Figure 5A , Control and degron ORP2 cells were co - plated , and after 1 day incubation in 5 % LPDS , cells were loaded with LDL ( + LDL ) and IAA ( + IAA ; ORP2 depletion ) for the indicated times . Intracellular cholesterol was labeled with D4H and imaged by widefield epifluorescence microscopy . Asterisk indicates region of ORP2 - depleted cells . CM , complete medium . Dashed lines separate ORP2 expressing and depleted cells . B , Quantification of A . Mean ± SD , n = 15 - 27 cells pooled from 2 independent experiments . Student ' s t - test . C , E , Control and degron ORP2 cells were co - plated , and after 1 day in 5 % LPDS , treated with 50 μg / ml LDL and IAA for the indicated times . Asterisk indicates an ORP2 - depleted cell . Double labeling of intracellular D4H and Lamp1 ( C ) or intracellular D4H and internalized integrin β1 ( E ) . Images were acquired by confocal microscopy . Red box indicates a peripheral region toward the leading edge in the control cell , and blue box a corresponding region in the ORP2 - depleted cell .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, Control and degron ORP2 cells were co-plated, and after 1 day incubation in 5 % LPDS, cells were loaded with LDL (+LDL) and IAA (+IAA; ORP2 depletion) for the indicated times. Intracellular cholesterol was labeled with D4H and imaged by widefield epifluorescence microscopy. Asterisk indicates region of ORP2-depleted cells. CM, complete medium. Dashed lines separate ORP2 expressing and depleted cells. B, Quantification of A. Mean ± SD, n = 15-27 cells pooled from 2 independent experiments. Student's t-test. C, E, Control and degron ORP2 cells were co-plated, and after 1 day in 5 % LPDS, treated with 50 μg/ml LDL and IAA for the indicated times. Asterisk indicates an ORP2-depleted cell. Double labeling of intracellular D4H and Lamp1 (C) or intracellular D4H and internalized integrin β1 (E). Images were acquired by confocal microscopy. Red box indicates a peripheral region toward the leading edge in the control cell, and blue box a corresponding region in the ORP2-depleted cell.


Figure 6B , Cells stably expressing NPC1 - mCherry were treated with control or OCRL siRNAs for 2 days , or transfected with GFP - ORP2 or - ORP2 - mHHK ( PI ( 4 , 5 ) P2 binding - deficient mutant ) for 1 day , stained with PI ( 4 , 5 ) P2 antibody and imaged by confocal microscopy . For FAK inhibition , GFP - ORP2 transfected cells were treated with 10 μM PF228 for 4 h . Dashed lines indicate cell edges . Arrowheads indicate colocalization of PI ( 4 , 5 ) P2 and NPC1 . C , Quantification of B . Values from Ctrl siRNA and Vector control were pooled and are shown as Ctrl siRNA / Vector ctrl . Mean ± SD , n = 20 - 43 cells pooled from 2 independent experiments . Student ' s t - test . D , E , Cells stably expressing NPC1 - mCherry were transfected with GFP - ORP2 or GFP - ORP2 - mHHK for 1 day and were indicated , treated with 10 μM PF228 for 4 h . For degron ORP2 cells , NPC1 - mCherry was transiently transfected for 2 days and cells incubated without ( - IAA ; ORP2 present ) or with IAA ( + IAA ; ORP2 depleted ) for 1 h . Arrowheads indicate tubular NPC1 organelles . Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured . Mean ± SD , n = 102 - 122 cells . Students ' s t - test . Please see also ctrl ( MovieEV4 ) and PF228 treated cell ( MovieEV5 ) , both videos 1 min recordings with 1 s frame rate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B, Cells stably expressing NPC1-mCherry were treated with control or OCRL siRNAs for 2 days, or transfected with GFP-ORP2 or -ORP2-mHHK (PI(4,5)P2 binding-deficient mutant) for 1 day, stained with PI(4,5)P2 antibody and imaged by confocal microscopy. For FAK inhibition, GFP-ORP2 transfected cells were treated with 10 μM PF228 for 4 h. Dashed lines indicate cell edges. Arrowheads indicate colocalization of PI(4,5)P2 and NPC1. C, Quantification of B. Values from Ctrl siRNA and Vector control were pooled and are shown as Ctrl siRNA/Vector ctrl. Mean ± SD, n = 20-43 cells pooled from 2 independent experiments. Student's t-test. D, E, Cells stably expressing NPC1-mCherry were transfected with GFP-ORP2 or GFP-ORP2-mHHK for 1 day and were indicated, treated with 10 μM PF228 for 4 h. For degron ORP2 cells, NPC1-mCherry was transiently transfected for 2 days and cells incubated without (-IAA; ORP2 present) or with IAA (+IAA; ORP2 depleted) for 1 h. Arrowheads indicate tubular NPC1 organelles. Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured. Mean ± SD, n = 102-122 cells. Students's t-test. Please see also ctrl (MovieEV4) and PF228 treated cell (MovieEV5), both videos 1 min recordings with 1 s frame rate.


Figure 7A , B , Control and degron ORP2 cells were co - plated , and after 1 day 5 % LPDS , treated with 50 μg / ml LDL and IAA ( + IAA ; ORP2 depletion ) for the indicated times and immunostained for pFAK to quantify its intensity . For rescue experiments , degron ORP2 cells were plated and transfected with GFP - ORP2 or - ORP2 - mHHK or - ORP2 - ∆ ELSK for 6 h . Cells expressing GFP - ORP2 constructs at levels similar to endo - GFP - ORP2 were used for quantifying pFAK intensity . Dashed lines indicate cell outlines . See also Fig EV6C . Asterisk indicates cells depleted of endogenous ORP2 . Images were acquired by confocal microscopy . Mean ± SD , n = 20 - 23 cells pooled from 2 independent experiments . Student ' s t - test . C , Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA . Cells were starved overnight in LPDS and loaded with 50 µg / mL LDL + / - IAA for 2 h . The proportion of FAK signal in the membrane fraction ( of total FAK in cytosol + membranes ) is presented ± SD . For no IAA n = 13 and for IAA n = 11 , 2 independent experiments . Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, B, Control and degron ORP2 cells were co-plated, and after 1 day 5 % LPDS, treated with 50 μg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron ORP2 cells were plated and transfected with GFP-ORP2 or -ORP2-mHHK or -ORP2-∆ELSK for 6 h. Cells expressing GFP-ORP2 constructs at levels similar to endo-GFP-ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20-23 cells pooled from 2 independent experiments. Student's t-test.C, Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/- IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol+membranes) is presented ± SD. For no IAA n=13 and for IAA n= 11, 2 independent experiments. Student's t-test.


Figure 1A . H3122 cells expressing endogenous EML4 - ALK V1 and HEK293 transfected with YFP - EML4 - ALK V1 WT and KD were stained for either anti - ALK or anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . B . Violin plots showing the number of EML4 - ALK V1 cytoplasmic foci per cell . Data represents 30 - 50 counts from three biological replicates . * * * P < 0 . 001 , * * * * P < 0 . 0001 in comparison to HEK293 YFP - EML4 - ALK - V1 KD by one - way ANOVA analysis . C . H2228 cells expressing endogenous EML4 - ALK V3 and HEK293 transfected with YFP - EML4 - ALK V3 WT and KD were stained for either anti - ALK or anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . D . Violin plots showing the number of EML4 - ALK V3 cytoplasmic foci per cell . Data represents 30 - 50 counts from four biological replicates . * * * P < 0 . 001 , * * * * P < 0 . 0001 in comparison to HEK293 YFP - EML4 - ALK - V3 KD by one - way ANOVA analysis . H . Whisker plot showing the calculated velocity of single events in the kymograph . Data represent 20 counts from 10 kymographs . n = 2 . Error bar represents SD of two biological replicates . * P < 0 . 05 in comparison to YFP EML4 - ALK V3 KD by unpaired t test .

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Figure 1A. H3122 cells expressing endogenous EML4-ALK V1 and HEK293 transfected with YFP-EML4-ALK V1 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B. Violin plots showing the number of EML4-ALK V1 cytoplasmic foci per cell. Data represents 30-50 counts from three biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V1 KD by one-way ANOVA analysis. C. H2228 cells expressing endogenous EML4-ALK V3 and HEK293 transfected with YFP-EML4-ALK V3 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. D. Violin plots showing the number of EML4-ALK V3 cytoplasmic foci per cell. Data represents 30-50 counts from four biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V3 KD by one-way ANOVA analysis. H. Whisker plot showing the calculated velocity of single events in the kymograph. Data represent 20 counts from 10 kymographs. n=2. Error bar represents SD of two biological replicates. *P< 0.05 in comparison to YFP EML4-ALK V3 KD by unpaired t test.


Figure 2A , B . H3122 and H2228 cells were stained for anti - ALK ( green ) , anti - GRB2 ( red ) , anti - SOS1 ( red ) , anti - pC - KITY721 ( red ) , anti - PI3K p85β or anti - pPLCγ2Y759 and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . C . HEK293 cells transfected with YFP - EM4L - ALK V1 WT or V3 WT for 48 hrs and treated with 5 % or 10 % 1 , 6 - hexanediol . Representative still images after 5 min ( taken from time - lapse movies ) are shown of cells treated with 10 % 1 , 6 - Hexanediol or DMSO . Scale bars , 10 μm ; the arrowheads indicate cytoplasmic foci . D . Box plot showing the number of cytoplasmic foci in DMSO and 5 % or 10 % Hexanediol ( 5 min ) . Data represent 5 - 10 counts from 3 independent experiments ( n = 3 ) . Error bar represents SD of three biological replicates . * * * * P < 0 . 0001 in comparison to YFP - EML4 - ALK V3 WT DMSO by one - way ANOVA analysis . E . Representative western blots of HEK293 transfected EML4 - ALK V1 WT and V3 WT for 48 hrs . Cells were treated with either 5 % or 10 % 1 , 6 - Hexanediol for 5 minutes . DMSO was used a control . Lysates were analysed for the phosphorylation and expression of the indicated antibodies . GAPDH was used as a loading control . Data representative of n = 2 experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, B. H3122 and H2228 cells were stained for anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.C. HEK293 cells transfected with YFP-EM4L-ALK V1 WT or V3 WT for 48 hrs and treated with 5% or 10% 1,6-hexanediol. Representative still images after 5 min (taken from time-lapse movies) are shown of cells treated with 10% 1,6-Hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci. D. Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5-10 counts from 3 independent experiments (n=3). Error bar represents SD of three biological replicates. ****P< 0.0001 in comparison to YFP-EML4-ALK V3 WT DMSO by one-way ANOVA analysis. E. Representative western blots of HEK293 transfected EML4-ALK V1 WT and V3 WT for 48 hrs. Cells were treated with either 5% or 10% 1,6-Hexanediol for 5 minutes. DMSO was used a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n=2 experiments.


Figure 3A , B , C , D . HEK293 cells were transfected with EML4 - ALK V1 WT or V3 WT or Kinase Dead ( KD ) for 48 hours . Cells were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown .

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Figure 3A, B, C, D. HEK293 cells were transfected with EML4-ALK V1 WT or V3 WT or Kinase Dead (KD) for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.


Figure 4B . HEK293 cells were transfected with EML4 - ALK V1 deletion 12N blade for 48 hours . Cells were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown .

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Figure 4B. HEK293 cells were transfected with EML4-ALK V1 deletion 12N blade for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.


Figure 5D . HEK293 cells were transfected with EML4 - ALK V3 ∆ JM 1058 - 1100 for 48 hours . Cells were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . E . Violin plot representing the number of cytoplasmic foci counted per cell from D . Data represent counts from at least 30 cells , n = 3 . * * * * P < 0 . 0001 in comparison to DMSO by one - way ANOVA .

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Figure 5D. HEK293 cells were transfected with EML4-ALK V3 ∆JM 1058-1100 for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. E. Violin plot representing the number of cytoplasmic foci counted per cell from D. Data represent counts from at least 30 cells, n=3. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.


Figure 6A - F HEK293 cells were transfected with A . YFP - EML4 - ALK V3 K1150M , C . R1275Q or E . F1174L constructs and treated with ALK inhibitors or DMSO for 4 hours before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . B , D , F . Violin plots representing the number of cytoplasmic foci counted per cell from A , C and E . Data represent counts from at least 30 cells , n = 3 or 4 . * * * * P < 0 . 0001 in comparison to DMSO by one - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-F HEK293 cells were transfected with A. YFP-EML4-ALK V3 K1150M, C. R1275Q or E. F1174L constructs and treated with ALK inhibitors or DMSO for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B, D, F. Violin plots representing the number of cytoplasmic foci counted per cell from A, C and E. Data represent counts from at least 30 cells, n=3 or 4. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.


Figure 7B . HEK293 cells transfected with YFP - EML4 - GyrB and either untreated ( DMSO ) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . C . Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP - EML4 - GyrB . Data represent counts from > 30 cells , n = 4 . * * * * P < 0 . 0001 in comparison to DMSO by unpaired t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B. HEK293 cells transfected with YFP-EML4-GyrB and either untreated (DMSO) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C. Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP-EML4-GyrB. Data represent counts from >30 cells, n= 4. ****P< 0.0001 in comparison to DMSO by unpaired t test.


Figure 1Phenotypic analysis . Wild - type , uvr8 , and rup1 rup2 seedlings were grown in 1 / 2 MS with or without UV - B ( 1 W / m2 ) for 2 weeks . Images are shown in ( A ) ; scale bars = 5 mm . Phenotypic analysis . Wild - type , uvr8 , and rup1 rup2 seedlings were grown in 1 / 2 MS with or without UV - B ( 1 W / m2 ) for 2 weeks . The lateral root density ( number of lateral roots / length of primary root ) ( B ) and average length of lateral roots ( each plant ) ( C ) of the indicated genotypes were measured . SDs ( n > 8 independent seedlings ) are indicated . Distribution of lateral root primordia stages . Seedlings of the indicated genotypes were grown in 1 / 2 MS treatment with or without UV - B ( 1 W / m2 ) for 2 weeks . SDs ( n > 8 ) are indicated . GUS staining of seedlings expressing DR5p : : GUS transgene in the WT , uvr8 and rup1 rup2 background . Seedlings were grown in 1 / 2 MS with or without UV - B for 2 weeks . Images of lateral root primordia ( E ) and lateral roots ( F ) are shown . Scale bars = 100 and 50 μm , respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Phenotypic analysis. Wild-type, uvr8, and rup1 rup2 seedlings were grown in 1/2 MS with or without UV-B (1 W/m2) for 2 weeks. Images are shown in (A); scale bars = 5 mm.Phenotypic analysis. Wild-type, uvr8, and rup1 rup2 seedlings were grown in 1/2 MS with or without UV-B (1 W/m2) for 2 weeks. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (each plant) (C) of the indicated genotypes were measured. SDs (n > 8 independent seedlings) are indicated.Distribution of lateral root primordia stages. Seedlings of the indicated genotypes were grown in 1/2 MS treatment with or without UV-B (1 W/m2) for 2 weeks. SDs (n > 8) are indicated.GUS staining of seedlings expressing DR5p::GUS transgene in the WT, uvr8 and rup1 rup2 background. Seedlings were grown in 1/2 MS with or without UV-B for 2 weeks. Images of lateral root primordia (E) and lateral roots (F) are shown. Scale bars = 100 and 50 μm, respectively.


Figure 2Phenotypic analysis . Seedlings of the indicated genotypes were grown in 1 / 2 MS with the addition of a series of concentrations of NAA under continuous white light or white plus UV - B ( 1 W / m2 ) light for 2 weeks . The lateral root density ( number of lateral roots / length of primary root ) ( A ) and average length of lateral roots ( B ) of the indicated genotypes were measured . UV - B inhibits the response of Arabidopsis seedlings to exogenously added NAA in a UVR8 - dependent manner , since the response to NAA was not repressed in uvr8 . Seedlings of the indicated genotypes were grown in LD ( 16 - h light / 8 - h dark ) conditions for 5 days , then transplanted to new plates containing 0 . 4 μM NAA and kept in continuous white light or white light plus UV - B ( 1 W / m2 ) for 7 days . Images are shown in ( C ) ; scale bars = 2 mm . The lateral root density ( number of lateral roots / length of primary root ) ( D ) and average length of lateral roots ( E ) of the indicated genotypes were measured .

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Figure 2Phenotypic analysis. Seedlings of the indicated genotypes were grown in 1/2 MS with the addition of a series of concentrations of NAA under continuous white light or white plus UV-B (1 W/m2) light for 2 weeks. The lateral root density (number of lateral roots/length of primary root) (A) and average length of lateral roots (B) of the indicated genotypes were measured.UV-B inhibits the response of Arabidopsis seedlings to exogenously added NAA in a UVR8-dependent manner, since the response to NAA was not repressed in uvr8. Seedlings of the indicated genotypes were grown in LD (16-h light/ 8-h dark) conditions for 5 days, then transplanted to new plates containing 0.4 μM NAA and kept in continuous white light or white light plus UV-B (1 W/m2) for 7 days. Images are shown in (C); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (D) and average length of lateral roots (E) of the indicated genotypes were measured.


Figure 3Phenotypic analysis . WT , GR - UVR8R338A / uvr8 and GR - UVR8W285F / uvr8 seedlings were grown in LD ( 16 - h light / 8 - h dark ) for 5 days , then transplanted to new medium with 0 . 4 μM NAA and with or without 20 μM DEX and kept in white light plus UV - B ( 1 W / m2 ) for 7 days . Images are shown in ( A ) ; scale bars = 2 mm . The lateral root density ( number of lateral roots / length of primary root ) ( B ) and average length of lateral roots ( C ) of indicated genotypes were measured . SDs ( n > 8 ) are indicated . UVR8 inhibited auxin responses under UV - B in a tissue - autonomous way . WT and uvr8 seedlings grown in LD for 5 days were used for reciprocal grafting . Seedlings were kept in LD for 7 days after grafting , then transplanted to new medium containing 0 . 4 μM NAA and kept in UV - B light ( 1 W / m2 ) for 10 days . Images are shown in ( D ) ; scale bar = 2 mm . The lateral root density ( number of lateral roots / length of primary root ) ( E ) and average length of lateral roots ( F ) of indicated genotypes were measured . SDs ( n > 8 ) are indicated .

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Figure 3Phenotypic analysis. WT, GR-UVR8R338A/uvr8 and GR-UVR8W285F/uvr8 seedlings were grown in LD (16-h light/ 8-h dark) for 5 days, then transplanted to new medium with 0.4 μM NAA and with or without 20 μM DEX and kept in white light plus UV-B (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of indicated genotypes were measured. SDs (n > 8) are indicated.UVR8 inhibited auxin responses under UV-B in a tissue-autonomous way. WT and uvr8 seedlings grown in LD for 5 days were used for reciprocal grafting. Seedlings were kept in LD for 7 days after grafting, then transplanted to new medium containing 0.4 μM NAA and kept in UV-B light (1 W/m2) for 10 days. Images are shown in (D); scale bar = 2 mm. The lateral root density (number of lateral roots/length of primary root) (E) and average length of lateral roots (F) of indicated genotypes were measured. SDs (n > 8) are indicated.


Figure 4Transcriptome analysis of gene expression regulated by auxin , UV - B , and UVR8 . ( A ) Heat map of UV - B - , UVR8 - , and auxin - regulated genes . The parameter measured by color key shows the Log - fold change . ( B ) Auxin signaling genes are up - regulated by auxin treatment but down - regulated by UV - B in a UVR8 - dependent manner . Three biological replicates were analyzed and final Log fold - change is shown . Quantitative RT - PCR results showing that UV - B represses the expression of auxin signaling genes . Six - day - old WT seedlings grown in continuous white light were transferred to UV - B light ( 2 W / m2 ) for the indicated period . Error bars are SD of three biological replicates . Quantitative RT - PCR analysis of auxin signaling gene expression in wild - type ( Col - 0 ) , uvr8 , and rup1 rup2 seedlings . Seedlings grown in continuous white light for 5 days were kept in white light ( D ) or transferred to white light plus UV - B ( E ) for 1 day with the addition of 1 μM IAA . Error bars are SD of three biological replicates . qPCR analysis of auxin signaling gene expression in roots of WT , uvr8 , and rup1 rup2 seedlings . Seedlings grown in continuous white light for 12 days were kept in white light ( F ) or transferred to white light plus UV - B ( G ) for 1 day with the addition of 0 . 4 μM NAA . Error bars are SD of three biological replicates .

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Figure 4Transcriptome analysis of gene expression regulated by auxin, UV-B, and UVR8. (A) Heat map of UV-B-, UVR8-, and auxin-regulated genes. The parameter measured by color key shows the Log-fold change. (B) Auxin signaling genes are up-regulated by auxin treatment but down-regulated by UV-B in a UVR8-dependent manner. Three biological replicates were analyzed and final Log fold-change is shown.Quantitative RT-PCR results showing that UV-B represses the expression of auxin signaling genes. Six-day-old WT seedlings grown in continuous white light were transferred to UV-B light (2 W/m2) for the indicated period. Error bars are SD of three biological replicates.Quantitative RT-PCR analysis of auxin signaling gene expression in wild-type (Col-0), uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 5 days were kept in white light (D) or transferred to white light plus UV-B (E) for 1 day with the addition of 1 μM IAA. Error bars are SD of three biological replicates.qPCR analysis of auxin signaling gene expression in roots of WT, uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 12 days were kept in white light (F) or transferred to white light plus UV-B (G) for 1 day with the addition of 0.4 μM NAA. Error bars are SD of three biological replicates.


Figure 5In vitro pull - down assay showing that UV - B treatment promotes the interaction between MYB73 and UVR8 . The bound proteins were eluted and analyzed by probing immunoblots with an anti - His antibody . In vitro pull - down assay showing that UV - B treatment promotes the interaction between MYB77 ( B ) and UVR8 . The bound proteins were eluted and analyzed by probing immunoblots with an anti - His antibody . BiFC assay showing that UVR8 interacts with MYB73 / MYB77 in white light condition . N . benthamiana was co - transformed with MYB73 - nYFP / MYB77 - nYFP and cCFP , nYFP , and UVR8 - cCFP , or MYB73 - nYFP / MYB77 - nYFP and UVR8 - cCFP . BF , bright field . Merge , overlay of the YFP and bright field images . Scale bar = 20 μm . Co - IP assays showing that MYB73 interact with UVR8 in a UV - B - dependent manner in whole seedlings and roots Input : immunoblots showing the levels of UVR8 , MYB73 - TAP in the total protein extract . UVR8 IP : the IP products precipitated by the UVR8 antibody . Total proteins ( Input ) or IP product were probed , on immunoblots , with an anti - UVR8 or anti - Myc antibody , which detects the TAP tag . Co - IP assays showing that MYB77 interact with UVR8 in a UV - B - dependent manner in whole seedlings and roots Input : immunoblots showing the levels of UVR8 , and MYB77 - TAP in the total protein extract . UVR8 IP : the IP products precipitated by the UVR8 antibody . Total proteins ( Input ) or IP product were probed , on immunoblots , with an anti - UVR8 or anti - Myc antibody , which detects the TAP tag .

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Figure 5In vitro pull-down assay showing that UV-B treatment promotes the interaction between MYB73 and UVR8. The bound proteins were eluted and analyzed by probing immunoblots with an anti-His antibody.In vitro pull-down assay showing that UV-B treatment promotes the interaction between MYB77 (B) and UVR8. The bound proteins were eluted and analyzed by probing immunoblots with an anti-His antibody.BiFC assay showing that UVR8 interacts with MYB73/MYB77 in white light condition. N. benthamiana was co-transformed with MYB73-nYFP/MYB77-nYFP and cCFP, nYFP, and UVR8-cCFP, or MYB73-nYFP/MYB77-nYFP and UVR8-cCFP. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bar = 20 μm.Co-IP assays showing that MYB73 interact with UVR8 in a UV-B-dependent manner in whole seedlings and roots Input: immunoblots showing the levels of UVR8, MYB73-TAP in the total protein extract. UVR8 IP: the IP products precipitated by the UVR8 antibody. Total proteins (Input) or IP product were probed, on immunoblots, with an anti-UVR8 or anti-Myc antibody, which detects the TAP tag.Co-IP assays showing that MYB77 interact with UVR8 in a UV-B-dependent manner in whole seedlings and roots Input: immunoblots showing the levels of UVR8 , and MYB77-TAP in the total protein extract. UVR8 IP: the IP products precipitated by the UVR8 antibody. Total proteins (Input) or IP product were probed, on immunoblots, with an anti-UVR8 or anti-Myc antibody, which detects the TAP tag.


Figure 6Phenotypic analysis . Seedlings of the indicated genotypes were grown in LD conditions ( 16 - h light / 8 - h dark ) for 5 days , and then transplanted to new medium containing 0 . 4 μM NAA and kept in continuous white light or white plus UV - B light ( 1 W / m2 ) for 7 days . Images are shown in ( A ) ; scale bars = 2 mm . Lateral root density ( number of lateral roots / length of primary root ) ( B ) and average length of lateral roots ( C ) of the indicated genotypes were measured . SDs ( n > 8 ) are indicated . Quantitative RT - PCR analysis of auxin signaling gene expression in roots of wild type ( Col - 0 ) , uvr8 , myb73 myb77 , and myb73 myb77 uvr8 . ( D ) Results under white light and ( E ) under white plus UV - B light . Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV - B for 1 day with the addition of 0 . 4 μM NAA , and roots were collected to analyze auxin signaling gene expression . Error bars represent SD of three biological replicates .

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Figure 6Phenotypic analysis. Seedlings of the indicated genotypes were grown in LD conditions (16-h light/ 8-h dark) for 5 days, and then transplanted to new medium containing 0.4 μM NAA and kept in continuous white light or white plus UV-B light (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. Lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of the indicated genotypes were measured. SDs (n > 8) are indicated.Quantitative RT-PCR analysis of auxin signaling gene expression in roots of wild type (Col-0), uvr8, myb73 myb77, and myb73 myb77 uvr8. (D) Results under white light and (E) under white plus UV-B light. Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV-B for 1 day with the addition of 0.4 μM NAA, and roots were collected to analyze auxin signaling gene expression. Error bars represent SD of three biological replicates.


Figure 7Electrophoretic mobility - shift assay ( EMSA ) showing that MYB73 / MYB77 binds to the MBSI motif in vitro . Cold probe was added as a competitor . EMSA showing that MYB73 / MYB77 binds to the promoter of IAA19 . Cold IAA19p or IAA19mp ( MBSI motif mutated ) was added as a competitor . EMSA results showing that monomeric UVR8 inhibits the DNA - binding activity of MYB73 ( C ) EMSA results showing that monomeric UVR8 inhibits the DNA - binding activity of MYB77 ( D ) . ChIP - qPCR analysis showed that UV - B inhibits the DNA - binding activity of MYB73 . ChIP - qPCR assays were performed using transgenic seedlings expressing 35S : : MYB73 - TAP , treated with or without UV - B for 5 h before harvesting samples . Chromatin fragments ( ~ 500 bp ) were immunoprecipitated by anti - Myc antibody , and the precipitated DNA was analyzed by qPCR using the primer pairs indicated . The IP / Input ratios are shown with SDs ( n = 3 ) . ChIP - qPCR showing that UV - B inhibits the DNA - binding activity of MYB73 in a UVR8 - dependent manner . The IP / Input ratios are shown with SDs ( n = 3 ) . ChIP - qPCR showing that UV - B inhibits the DNA - binding activity of MYB73 in roots ( G ) in a UVR8 - dependent manner ( H ) . The IP / Input ratios are shown with SDs ( n = 3 ) . ChIP - qPCR showing that UV - B inhibits MYB77 from binding to the indicated promoters in a UVR8 - dependent manner . The IP / Input ratios are shown with SDs ( n = 3 ) .

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Figure 7Electrophoretic mobility-shift assay (EMSA) showing that MYB73/MYB77 binds to the MBSI motif in vitro. Cold probe was added as a competitor.EMSA showing that MYB73/MYB77 binds to the promoter of IAA19. Cold IAA19p or IAA19mp (MBSI motif mutated) was added as a competitor.EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB73(C)EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB77(D).ChIP-qPCR analysis showed that UV-B inhibits the DNA-binding activity of MYB73. ChIP-qPCR assays were performed using transgenic seedlings expressing 35S::MYB73-TAP, treated with or without UV-B for 5 h before harvesting samples. Chromatin fragments (~500 bp) were immunoprecipitated by anti-Myc antibody, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/Input ratios are shown with SDs (n=3). ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in roots (G) in a UVR8-dependent manner (H). The IP/Input ratios are shown with SDs (n=3).ChIP-qPCR showing that UV-B inhibits MYB77 from binding to the indicated promoters in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).


Figure 1C HEK293 FT cells were transfected with expression plasmids for the indicated proteins . The cells were lysed and subjected to immunoprecipitation with anti - Flag antibodies , and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated .

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Figure 1C HEK293 FT cells were transfected with expression plasmids for the indicated proteins. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated.


Figure 2C Proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting . In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs ( E , Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs F , Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein .

O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2C Proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs (E, Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs F, Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


Figure 3In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . J In vitro translation ( IVT ) / biotinylation reactions using Hela cell lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . Bottom , the indicated proteins were prepared by in vitro translation using Hela cell lysates and analyzed by αFlag western blotting .

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Figure 3In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.J In vitro translation (IVT)/biotinylation reactions using Hela cell lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein. Bottom, the indicated proteins were prepared by in vitro translation using Hela cell lysates and analyzed by αFlag western blotting.


Figure 4C - F Mouse kidney was lysed , treated with or without RNase I , and RNA was isolated . The RNA was reverse transcribed with the indicated primers for ( C , E ) Hifa and ( D , F ) Xbp1u mRNA , and then analyzed by Real Time PCR for disomes using the indicated primers . ∆ CT = ( ∆ CT = CT - RNase - CT + RNase ) . Biological replicates ( from three males ) , with mean ± SD are shown . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed t - test .

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Figure 4C-F Mouse kidney was lysed, treated with or without RNase I, and RNA was isolated. The RNA was reverse transcribed with the indicated primers for (C,E) Hifa and (D,F) Xbp1u mRNA, and then analyzed by Real Time PCR for disomes using the indicated primers. ∆CT = (∆CT = CT-RNase - CT+RNase). Biological replicates (from three males), with mean ± SD are shown. * P < 0.05, ** P < 0.01, by unpaired two-tailed t-test.


Figure 5A HEK293 FT cells were transfected with expression plasmids for the indicated proteins . The cells were lysed and subjected to immunoprecipitation with anti - Flag antibodies , and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated . ZFmut = C36S / C42S . B In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated substrate . Bottom , the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting . C HEK293 FT cells were transfected with expression plasmids for the indicated proteins . Tib = Tibetan . The cells were lysed and subjected to immunoprecipitation with anti - Flag antibodies , and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated . E In vitro translation / biotinylation reactions using reticulocyte lysates and construct 1 ( left ) or construct 7 ( right ) . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated substrate . Bottom , the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting .

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Figure 5A HEK293 FT cells were transfected with expression plasmids for the indicated proteins. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated. ZFmut = C36S/C42S.B In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated substrate. Bottom, the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.C HEK293 FT cells were transfected with expression plasmids for the indicated proteins. Tib = Tibetan. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated.E In vitro translation/biotinylation reactions using reticulocyte lysates and construct 1 (left) or construct 7 (right). Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated substrate. Bottom, the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.


Figure 1A 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 . 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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Figure 1A 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.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.


Figure 2Changes 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 . 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 ) . 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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Figure 2Changes 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.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).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.


Figure 3B 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 . 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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Figure 3B 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.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.


Figure 4Changes 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 . 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 . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Changes 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.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.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 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 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.


Figure 5A 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 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 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 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A 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 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 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 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 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 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 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).


Figure 6KGN 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 ) . 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 population at S phase ( C and D ) 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 ) . Efficient silencing of FOXL2 using specific siRNAs was confirmed by western blotting . E , F KGN cells were transfected with control siRNA or siFOXL2 for 24 h . Then , KGN cells were further transfected with control miRNA or miR - 1236 ( E ) or with control anti - miRNA or anti - miR - 1236 ( F ) for 48 h , and Transwell - migration assays were performed . The migrated cells were imaged under a bright - field microscope ( 100 × magnification , scale bar = 100 µm ) . The results are from three independent experiments and represent fold - changes in the average number of cells / field ( mean ± SEM ) . Different letters denote statistically significant differences ( p < 0 . 01 ; Student - Newman - Keuls test ) . G , H COV434 cells were transfected with miR - control or miR - 1236 for 48 h , after which cell viabilities ( G ) and migration abilities ( H ) were measured . The data are presented as the mean ± SEM of three independent experiments . Immunoblots showing no change in FOXL2 protein are presented in the top panel , and images of migrated cells are presented in the right panel . The migrated cells were imaged under a bright - field microscope ( 100 × magnification , scale bar = 100 µm ) . I - K The properties of miR - 1236 - / + and miR - 1236 - / - KGN cells versus control KGN cells were assessed by measuring cell viability ( I ) , cell proliferation ( J ) , and cell migration ( K ) . The data are presented as the mean ± SEM of three independent experiments . The migrated cells were imaged under a bright - field microscope ( 100 × magnification ; top of Fig . 6K ) . Scale bar = 100 µm . L No difference in the cell - migration activities of control and two independent miR - 1236 - / - ( # 1 and # 2 ) COV434 cell lines . The migrated cells were imaged under a bright - field microscope ( 100 × magnification , scale bar = 100 µm ; top ) and the results ( bottom ) represent fold - changes in the average number of cells / field . The data are presented as the mean ± SEM of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6KGN 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).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 population at S phase (C and D) 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). Efficient silencing of FOXL2 using specific siRNAs was confirmed by western blotting.E, F KGN cells were transfected with control siRNA or siFOXL2 for 24 h. Then, KGN cells were further transfected with control miRNA or miR-1236 (E) or with control anti-miRNA or anti-miR-1236 (F) for 48 h, and Transwell-migration assays were performed. The migrated cells were imaged under a bright-field microscope (100 × magnification, scale bar = 100 µm). The results are from three independent experiments and represent fold-changes in the average number of cells/field (mean ± SEM). Different letters denote statistically significant differences (p < 0.01; Student-Newman-Keuls test).G, H COV434 cells were transfected with miR-control or miR-1236 for 48 h, after which cell viabilities (G) and migration abilities (H) were measured. The data are presented as the mean ± SEM of three independent experiments. Immunoblots showing no change in FOXL2 protein are presented in the top panel, and images of migrated cells are presented in the right panel. The migrated cells were imaged under a bright-field microscope (100 × magnification, scale bar = 100 µm).I-K The properties of miR-1236-/+ and miR-1236-/- KGN cells versus control KGN cells were assessed by measuring cell viability (I), cell proliferation (J), and cell migration (K). The data are presented as the mean ± SEM of three independent experiments. The migrated cells were imaged under a bright-field microscope (100 × magnification; top of Fig. 6K). Scale bar = 100 µm.L No difference in the cell-migration activities of control and two independent miR-1236-/- (#1 and #2) COV434 cell lines. The migrated cells were imaged under a bright-field microscope (100× magnification, scale bar = 100 µm; top) and the results (bottom) represent fold-changes in the average number of cells/field. The data are presented as the mean ± SEM of three independent experiments.


Figure 7The effect of miR - 1236 KO on AGCT metastasis was assessed , using an in vivo xenograft mice model . ( A ) Representative images of tumor nodules ( white arrows ) formed in the intestines of nude mice xenografted with control or miR - 1236 - / - KGN cells are shown ( left and right ; scale bar = 5 mm ) . Hematoxylin and eosin staining confirmed the pathological characteristics of the metastasized GCT nodules ( middle , black arrows ; scale bar = 50 µm ) . The black dashed circles are metastasized nodules of xenografted KGN cells found in mouse intestines . ( B ) The number of tumor nodules formed in the intestines was counted in control ( n = 8 ) and miR - 1236 - / - ( n = 8 ) mice . ( C ) Allele - specific real - time RT - PCR analysis of the WT and 402C > G variant FOXL2 mRNAs was performed using RNA extracted from tumor nodules from control or miR - 1236 - / - mice . The p values were analysed by unpaired , two - tailed Student ' s t - test ( * * p < 0 . 01 , * * * p < 0 . 001 ) . D - F Box - and - whisker plots showing the relative expression of miR - 1236 ( D ) , variant FOXL2 mRNA ( E ) , and WT FOXL2 mRNA ( F ) , respectively , in 32 patients with non - metastasized AGCTs and 14 patients with metastasized AGCTs . X - axis indicates patient subgroups depending on whether they exhibit metastasized AGCTs ( meta ) or not ( non - meta ) . The relative miR - 1236 levels were measured using a TaqMan ® microRNA RT - qPCR assay , with expression normalized to RNU6B . The levels of 402C > G or WT FOXL2 mRNA were determined by real - time RT - PCR , and the data were normalized to paired - gDNA levels . The relative levels of miR - 1236 and FOXL2 mRNAs were quantified by setting the levels of AGCT # 1 to 1 . Real - time RT - PCR was performed in triplicate for each specimen . 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 . Comparisons between groups were performed using Student ' s t - tests , and p values are presented . G , H The estimated regression line superimposed on the scatter plot of miR - 1236 levels with 402C > G FOXL2 mRNA ( G ) or WT FOXL2 mRNA ( H ) in AGCT samples ( n = 46 ) is shown , along with correlation coefficient ( r ) and p values .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7The effect of miR-1236 KO on AGCT metastasis was assessed, using an in vivo xenograft mice model. (A) Representative images of tumor nodules (white arrows) formed in the intestines of nude mice xenografted with control or miR-1236-/- KGN cells are shown (left and right; scale bar = 5 mm). Hematoxylin and eosin staining confirmed the pathological characteristics of the metastasized GCT nodules (middle, black arrows; scale bar = 50 µm). The black dashed circles are metastasized nodules of xenografted KGN cells found in mouse intestines. (B) The number of tumor nodules formed in the intestines was counted in control (n = 8) and miR-1236-/- (n = 8) mice.(C) Allele-specific real-time RT-PCR analysis of the WT and 402C>G variant FOXL2 mRNAs was performed using RNA extracted from tumor nodules from control or miR-1236-/- mice. The p values were analysed by unpaired, two-tailed Student's t-test (**p < 0.01, ***p < 0.001).D-F Box-and-whisker plots showing the relative expression of miR-1236 (D), variant FOXL2 mRNA (E), and WT FOXL2 mRNA (F), respectively, in 32 patients with non-metastasized AGCTs and 14 patients with metastasized AGCTs. X- axis indicates patient subgroups depending on whether they exhibit metastasized AGCTs (meta) or not (non-meta). The relative miR-1236 levels were measured using a TaqMan® microRNA RT-qPCR assay, with expression normalized to RNU6B. The levels of 402C>G or WT FOXL2 mRNA were determined by real-time RT-PCR, and the data were normalized to paired-gDNA levels. The relative levels of miR-1236 and FOXL2 mRNAs were quantified by setting the levels of AGCT #1 to 1. Real-time RT-PCR was performed in triplicate for each specimen. 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. Comparisons between groups were performed using Student's t-tests, and p values are presented.G, H The estimated regression line superimposed on the scatter plot of miR-1236 levels with 402C>G FOXL2 mRNA (G) or WT FOXL2 mRNA (H) in AGCT samples (n = 46) is shown, along with correlation coefficient (r) and p values.


Figure 1B . Cell lines generated from WT , null , and mutant p53 esophagi were assessed by Western blot analysis for p53 protein expression . Relative intensity densitometry of Rab11 - FIP1 results are shown at the bottom . C . Organoids generated from WT , mutant , and null p53 cell lines . Scale bar : 100μm . D . Quantification of the portion of organoids exhibiting a solid , intermediate or stratified morphology ( > 37 organoids from 2 - 3 cell lines per group ) . E - G . Cells were subcutaneously injected in the lower flanks of nude mice ( n = 4 flanks / cell line . WT p53 2 cell lines , Mutant p53 - 3 cell lines , p53 null - 3 cell lines ) . E . The number of tumors formed in each group was quantified . F . Tumor growth was measured at the indicated time points post - implantation . Graph represents total tumor size per group . G . Average tumor weight following dissection ( n = 2 biological replicates for WT p53 cell lines , n = 3 biological replicates for mutant p53 cell lines , n = 3 biological replicates for p53 null cell lines , n = 4 technical replicates injected per cell line , calculated as average tumor weight per cell line ) . Error bars represent ± S . E . M . * p = 0 . 02 Mutant p53 vs WT p53 , * p = 0 . 02 Mutant p53 vs Null p53 ( One - way Anova ) .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Cell lines generated from WT, null, and mutant p53 esophagi were assessed by Western blot analysis for p53 protein expression. Relative intensity densitometry of Rab11-FIP1 results are shown at the bottom.C. Organoids generated from WT, mutant, and null p53 cell lines. Scale bar: 100μm. D. Quantification of the portion of organoids exhibiting a solid, intermediate or stratified morphology (> 37 organoids from 2-3 cell lines per group). E-G. Cells were subcutaneously injected in the lower flanks of nude mice (n= 4 flanks/cell line. WT p53 2 cell lines, Mutant p53-3 cell lines, p53 null-3 cell lines). E. The number of tumors formed in each group was quantified. F. Tumor growth was measured at the indicated time points post-implantation. Graph represents total tumor size per group. G. Average tumor weight following dissection (n=2 biological replicates for WT p53 cell lines, n=3 biological replicates for mutant p53 cell lines, n=3 biological replicates for p53 null cell lines, n=4 technical replicates injected per cell line, calculated as average tumor weight per cell line). Error bars represent ±S.E.M. *p=0.02 Mutant p53 vs WT p53, *p=0.02 Mutant p53 vs Null p53 (One-way Anova).


Figure 2B . Relative fold change in expression level of endocytic recycling genes in invasive vs . non - invasive cells , and in mutant vs . WT p53 cells based on microarray and RNA - seq analysis , respectively . C . qPCR analysis of recycling - related gene expression in mutant p53 relative to WT p53 cells ( red and black bars , respectively ) . Graphs represent mean ± SEM ( n = 3 biological replicates for Rab11 - FIP1 , Rab25 , Myo5b and Clic3 ; n = 2 biological replicates for Rab5 ) . Rab11 - FIP1 * p = 0 . 0109 , Rab25 * * p = 0 . 0021 , Myo5b * * * p = 0 . 0001 , Clic3 * * p = 0 . 0051 , Rab5 no significant differences ( n . s . ) . ( Unpaired t - test ) .

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Figure 2B. Relative fold change in expression level of endocytic recycling genes in invasive vs. non-invasive cells, and in mutant vs. WT p53 cells based on microarray and RNA-seq analysis, respectively.C. qPCR analysis of recycling-related gene expression in mutant p53 relative to WT p53 cells (red and black bars, respectively). Graphs represent mean ± SEM (n=3 biological replicates for Rab11-FIP1, Rab25, Myo5b and Clic3; n=2 biological replicates for Rab5). Rab11-FIP1 *p=0.0109, Rab25 **p=0.0021, Myo5b ***p=0.0001, Clic3 **p=0.0051, Rab5 no significant differences (n.s.). (Unpaired t-test).