixed and labelled with the indicated antibodies . Co - localization analysis was performed by using ImageJ Jacop plugin and mean ± SEM of Mander ' s coefficients of ULK1 , expressed as percentage , is reported ( bottom ) . White arrowheads point at co - localization events between ULK1 and LC3B ; white arrows track puncta not co - localizing ; N , nucleus . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * , P < 0 , 05 ; * * , P < 0 . 01 ; * * * * , P < 0 . 0001 . ( n = 3 independent experiments , n ≥ 16 fields analysed ) . Scale bar 5 μm .

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

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


HEK293 cells stably expressing either GFP - ATG13 ( B ) were transiently transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) , together with CFP - LC3B plasmid ( visualized in red ) and cultured in starved conditions for 40 ' . Wide - field live - cell imaging of starved cells were taken by cellSens microscope . Representative images of lifespan of ATG13 ( B ) are reported . Images of ATG13 ( B ) forming association with LC3B - II are also shown . The appearance of LC3B on ATG13 ( B ) ( bottom right ) ( C ) puncta were quantified and reported . All values are expressed in seconds ( sec ) as mean ± SEM . Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset , white arrowheads and white arrows show omegasomes and autophagosomes as indicated . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * , P < 0 . 01 . ( n = 3 independent experiments , n = 50 events / condition analysed ) . Scale bar 1 μm .

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

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


HEK293 cells stably expressing either GFP - DFCP1 ( C ) were transiently transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) , together with CFP - LC3B plasmid ( visualized in red ) and cultured in starved conditions for 40 ' . Wide - field live - cell imaging of starved cells were taken by cellSens microscope . Representative images of lifespan of both ATG13 ( C ) are reported . Images of DFCP1 ( C ) forming association with LC3B - II are also shown . The appearance of LC3B on on DFCP1 ( bottom right ) ( C ) puncta were quantified and reported . All values are expressed in seconds ( sec ) as mean ± SEM . Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset , white arrowheads and white arrows show omegasomes and autophagosomes as indicated . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * , P < 0 . 01 . ( n = 3 independent experiments , n = 50 events / condition analysed ) . Scale bar 1 μm .

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

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


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

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

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


E ) HeLa cells stably expressing GFP - ATG13 and CFP - ATG16 were transiently co - transfected with DsRed - Rab7 and TFG siRNA ( siTFG ) . Cells were cultured in starved conditions for 1h . Representative electron micrographs ( x3900 ) and merge of the three fluorophores CLEM are shown . Micrographs at higher magnification ( x13500 ) of a region showing the three fluorophores split and merged is reported . Scale bars : 5 μm upper panels , 1 μm bottom panels .

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

E) HeLa cells stably expressing GFP-ATG13 and CFP-ATG16 were transiently co-transfected with DsRed-Rab7 and TFG siRNA (siTFG). Cells were cultured in starved conditions for 1h. Representative electron micrographs (x3900) and merge of the three fluorophores CLEM are shown. Micrographs at higher magnification (x13500) of a region showing the three fluorophores split and merged is reported. Scale bars: 5 μm upper panels, 1 μm bottom panels.


G ) Control ( TFG - WT ) and patient ' s ( TFG - R106C ) fibroblasts were grown in fed or starvation conditions for 1h . Cells were fixed , permeabilized , and labelled with anti - ULK ( red ) antibody . Hoechst was used to stain nuclei . The average of ULK1 puncta number per cell is shown ( bottom ) . All data are expressed as the mean value ± SEM . Statistical analysis was performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * , P < 0 . 05 ; * * * , P < 0 , 001 ; * * * * ; P < 0 , 0001 . ( n = 3 independent experiments , n ≥ 14 fields analysed ) . Scale bar 5 μm .

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

G) Control (TFG-WT) and patient's (TFG-R106C) fibroblasts were grown in fed or starvation conditions for 1h. Cells were fixed, permeabilized, and labelled with anti-ULK (red) antibody. Hoechst was used to stain nuclei. The average of ULK1 puncta number per cell is shown (bottom). All data are expressed as the mean value ± SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0.05; ***, P<0,001; ****; P<0,0001. (n=3 independent experiments, n≥14 fields analysed). Scale bar 5 μm.


HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3 ' UTR TFG siRNA , starved for 1h , fixed , permeabilized and immunolabelled with ULK1 ( red ) Hoechst was used to stain nuclei . ULK1 puncta number ( G ) was analysed and reported as mean ± SEM in the graph ( right ) ( n = 3 independent experiments , n = 24 fields analysed ) . Values of Mander ' s coefficient for ULK1 , expressed as percentage , are reported as mean ± SEM ( bottom graph ) . ( n = 3 independent experiments , n = 19 fields analysed ) White arrowheads point at co - localization events between ULK1 and ERGIC . Statistical analyses were performed by unpaired Student ' s t ‐ test . * * , P < 0 , 01 ; * * * , 0 , 001 . Scale bar 5 μm .

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

HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) Hoechst was used to stain nuclei. ULK1 puncta number (G) was analysed and reported as mean ± SEM in the graph (right) (n=3 independent experiments, n=24 fields analysed). Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm.


HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3 ' UTR TFG siRNA , starved for 1h , fixed , permeabilized and immunolabelled with ULK1 ( red ) alone or ULK1 ( red ) and ERGIC53 ( green ) ( H ) . Hoechst was used to stain nuclei . analysed ) . Co - localization analyses ( H ) were performed by Jacop plugin . Values of Mander ' s coefficient for ULK1 , expressed as percentage , are reported as mean ± SEM ( bottom graph ) . ( n = 3 independent experiments , n = 19 fields analysed ) White arrowheads point at co - localization events between ULK1 and ERGIC . Statistical analyses were performed by unpaired Student ' s t ‐ test . * * , P < 0 , 01 ; * * * , 0 , 001 . Scale bar 5 μm .

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

HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) alone or ULK1 (red) and ERGIC53 (green) (H). Hoechst was used to stain nuclei. analysed). Co-localization analyses (H) were performed by Jacop plugin. Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm.


M ) HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were co - transfected with HA - LC3C and Myc - ULK1 plasmids as indicated . Cells were starved for 30 ' and protein extracts were immunoprecipitated using an anti - HA antibody or IgG as negative control . Protein extracts were analysed by WB to detect ULK1 , LC3C , TFG and ACTIN as indicated .

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

M) HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were co-transfected with HA-LC3C and Myc-ULK1 plasmids as indicated. Cells were starved for 30' and protein extracts were immunoprecipitated using an anti-HA antibody or IgG as negative control. Protein extracts were analysed by WB to detect ULK1, LC3C, TFG and ACTIN as indicated.


Seedling phenotype under GA treatment . After germination , Five - day - old seedlings with the same growth stage were transferred to 0 . 5 MS liquid media without or with different concentrations of GA3 . PA from soybean was added to the media at a final concentration of 20 μM . Pictures were taken 7 days after transfer . The horizontal red line separates different plants and the vertical red scale bar represents 2 cm .

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

Seedling phenotype under GA treatment. After germination, Five-day-old seedlings with the same growth stage were transferred to 0.5 MS liquid media without or with different concentrations of GA3. PA from soybean was added to the media at a final concentration of 20 μM. Pictures were taken 7 days after transfer. The horizontal red line separates different plants and the vertical red scale bar represents 2 cm.


Immunoblotting of His - tagged PLDα6 ( arrow ) expressed in E . coli as separated on an 10 % SDS - PAGE and blotted to a membrane .

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

Immunoblotting of His-tagged PLDα6 (arrow) expressed in E. coli as separated on an 10% SDS-PAGE and blotted to a membrane.


Lipid hydrolyzing activity assayed in the presence of different phospholipids using purified PLDα6 from E . coli . Solid bars are activities assayed using purified PLDα6 whereas open bars were empty vector control that used an equal volume of eluents from bacteria containing the empty vector that was identically processed as those expressing PLDα6 . Values are means ± SD ( n = 3 biological replicates ) .

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Lipid hydrolyzing activity assayed in the presence of different phospholipids using purified PLDα6 from E. coli. Solid bars are activities assayed using purified PLDα6 whereas open bars were empty vector control that used an equal volume of eluents from bacteria containing the empty vector that was identically processed as those expressing PLDα6. Values are means ± SD (n = 3 biological replicates).


Total lipid levels in WT , pldα6 and COM without and with 10 μM GA3 . Leaf samples ( 15 seedling each ) from 4 - leaf stage rice ( Dongjin background ) were collected and lipids were extracted and profiled using ESI - tandem mass spectrometry . Values are means ± SD ( n = 3 biological replicates ) ) . MGDG , monogalactosyldiacylglycerol ; DGDG , digalactosyldiacylglycerol ; PC , phosphatidylcholine ; PE , phosphatidylethanolamine ; PG , phosphatidylglycerol ; PS , phosphatidylserine ; PA , phosphatidic acid .

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Total lipid levels in WT, pldα6 and COM without and with 10 μM GA3. Leaf samples (15 seedling each) from 4-leaf stage rice (Dongjin background) were collected and lipids were extracted and profiled using ESI-tandem mass spectrometry. Values are means ± SD (n = 3 biological replicates)). MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PA, phosphatidic acid.


Phospholipid species in WT , pldα6 and COM . Leaf samples ( 15 seedling each ) from 4 - leaf stage rice ( Dongjin background ) were collected and the different phospholipid species including carbon number and double bond number were determined . Values are means ± SD ( n = 3 biological replicates ) . GA3 was dissolved in ethanol and seedlings treated with the same ethanol concentration were used as control .

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Phospholipid species in WT, pldα6 and COM. Leaf samples (15 seedling each) from 4-leaf stage rice (Dongjin background) were collected and the different phospholipid species including carbon number and double bond number were determined. Values are means ± SD (n = 3 biological replicates). GA3 was dissolved in ethanol and seedlings treated with the same ethanol concentration were used as control.


Immunoblotting of His - GID1 proteins using constructs shown in ( D ) . Proteins were separated by SDS - PAGE , followed by immunoblotting with anti - His - tag antibodies . The PA binding activity of different truncation mutations were analyzed by fat - immunoblotting . The red arrowheads indicate truncated proteins with different molecular weights .

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Immunoblotting of His-GID1 proteins using constructs shown in (D). Proteins were separated by SDS-PAGE, followed by immunoblotting with anti-His-tag antibodies. The PA binding activity of different truncation mutations were analyzed by fat-immunoblotting. The red arrowheads indicate truncated proteins with different molecular weights.


Immunoblotting of His - GID1 mutants and lipid - immunoblotting of PA binding by GID1 mutant proteins on a filter .

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Immunoblotting of His-GID1 mutants and lipid-immunoblotting of PA binding by GID1 mutant proteins on a filter.


PLDα6 - GFP distribution in rice protoplasts with different concentrations of GA3 or IAA . Protoplasts from 12 - day old rice ( ZH11 background ) leaf sheath tissue were collected . Rice protoplasts were transfected with pM999 - PLDα6 for 12 hours . After that , GA3 or IAA was added to protoplasts and 2 hours later , confocal images of protoplasts are shown . pM999 - GFP refers to the empty vector with GFP only that was transformed as control and Ghd7 - RFP was a nucleus marker . Scale bar = 10 μm .

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PLDα6-GFP distribution in rice protoplasts with different concentrations of GA3 or IAA. Protoplasts from 12-day old rice (ZH11 background) leaf sheath tissue were collected. Rice protoplasts were transfected with pM999-PLDα6 for 12 hours. After that, GA3 or IAA was added to protoplasts and 2 hours later, confocal images of protoplasts are shown. pM999-GFP refers to the empty vector with GFP only that was transformed as control and Ghd7-RFP was a nucleus marker. Scale bar = 10 μm.


Immunoblotting of PLDα6 in subcellular fractions . Total ( T ) , soluble ( S ) and nuclear ( N ) proteins were isolated from PLDα6 : GFP expressed in rice protoplasts treated with 10 μM GA3 or IAA . Equal amount of each sample was loaded for SDS - PAGE and blotting .

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Immunoblotting of PLDα6 in subcellular fractions. Total (T), soluble (S) and nuclear (N) proteins were isolated from PLDα6:GFP expressed in rice protoplasts treated with 10 μM GA3 or IAA. Equal amount of each sample was loaded for SDS-PAGE and blotting.


Subcellular location of GID1 in WT and pldα6 protoplasts with or without GA3 treatments . Cells for 12 hours after transformation were treated with or without 10 μM GA3 for two hours . Bars = 10 μm .

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Subcellular location of GID1 in WT and pldα6 protoplasts with or without GA3 treatments. Cells for 12 hours after transformation were treated with or without 10 μM GA3 for two hours. Bars = 10 μm.


Immunoblotting of subcellular fractions of GID1 expressed in WT and pldα6 protoplasts . After 2 - hour treatment with 10 μM GA3 , total ( T ) , soluble ( S ) and nuclear ( N ) protein fractions were isolated from 10 samples of protoplasts . Equal amount of each sample was loaded for SDS - PAGE and immunoblotting .

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Immunoblotting of subcellular fractions of GID1 expressed in WT and pldα6 protoplasts. After 2-hour treatment with 10 μM GA3, total (T), soluble (S) and nuclear (N) protein fractions were isolated from 10 samples of protoplasts. Equal amount of each sample was loaded for SDS-PAGE and immunoblotting.


Protoplasts of WT and pldα6 from 12 - day old stage leaf sheath were collected and transfected with a pM999 - SLR1 construct , incubated for 12 hours and then treated with 10 μM GA3 for 0 , 3 , and 9 hours . The first column , fluorescence from GFP ; the second , red fluorescence from the nucleus marker Ghd7 ; the third , bright field , and the fourth , overlay of the three channels . All confocal images were scanned using similar laser gain and offset settings . Bars = 10 μm .

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Protoplasts of WT and pldα6 from 12-day old stage leaf sheath were collected and transfected with a pM999-SLR1 construct, incubated for 12 hours and then treated with 10 μM GA3 for 0, 3, and 9 hours. The first column, fluorescence from GFP; the second, red fluorescence from the nucleus marker Ghd7; the third, bright field, and the fourth, overlay of the three channels. All confocal images were scanned using similar laser gain and offset settings. Bars = 10 μm.


Immunoblotting of SLR1 - GFP proteins from protoplast cells . Equal amounts of proteins from each sample were used for SDS - PAGE , followed by immunoblotting with anti - GFP antibodies .

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Immunoblotting of SLR1-GFP proteins from protoplast cells. Equal amounts of proteins from each sample were used for SDS-PAGE, followed by immunoblotting with anti-GFP antibodies.


A Bar graph and box - and - whisker plots are presented , which show the allelic proportions of WT FOXL2 mRNA and 402C > G FOXL2 mRNA in AGCT tissues from 46 patients analyzed by pyrosequencing . The box plot represents the minimum value , first quartile , median , third quartile and maximum value of a data set . X - axis indicates mRNAs of WT FOXL2 and 402C > G FOXL2 . The whiskers extend to the most extreme data points not considered outliers , and the outliers are represented as dots . Comparisons between groups were performed using Student ' s t - test , and p values are presented .

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A Bar graph and box-and-whisker plots are presented, which show the allelic proportions of WT FOXL2 mRNA and 402C>G FOXL2 mRNA in AGCT tissues from 46 patients analyzed by pyrosequencing. The box plot represents the minimum value, first quartile, median, third quartile and maximum value of a data set. X-axis indicates mRNAs of WT FOXL2 and 402C>G FOXL2. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots. Comparisons between groups were performed using Student's t-test, and p values are presented.


B The relative abundances of WT and variant FOXL2 mRNA were analyzed in KGN and COV434 cells by pyrosequencing ( left graph ) , allele - specific RT - PCR ( middle graph ) and real - time RT - PCR ( right graph ) . gDNA was detected as a positive control . The relative abundances of the variant FOXL2 mRNA were normalized to that of WT mRNA ( set to 1 ) . FOXL2 mRNA levels detected by real - time RT - PCR were normalized to matching gDNA levels . The pyrosequencing data are presented from two independent experiments . The allele - specific semi - quantitative and real - time RT - PCR data are presented as the mean ± SEM from three independent experiments . The p values were analyzed by unpaired , two - tailed Student ' s t - test ( * * * p < 0 . 001 ) . n . d . not detected .

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B The relative abundances of WT and variant FOXL2 mRNA were analyzed in KGN and COV434 cells by pyrosequencing (left graph), allele-specific RT-PCR (middle graph) and real-time RT-PCR (right graph). gDNA was detected as a positive control. The relative abundances of the variant FOXL2 mRNA were normalized to that of WT mRNA (set to 1). FOXL2 mRNA levels detected by real-time RT-PCR were normalized to matching gDNA levels. The pyrosequencing data are presented from two independent experiments. The allele-specific semi-quantitative and real-time RT-PCR data are presented as the mean ± SEM from three independent experiments. The p values were analyzed by unpaired, two-tailed Student's t-test (***p < 0.001). n.d. not detected.


C RNA - decay rates of WT and 402C > G FOXL2 mRNAs in KGN cells were determined after treatment with 5 µg / mL ActD for the indicated times . The estimated half - lives of each transcript are presented . The data are presented as the mean ± SEM from three independent experiments .

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C RNA-decay rates of WT and 402C>G FOXL2 mRNAs in KGN cells were determined after treatment with 5 µg/mL ActD for the indicated times. The estimated half-lives of each transcript are presented. The data are presented as the mean ± SEM from three independent experiments.


Changes in WT and variant FOXL2 mRNA expression were assessed by RT - PCR ( top ) and real - time RT - PCR ( bottom ) ( A ) after transfecting KGN cells with anti - miRNAs for 48 h .

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Changes in WT and variant FOXL2 mRNA expression were assessed by RT-PCR (top) and real-time RT-PCR (bottom) (A) after transfecting KGN cells with anti-miRNAs for 48 h.


Changes in WT and variant FOXL2 mRNA expression were assessed by western blot analysis ( B ) after transfecting KGN cells with anti - miRNAs for 48 h .

Changes in WT and variant FOXL2 mRNA expression were assessed by western blot analysis (B) after transfecting KGN cells with anti-miRNAs for 48 h.


C FOXL2 protein levels after transfection of control or miR - 1236 were assessed in KGN and COV434 cells .

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C FOXL2 protein levels after transfection of control or miR-1236 were assessed in KGN and COV434 cells.


D The mRNA levels of WT and variant FOXL2 in control , miR - 1236 - / + , and miR - 1236 - / - KGN cells , with or without miR - 1236 transfection , were determined by RT - PCR ( top ) or real - time RT - PCR ( bottom ) .

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D The mRNA levels of WT and variant FOXL2 in control, miR-1236-/+, and miR-1236-/- KGN cells, with or without miR-1236 transfection, were determined by RT-PCR (top) or real-time RT-PCR (bottom).


E FOXL2 protein expression in control , miR - 1236 - / + , and miR - 1236 - / - KGN cells , and two independent miR - 1236 - / - ( # 1 and # 2 ) COV434 cell lines were determined by western blotting .

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E FOXL2 protein expression in control, miR-1236-/+, and miR-1236-/- KGN cells, and two independent miR-1236-/- (#1 and #2) COV434 cell lines were determined by western blotting.


B Luciferase activity of the reporter constructs shown in ( A ) were measured in KGN cells after transfection with an miR - 1236 mimic for 48 h . The black arrow indicates the position of 402C > G mutation site .

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B Luciferase activity of the reporter constructs shown in (A) were measured in KGN cells after transfection with an miR-1236 mimic for 48 h. The black arrow indicates the position of 402C>G mutation site.


D Luciferase activities were measured in KGN cells , using the reporter constructs shown in ( C ) , after transfection with a control miRNA or an miR - 1236 mimic for 48 h .

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D Luciferase activities were measured in KGN cells, using the reporter constructs shown in (C), after transfection with a control miRNA or an miR-1236 mimic for 48 h.


E , F miR - 1236 mutants , in which the C that pairs with G402 of the FOXL2 mutant was substituted with either G ( miR - 1236 - G ) ( E ) or U ( miR - 1236 - U ) ( F ) , were cotransfected into KGN cells with one of the reporter constructs described above . Luciferase activities were subsequently determined . Arrows indicate the mismatched sites .

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E, F miR-1236 mutants, in which the C that pairs with G402 of the FOXL2 mutant was substituted with either G (miR-1236-G) (E) or U (miR-1236-U) (F), were cotransfected into KGN cells with one of the reporter constructs described above. Luciferase activities were subsequently determined. Arrows indicate the mismatched sites.


G In vitro annealing kinetics of miR - 1236 with 230 nt - long transcripts of WT or variant FOXL2 . 32P - labeled miR - 1236 ( 0 . 5 nM ) was incubated with increasing concentrations of synthetic FOXL2 transcripts ( 0 , 2 . 5 , 12 . 5 , 25 , or 50 nM ) . FOXL2 mRNA - miR - 1236 complexes were resolved on a 6 % native gel and detected by autoradiography ( left ) . The predicted Kds for the WT and 402C > G FOXL2 transcripts are presented in the right graph .

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G In vitro annealing kinetics of miR-1236 with 230 nt-long transcripts of WT or variant FOXL2. 32P-labeled miR-1236 (0.5 nM) was incubated with increasing concentrations of synthetic FOXL2 transcripts (0, 2.5, 12.5, 25, or 50 nM). FOXL2 mRNA-miR-1236 complexes were resolved on a 6% native gel and detected by autoradiography (left). The predicted Kds for the WT and 402C>G FOXL2 transcripts are presented in the right graph.


Changes in WT and variant FOXL2 mRNA - expression levels were assessed by real - time RT - PCR ( A ) after transfecting KGN cells with siRNAs against AGO mRNAs for 48 h . The data ( mean ± SEM ) are from three independent experiments , performed in triplicate .

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Changes in WT and variant FOXL2 mRNA-expression levels were assessed by real-time RT-PCR (A) after transfecting KGN cells with siRNAs against AGO mRNAs for 48 h. The data (mean ± SEM) are from three independent experiments, performed in triplicate.


Changes in WT and variant FOXL2 mRNA - expression levels were assessed by western blot analysis ( B ) after transfecting KGN cells with siRNAs against AGO mRNAs for 48 h . The data ( mean ± SEM ) are from three independent experiments , performed in triplicate .

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Changes in WT and variant FOXL2 mRNA-expression levels were assessed by western blot analysis (B) after transfecting KGN cells with siRNAs against AGO mRNAs for 48 h. The data (mean ± SEM) are from three independent experiments, performed in triplicate.


C The mRNA levels of WT ( left ) and variant FOXL2 ( right ) were determined in KGN cells by real - time RT - PCR , after transfecting a control miRNA or miR - 1236 . The data ( mean ± SEM ) are from three independent experiments , performed in triplicate .

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C The mRNA levels of WT (left) and variant FOXL2 (right) were determined in KGN cells by real-time RT-PCR, after transfecting a control miRNA or miR-1236. The data (mean ± SEM) are from three independent experiments, performed in triplicate.


D The mRNA levels of WT and the variant FOXL2 in control ( left ) and miR - 1236 - / - KGN cells ( right ) after transfecting siRNAs against AGO mRNAs were determined by allele - specific real - time RT - PCR . The data ( mean ± SEM ) are from three independent experiments , performed in triplicate .

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D The mRNA levels of WT and the variant FOXL2 in control (left) and miR-1236-/- KGN cells (right) after transfecting siRNAs against AGO mRNAs were determined by allele-specific real-time RT-PCR. The data (mean ± SEM) are from three independent experiments, performed in triplicate.


E 293T cells were transfected with an miR - 1236 mimic ( 50 nM ) for 24 h , followed by cotransfection with expression vectors encoding FLAG / HA - tagged variants of the indicated human AGOs and pGL3c - CDS - MT for 24 h . The empty p3XFLAG - CMV - 10 vector was used as control . Co - immunoprecipitated mRNAs were reverse transcribed , and the cDNA products were used for allele - specific real - time PCR analysis of the FOXL2 variant and GAPDH mRNAs ( top ) . The level of variant FOXL2 mRNA immunoprecipitated using FLAG - tagged AGO proteins was normalized using the level of GAPDH mRNA from the same lysates . The immunoprecipitated - AGO proteins were detected by western blotting ( bottom ) . The data ( mean ± SEM ) are from three independent experiments .

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E 293T cells were transfected with an miR-1236 mimic (50 nM) for 24 h, followed by cotransfection with expression vectors encoding FLAG/HA-tagged variants of the indicated human AGOs and pGL3c-CDS-MT for 24 h. The empty p3XFLAG-CMV-10 vector was used as control. Co-immunoprecipitated mRNAs were reverse transcribed, and the cDNA products were used for allele-specific real-time PCR analysis of the FOXL2 variant and GAPDH mRNAs (top). The level of variant FOXL2 mRNA immunoprecipitated using FLAG-tagged AGO proteins was normalized using the level of GAPDH mRNA from the same lysates. The immunoprecipitated-AGO proteins were detected by western blotting (bottom). The data (mean ± SEM) are from three independent experiments.