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

O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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).


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

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

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


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

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


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

O O B-CONTROLLED_VAR 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

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


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

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


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

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


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

O O B-CONTROLLED_VAR 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

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


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

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

O O O O O O O O O O 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

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


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

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

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 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

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


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

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 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

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


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

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

O O O 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. Representative images of epithelial cells taken from egg chambers with aspect ratios of approximately 1.4 (A).


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

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

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

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


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

O O O O O O O O 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 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

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


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

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

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


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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Pak - shRNA egg chambers . Mann Whitney w1118 versus Pak shRNA p = 0 . 0094 . Neither Fat2 - shRNA nor Pak - shRNA distributions are stastically different from random ( Wilcoxon Signed Rank , Theoretical Mean of 45 ) . Number of analyzed : w1118 n = 97 , Fat2 - shRNA n = 44 , Pak - shRNA n = 39 . Egg chambers analyzed : w1118 n = 40 , Fat2 - shRNA n = 21 , Pak - shRNA n =

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

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


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

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


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

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


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

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


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

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


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

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


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

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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 apical cortex of follicle during egg chamber elongation showing a transition from to tissue - level organization during stage 6 . ( B ) localization Sqh - mCherry is shown relative to adherens junction marked by Shg : :

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


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

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


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

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


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

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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 were treated as in B ) then fixed stained with ULK1 ( green ) ( F ) or ATG13 ( red ) antibodies ( G ) respectively . Hoechst was used to stain nuclei . Graphs ( right ) show average of both ULK1 ( F ) ( n = 3 independent experiments , n = 11 fields analysed ) or ATG13 ( n = 3 independent experiments , n ≥ 16 fields analysed ) ( G ) number per both ULK1 ( n = 3 independent experiments ) ( F ) or ATG13 ( n = 3 independent experiments ) ( G ) fold of increase of average respectively . arrows indicate most representative of analysed conditions . data are reported as mean value ± . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison . * * , P < 0 , 01 ; * * * * , P < 0 . 0001 or by unpaired Student ' s t ‐ when two groups were compared . * , P < 0 . 05 ; * * * * , P < 0 . 0001 . Scale bar 5 μm .

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

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

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

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


A ) HeLa were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) grown in fed ( FED ) or starvation conditions ( STV ) in presence or absence of CLQ for 1h . were then fixed stained using LC3B - II ( red ) antibody . of LC3B number is reported ( bottom ) . data are expressed as mean value ± . Statistical were performed by two - way ANOVA followed by Tukey ' s multiple comparison . * * * * , P < 0 . 0001 . ( n = 4 independent experiments , n ≥ 25 fields analysed ) . Scale bar 5 μm .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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

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

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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 stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3 ' UTR TFG siRNA , starved for 1h , fixed , permeabilized immunolabelled with ULK1 ( red ) alone or ULK1 ( red ) 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 , are reported as mean ± ( bottom graph ) . ( n = 3 independent experiments , n = 19 fields analysed ) arrowheads point at co - localization events between ULK1 ERGIC . Statistical analyses were performed by unpaired Student ' s t ‐ . * * , P < 0 , 01 ; * * * , 0 , 001 . Scale bar 5 μm .

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

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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 same growth stage were transferred to 0 . 5 MS liquid media without or with different concentrations of GA3 . PA from soybean was added to media at a final of 20 μM . Pictures were taken 7 days after transfer . horizontal red separates different vertical red scale bar represents 2 cm .

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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 blotted to a .

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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 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 containing 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 in WT , pldα6 COM without with 10 μM GA3 . Leaf samples ( 15 seedling each ) from 4 - leaf stage rice ( Dongjin background ) were collected lipids were extracted 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 COM . Leaf samples ( 15 seedling each ) from 4 - leaf stage rice ( Dongjin background ) were collected different phospholipid species including carbon number double bond number were determined . Values are means ± SD ( n = 3 biological replicates ) . GA3 was dissolved in ethanol seedlings treated with same ethanol 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 using constructs shown in ( D ) . were separated by SDS - PAGE , followed by immunoblotting with anti - His - tag antibodies . PA binding activity of different truncation mutations were analyzed by fat - immunoblotting . red arrowheads indicate truncated 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 lipid - immunoblotting of PA binding by GID1 mutant 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 2 hours later , confocal of protoplasts are shown . pM999 - GFP refers to empty vector with GFP only that was transformed as control 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 ) ( N ) 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 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 pldα6 protoplasts with or without GA3 treatments . 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 pldα6 protoplasts . After 2 - hour treatment with 10 μM GA3 , total ( T ) , soluble ( S ) ( N ) fractions were isolated from 10 samples of protoplasts . Equal amount of each sample was loaded for SDS - PAGE 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 pldα6 from 12 - day old stage leaf sheath were collected transfected with a pM999 - SLR1 construct , incubated for 12 hours then treated with 10 μM GA3 for 0 , 3 , 9 hours . first column , from GFP ; second , red from nucleus marker Ghd7 ; third , bright field , fourth , overlay of three channels . confocal were scanned using similar gain 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 from protoplast . Equal amounts of 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 box - - whisker plots are presented , which show allelic proportions of WT FOXL2 mRNA 402C > G FOXL2 mRNA in AGCT tissues from 46 analyzed by pyrosequencing . box plot represents minimum value , first quartile , median , third quartile maximum value of a data set . X - axis indicates mRNAs of WT FOXL2 402C > G FOXL2 . whiskers extend to most extreme data points not considered outliers , outliers are represented as dots . Comparisons between groups were performed using Student ' s t - , 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 relative abundances of WT variant FOXL2 mRNA were analyzed in KGN COV434 by pyrosequencing ( left graph ) , allele - specific RT - PCR ( middle graph ) real - time RT - PCR ( right graph ) . gDNA was detected as a positive control . relative abundances of variant FOXL2 mRNA were normalized to that of WT mRNA ( set to 1 ) . FOXL2 mRNA detected by real - time RT - PCR were normalized to matching gDNA . pyrosequencing data are presented from two independent experiments . allele - specific semi - quantitative real - time RT - PCR data are presented as mean ± from three independent experiments . p values were analyzed by unpaired , two - Student ' s t - ( * * * 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 - decay rates of WT 402C > G FOXL2 mRNAs in KGN were determined after treatment with 5 µg / mL ActD for indicated times . estimated half - lives of each transcript are presented . data are presented as mean ± 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 variant FOXL2 mRNA expression were assessed by RT - PCR ( top ) real - time RT - PCR ( bottom ) ( A ) after transfecting KGN 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 variant FOXL2 mRNA expression were assessed by western blot ( B ) after transfecting KGN with anti - miRNAs for 48 h .

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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 after transfection of control or miR - 1236 were assessed in KGN COV434 .

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


D mRNA of WT variant FOXL2 in control , miR - 1236 - / + , miR - 1236 - / - KGN , 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 expression in control , miR - 1236 - / + , miR - 1236 - / - KGN , two independent miR - 1236 - / - ( # 1 # 2 ) COV434 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 reporter constructs shown in ( A ) were measured in KGN after transfection with an miR - 1236 mimic for 48 h . black arrow indicates 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 , using 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 C that pairs with G402 of FOXL2 mutant was substituted with either G ( miR - 1236 - G ) ( E ) or U ( miR - 1236 - U ) ( F ) , were cotransfected into KGN with one of reporter constructs described above . Luciferase activities were subsequently determined . Arrows indicate 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 detected by autoradiography ( left ) . predicted Kds for WT 402C > G FOXL2 transcripts are presented in 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 variant FOXL2 mRNA - expression were assessed by real - time RT - PCR ( A ) after transfecting KGN with siRNAs against AGO mRNAs for 48 h . data ( mean ± ) 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 variant FOXL2 mRNA - expression were assessed by western blot ( B ) after transfecting KGN with siRNAs against AGO mRNAs for 48 h . data ( mean ± ) 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 mRNA of WT ( left ) variant FOXL2 ( right ) were determined in KGN by real - time RT - PCR , after transfecting a control miRNA or miR - 1236 . data ( mean ± ) 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 mRNA of WT variant FOXL2 in control ( left ) miR - 1236 - / - KGN ( right ) after transfecting siRNAs against AGO mRNAs were determined by allele - specific real - time RT - PCR . data ( mean ± ) 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 were transfected with an miR - 1236 mimic ( 50 nM ) for 24 h , followed by cotransfection with expression vectors encoding FLAG / HA - tagged variants of indicated human AGOs pGL3c - CDS - MT for 24 h . empty p3XFLAG - CMV - 10 vector was used as control . Co - immunoprecipitated mRNAs were reverse transcribed , cDNA products were used for allele - specific real - time PCR of FOXL2 variant GAPDH mRNAs ( top ) . level of variant FOXL2 mRNA immunoprecipitated using FLAG - tagged AGO was normalized using level of GAPDH mRNA from same lysates . immunoprecipitated - AGO were detected by western blotting ( bottom ) . data ( mean ± ) 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.


F In vivo association of AGO3 - mediated miRISC formation with FOXL2 mRNAs are shown . Following transfection of a control miRNA or miR - 1236 - G mutant into KGN , AGO3 - mediated RISC - associated RNAs were isolated by immunoprecipitation with an anti - AGO3 antibody . IgG was used as a control . co - immunoprecipitated mRNAs were reverse transcribed using a FOXL2 - 430 - R primer binding downstream of 402C > G site . cDNA products were used for FOXL2 allele - specific PCR with FOXL2 - 279F primer ( Appendix Fig . S1 ) , a representative result ( top left ) is shown . Quantitative real - time RT - PCR results that examined FOXL2 mRNAs , normalized using level of GAPDH mRNA ( bottom left ) , are also presented . Western blot of immunoprecipitated AGO3 inputs are shown in right panel . data are presented as mean ± of two independent experiments .

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F In vivo association of AGO3-mediated miRISC formation with FOXL2 mRNAs are shown. Following transfection of a control miRNA or the miR-1236-G mutant into KGN cells, AGO3-mediated RISC-associated RNAs were isolated by immunoprecipitation with an anti-AGO3 antibody. IgG was used as a control. The co-immunoprecipitated mRNAs were reverse transcribed using a FOXL2-430-R primer binding downstream of the 402C>G site. The cDNA products were used for FOXL2 allele-specific PCR analysis with the FOXL2-279F primer (Appendix Fig. S1), and a representative result (top left) is shown. Quantitative real-time RT-PCR results that examined FOXL2 mRNAs, normalized using the level of GAPDH mRNA (bottom left), are also presented. Western blot analysis of immunoprecipitated AGO3 and inputs are shown in the right panel. The data are presented as the mean ± SEM of two independent experiments.


G - seq was performed to determine AGO - expression ( transcripts per million ) from tissues from 20 independent AGCT . X - axis represents mRNAs of AGO1 to 4 . box plot represents minimum value , first quartile , median , third quartile maximum value of a data set . whiskers extend to most extreme data points not considered outliers , outliers are represented as dots .

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G RNA-seq analysis was performed to determine AGO-expression levels (transcripts per million) from the individual tissues from 20 independent AGCT patients. X-axis represents mRNAs of AGO1 to 4. The box plot represents the minimum value, first quartile, median, third quartile and maximum value of a data set. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots.


A Changes in WT variant FOXL2 mRNA in KGN were assessed by real - time RT - PCR after transfecting siRNAs against indicated factors for 48 h . data are presented as mean ± from three independent experiments , performed in triplicate .

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A Changes in WT and variant FOXL2 mRNA levels in KGN cells were assessed by real-time RT-PCR after transfecting siRNAs against the indicated factors for 48 h. The data are presented as the mean ± SEM from three independent experiments, performed in triplicate.


B FOXL2 - expression were determined by western blotting after transfecting KGN with control , DHX9 , or GW182 siRNAs for 48 h . of FOXL2 expression is presented in bottom panel . data are presented as mean ± from three independent experiments . p values were analyzed by unpaired , two - Student ' s t - ( * * p < 0 . 01 , * * * p < 0 . 001 ) .

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B FOXL2 protein-expression levels were determined by western blotting after transfecting KGN cells with control, DHX9, or GW182 siRNAs for 48 h. Quantification of FOXL2 protein expression is presented in the bottom panel. The 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 < 0.01, ***p < 0.001).


C Relative binding affinities of DHX9 GW182 to AGOs . 293T were transfected with expression vectors encoding indicated FLAG / HA - tagged AGOs , extracts were prepared immunoprecipitated with an anti - FLAG antibody , followed by immunoblot analyses ( top ) . empty p3XFLAG - CMV - 10 vector was used as a control . band intensities of immunoprecipitated DHX9 GW182 were quantified normalized following pull - down with indicated AGOs ( bottom ) . data are presented as mean ± from three independent experiments . * # indicate statistically significant differences in respective amounts of DHX9 or GW182 bound to AGO1 . p values were analysed by unpaired , two - Student ' s t - ( p < 0 . 05 ) .

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C Relative binding affinities of DHX9 and GW182 to AGOs. 293T cells were transfected with expression vectors encoding the indicated FLAG/HA-tagged AGOs, and cell extracts were prepared and immunoprecipitated with an anti-FLAG antibody, followed by immunoblot analyses (top). The empty p3XFLAG-CMV-10 vector was used as a control. The band intensities of immunoprecipitated DHX9 and GW182 were quantified and normalized following pull-down with the indicated AGOs (bottom). The data are presented as the mean ± SEM from three independent experiments. * and # indicate statistically significant differences in the respective amounts of DHX9 or GW182 bound to AGO1. The p values were analysed by unpaired, two-tailed Student's t-test (p < 0.05).


D Association of endogenous miR - 1236 with RISC components in KGN was determined via pulldown assays using immobilized 2 ′ - O - methylated oligonucleotides ( 2 ′ - O - Me oligos ) complementary to miR - 1236 followed with a pulldown using streptavidin - coupled Dynabeads western blot analyses ( top ) . Relative of bound compared with from input is presented as fold enrichment ( bottom ) . Efficient pulldown of endogenous miR - 1236 using 2 ′ - O - Me oligos was confirmed with depleted miR - 1236 in discarded supernatant following pulldown ( Appendix Fig S8 ) . As a control , 2 ′ - O - Me oligos not complementary to miR - 1236 were used . data are means ± from three independent experiments . p values were analyzed by unpaired , two - Student ' s t - ( * p < 0 . 05 , * * p < 0 . 01 ) .

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D Association of endogenous miR-1236 with RISC components in KGN cells was determined via pulldown assays using immobilized 2′-O-methylated oligonucleotides (2′-O-Me oligos) complementary to miR-1236 followed with a pulldown using streptavidin-coupled Dynabeads and western blot analyses (top). Relative quantification of bound proteins compared with proteins from the input is presented as fold enrichment (bottom). Efficient pulldown of endogenous miR-1236 using the 2′-O-Me oligos was confirmed with depleted miR-1236 in the discarded supernatant following the pulldown (Appendix Fig S8). As a control, 2′-O-Me oligos not complementary to miR-1236 were used. The data are the means ± SEM from three independent experiments. The p values were analyzed by unpaired, two-tailed Student's t-test (*p < 0.05, **p < 0.01).


E Following transfection of control siRNA or siDHX9 into KGN , AGO3 - mediated RISC - associated RNAs were immunoprecipitated using an anti - AGO3 antibody . IgG was used as a control . Co - immunoprecipitated mRNAs were reverse transcribed using a FOXL2 - 430 - R primer binding downstream of 402C > G site . cDNA products were used for FOXL2 allele - specific PCR with a FOXL2 - 279F primer ( Appendix Fig S1A ) , a representative result obtained by RT - PCR ( top ) is shown . Quantitative real - time RT - PCR results ( middle ) are also presented as fold enrichment of FOXL2 mRNAs normalized using level of GAPDH mRNA . Western blots of immunoprecipitated AGO3 inputs are shown in bottom panel . data are presented as mean ± from three independent experiments . Different letters denote statistically significant differences ( p < 0 . 05 ; Student - Newman - Keuls ) .

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E Following transfection of control siRNA or siDHX9 into KGN cells, AGO3-mediated RISC-associated RNAs were immunoprecipitated using an anti-AGO3 antibody. IgG was used as a control. Co-immunoprecipitated mRNAs were reverse transcribed using a FOXL2-430-R primer binding downstream of the 402C>G site. The cDNA products were used for FOXL2 allele-specific PCR analysis with a FOXL2-279F primer (Appendix Fig S1A), and a representative result obtained by RT-PCR (top) is shown. Quantitative real-time RT-PCR results (middle) are also presented as fold enrichment of FOXL2 mRNAs normalized using the level of GAPDH mRNA. Western blots of immunoprecipitated AGO3 and the inputs are shown in the bottom panel. The data are presented as the mean ± SEM from three independent experiments. Different letters denote statistically significant differences (p < 0.05; Student-Newman-Keuls test).


F We examined whether DHX9 affected association between miR - 1236 AGOs . After transfecting KGN with control siRNA or siDHX9 , total AGOs - mediated RISC - associated RNAs were isolated following immunoprecipitations using anti - AGO3 or anti - AGO2 antibodies . AGOs - immunoprecipitated RNAs were extracted using an acidic phenol : chloroform mixture ( 5 : 1 , pH 4 . 3 ) precipitated with isopropanol using 10 % of 3 M NaOAc ( pH 5 . 2 ) . enrichment of miR - 1236 within miRISCs was detected using TaqMan ® microRNA in immunoprecipitated RNAs normalized using level of total miR - 1236 . data ( means ± ) are presented as fold enrichment calculated from three independent experiments . Different letters denote statistically significant differences ( p < 0 . 05 ; Student - Newman - Keuls ) .

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F We examined whether DHX9 affected the association between miR-1236 and AGOs. After transfecting KGN cells with control siRNA or siDHX9, the total RNA and AGOs-mediated RISC-associated RNAs were isolated following immunoprecipitations using anti-AGO3 or anti-AGO2 antibodies. The AGOs-immunoprecipitated RNAs were extracted using an acidic phenol:chloroform mixture (5:1, pH 4.3) and precipitated with isopropanol using 10% of 3 M NaOAc (pH 5.2). The enrichment of miR-1236 within miRISCs was detected using the TaqMan® microRNA assay in the immunoprecipitated RNAs and normalized using the level of total miR-1236. The data (means ± SEM) are presented as the fold enrichment calculated from three independent experiments. Different letters denote statistically significant differences (p < 0.05; Student-Newman-Keuls test).


G , H Luciferase activities of reporter constructs presented in Fig 3A C were measured in KGN after transfecting miR - 1236 mimic , indicated siRNAs , either pGL3c - CDS - FOXL2 MT or pGL3c - UTR - FOXL2 MT for 48 h . data are expressed as means ± from three independent experiments were performed in triplicate . p values were analyzed by unpaired , two - Student ' s t - ( * * * p < 0 . 001 ) .

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G, H Luciferase activities of the reporter constructs presented in Fig 3A and C were measured in KGN cells after transfecting the miR-1236 mimic, indicated siRNAs, and either pGL3c-CDS-FOXL2 MT or pGL3c-UTR-FOXL2 MT for 48 h. The data are expressed as the means ± SEM from three independent experiments and were performed in triplicate. The p values were analyzed by unpaired, two-tailed Student's t-test (***p < 0.001).


KGN were transfected with 200 nM of scrambled control or FOXL2 - specific siRNAs for 24 h . Then , KGN were further transfected with 20 nM of control miRNA , miR - 1236 , control anti - miRNA , or anti - miR - 1236 . proportion of annexin - V - positive apoptotic ( A B ) were analyzed by flow cytometry . data are presented as mean ± of three independent experiments . Different letters denote statistically significant differences ( p < 0 . 0001 ; Student - Newman - Keuls ) .

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KGN cells were transfected with 200 nM of scrambled control or FOXL2-specific siRNAs for 24 h. Then, KGN cells were further transfected with 20 nM of control miRNA, miR-1236, control anti-miRNA, or anti-miR-1236. The proportion of annexin-V-positive apoptotic cells (A and B) were analyzed by flow cytometry. The data are presented as the mean ± SEM of three independent experiments. Different letters denote statistically significant differences (p < 0.0001; Student-Newman-Keuls test).