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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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 .

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

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


G ) Control ( TFG - WT ) ' 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 .

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

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


HeLa 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 .

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

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 .

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

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 .

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

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


Seedling phenotype under GA treatment . After germination , Five - day - old seedlings with 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 .

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

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 .

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

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 ) .

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

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 .

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

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 .

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

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 .

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 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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

Immunoblotting of 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 .

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

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 .

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

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 .

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 O O O O O O O O O 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 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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 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

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 .

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

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 .

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

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 .

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

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 .

O O O O 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

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 .

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

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 ) .

O B-MEASURED_VAR 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 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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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).


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 . population at S phase ( C D ) 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 ) . Efficient silencing of FOXL2 using specific siRNAs was confirmed by western blotting .

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

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 were transfected with control siRNA or siFOXL2 for 24 h . Then , KGN were further transfected with control miRNA or miR - 1236 ( E ) or with control anti - miRNA or anti - miR - 1236 ( F ) for 48 h , Transwell - migration assays were performed . migrated were imaged under a bright - field microscope ( 100 × magnification , scale bar = 100 µm ) . results are from three independent experiments represent fold - changes in average number of / field ( mean ± ) . Different letters denote statistically significant differences ( p < 0 . 01 ; Student - Newman - Keuls ) .

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

E, 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 were transfected with miR - control or miR - 1236 for 48 h , after which viabilities ( G ) migration abilities ( H ) were measured . data are presented as mean ± of three independent experiments . Immunoblots showing no change in FOXL2 are presented in top panel , of migrated are presented in right panel . migrated were imaged under a bright - field microscope ( 100 × magnification , scale bar = 100 µm ) .

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

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 properties of miR - 1236 - / + miR - 1236 - / - KGN versus control KGN were assessed by measuring viability ( I ) , proliferation ( J ) , migration ( K ) . data are presented as mean ± of three independent experiments . migrated were imaged under a bright - field microscope ( 100 × magnification ; top of Fig . 6K ) . Scale bar = 100 µm .

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

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 - migration activities of control two independent miR - 1236 - / - ( # 1 # 2 ) COV434 lines . migrated were imaged under a bright - field microscope ( 100 × magnification , scale bar = 100 µm ; top ) results ( bottom ) represent fold - changes in average number of / field . data are presented as mean ± of three independent experiments .

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

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.


effect of miR - 1236 KO on AGCT metastasis was assessed , using an in vivo xenograft mice model . ( A ) Representative of nodules ( arrows ) formed in intestines of nude mice xenografted with control or miR - 1236 - / - KGN are shown ( left right ; scale bar = 5 mm ) . Hematoxylin eosin confirmed pathological characteristics of metastasized GCT nodules ( middle , black arrows ; scale bar = 50 µm ) . black dashed circles are metastasized nodules of xenografted KGN found in mouse intestines . ( B ) number of nodules formed in intestines was counted in control ( n = 8 ) miR - 1236 - / - ( n = 8 ) mice .

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

The 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 of WT 402C > G variant FOXL2 mRNAs was performed using extracted from nodules from control or miR - 1236 - / - mice . p values were analysed by unpaired , two - Student ' s t - ( * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

(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 - - whisker plots showing relative expression of miR - 1236 ( D ) , variant FOXL2 mRNA ( E ) , WT FOXL2 mRNA ( F ) , respectively , in 32 with non - metastasized AGCTs 14 with metastasized AGCTs . X - axis indicates subgroups depending on whether they exhibit metastasized AGCTs ( meta ) or not ( non - meta ) . relative miR - 1236 were measured using a TaqMan ® microRNA RT - qPCR , with expression normalized to RNU6B . of 402C > G or WT FOXL2 mRNA were determined by real - time RT - PCR , data were normalized to paired - gDNA . relative of miR - 1236 FOXL2 mRNAs were quantified by setting of AGCT # 1 to 1 . Real - time RT - PCR was performed in triplicate for each specimen . 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 . Comparisons between groups were performed using Student ' s t - , p values are presented .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 estimated regression superimposed on scatter plot of miR - 1236 with 402C > G FOXL2 mRNA ( G ) or WT FOXL2 mRNA ( H ) in AGCT samples ( n = 46 ) is shown , along with correlation coefficient ( r ) p values .

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

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.


( A ) Dose - response curves ( normalized growth rate as a function of drug ) for different . Growth rate was measured via optical density measurements over time used : Trimethoprim ( TMP ) , tetracycline ( TET ) , chloramphenicol ( CHL ) , ciprofloxacin ( CPR ) , lincomycin ( LIN ) , nitrofurantoin ( NIT ) , mecillinam ( MEC ) . TMP dose - response curve ( blue ) is by far shallowest . Lines are fits of Hill function $ \ frac { g \ left ( c \ right ) } { g ( 0 ) } = \ frac { 1 } { 1 + \ left ( \ frac { c } { IC _ { 50 } } \ right ) ^ { n } } \ $ to data . Drug concentrations were arbitrarily rescaled to better visualize dose - response curve steepness ; Error bars show standard deviation of 12 biological replicates .

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

(A) Dose-response curves (normalized growth rate as a function of drug concentration) for different antibiotics. Growth rate was measured via optical density measurements over time Antibiotics used: Trimethoprim (TMP), tetracycline (TET), chloramphenicol (CHL), ciprofloxacin (CPR), lincomycin (LIN), nitrofurantoin (NIT), and mecillinam (MEC). The TMP dose-response curve (dark blue) is by far the shallowest. Lines are fits of the Hill function $\frac{g\left( c \right)}{g(0)} = \frac{1}{1 + \left( \frac{c}{IC_{50}} \right)^{n}}\ $to the data. Drug concentrations were arbitrarily rescaled to better visualize dose-response curve steepness; Error bars show standard deviation of 12 biological replicates.


( C ) Density scatterplot showing growth response to TMP versus normalized drug - free growth rate for 3 , 913 gene deletion strains these are essentially viable gene deletion strains in E . coli , no selection of strains was made . These gene deletion strains exhibit diverse growth rates , offering an unbiased way to relation between drug - free growth rate response to . Response is defined as growth rate in presence of TMP normalized to drug - free growth rate of respective deletion . TMP was used at a fixed that inhibits wild type growth by about 30 % Spearman correlation coefficient ρs is shown .

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

(C) Density scatterplot showing growth response to TMP versus normalized drug-free growth rate for 3,913 gene deletion strains these are essentially all viable gene deletion strains in E. coli, no selection of strains was made. These gene deletion strains exhibit diverse growth rates, offering an unbiased way to test the relation between the drug-free growth rate and the response to antibiotics. Response is defined as growth rate in the presence of TMP normalized to the drug-free growth rate of the respective deletion strain. TMP was used at a fixed concentration that inhibits wild type growth by about 30% Spearman correlation coefficient ρs is shown.


( D ) Bar chart showing negative Spearman correlation coefficients − ρs compared across Error bars show bootstrap standard error of ρs . TMP ( blue ) exhibits by far strongest negative correlation , indicating existence of a particularly strong growth - mediated negative feedback loop for this antibiotic .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar chart showing negative Spearman correlation coefficients  − ρs compared across antibiotics Error bars show bootstrap standard error of ρs. TMP (blue) exhibits by far the strongest negative correlation, indicating the existence of a particularly strong growth-mediated negative feedback loop for this antibiotic.


( A ) Growth rate under glucose limitation achieved by adding non - metabolizable structural glucose analog α - methyl glucoside ( αMG ) at different ratios to glucose in a minimal medium Data information : Error bars in A , show standard deviation from 6 , 12 , 18 biological replicates , respectively ; day - to - day reproducibility of growth rate measurements is

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

(A) Growth rate under glucose limitation achieved by adding the non-metabolizable structural glucose analog α-methyl glucoside (αMG) at different ratios to glucose in a minimal medium Data information: Error bars in A, show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of growth rate measurements is high


( B ) Normalized growth rate ( gray scale ) from a checkerboard in a two - dimensional gradient of TMP αMG . Dashed black shows contour of 90 % growth inhibition ( IC90 ) . Red arrow shows increase in IC90 as growth is lowered . Inset : Normalized growth rate as a function of TMP along column marked in blue .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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) Normalized growth rate (gray scale) from a checkerboard assay in a two-dimensional concentration gradient of TMP and αMG. Dashed black line shows contour line of 90% growth inhibition (IC90 line). Red arrow shows increase in IC90 as growth is lowered. Inset: Normalized growth rate as a function of TMP concentration along the column marked in blue.


( C ) Fold - change in IC90 at αMG / glucose 2 . 5 in assays as in B for different Lowering growth rate increases IC90 for TMP but not for other . Data information : Error bars in C show standard deviation from three neighboring αMG / glucose

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fold-change in IC90 at αMG/glucose ratio 2.5 in assays as in B for different antibiotics Lowering growth rate increases IC90 for TMP but not for other antibiotics. Data information: Error bars in C show standard deviation from three neighboring αMG/glucose ratios


( D ) Growth rate in rich medium ( LB ) under different of overexpression of a gratuitous from a T5 - lac promoter ; overexpression burden is controlled by IPTG Data information : Error bars in D , show standard deviation from 6 , 12 , 18 biological replicates , respectively ; day - to - day reproducibility of growth rate measurements is

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth rate in rich medium (LB) under different levels of overexpression of a gratuitous protein from a T5-lac promoter; overexpression burden is controlled by IPTG concentration Data information: Error bars in D, show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of growth rate measurements is high


( E ) As B but for growth rate reduction by overexpression in a two - dimensional gradient of TMP IPTG .

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

(E) As B but for growth rate reduction by protein overexpression in a two-dimensional concentration gradient of TMP and IPTG.


( F ) Fold - change in IC90 at 1 . 25 mM IPTG in assays as in E for different Overexpression of unnecessary increases IC90 for TMP by almost five - fold ; no comparable increase occurs for other . Data information : Error bars in F show standard deviation from IPTG concentrations centered at 2 . 5 1 . 25 mM , respectively . CHL was not used in overexpression in F since plasmid used for overexpression has a CHL - resistance

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

(F) Fold-change in IC90 at 1.25 mM IPTG in assays as in E for different antibiotics Overexpression of unnecessary protein increases IC90 for TMP by almost five-fold; no comparable increase occurs for other antibiotics. Data information: Error bars in F show standard deviation from IPTG concentrations centered at 2.5 and 1.25 mM, respectively. CHL was not used in the protein overexpression assay in F since the plasmid used for overexpression has a CHL-resistance marker


( G ) Growth rate in minimal medium containing different carbon sources Glucose ( GLU ) , fructose ( FRU ) , mannose ( MAN ) , galactose ( GAL ) , glycerol ( GLY ) . Data information : Error bars in show standard deviation from 6 , 12 , 18 biological replicates , respectively ; day - to - day reproducibility of growth rate measurements is

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

(G) Growth rate in minimal medium containing different carbon sources Glucose (GLU), fructose (FRU), mannose (MAN), galactose (GAL), and glycerol (GLY). Data information: Error bars in show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of growth rate measurements is high


( H ) Normalized growth rates ( gray scale ) on different carbon sources ( x - axis ) at different TMP concentrations ( y - axis ) .

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

(H) Normalized growth rates (gray scale) on different carbon sources (x-axis) at different TMP concentrations (y-axis).


( I ) Fold - change in IC90 in assays as in H for different Data information : Error bars in I show standard deviation from three biological replicates .

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

(I) Fold-change in IC90 in assays as in H for different antibiotics Data information: Error bars in I show standard deviation from three biological replicates.


( A ) TMP dose - response curve in minimal medium with glucose as carbon source at lower drug - free growth rate due to glucose limitation , achieved by increasing αMG / glucose from 0 ( black ) to 5 ( gray ) . Glucose limitation results in a steeper dose - response curve . Lines show Hill function fits ( B ) Steepness of TMP dose - response curves ( dose - sensitivity n ) versus drug - free growth rate at different αMG concentrations Numbers next to data points show αMG / glucose . Data information : Growth rate error bars show standard deviation of three replicates ; vertical error bars in B show standard deviation of parameter estimates from Hill function fit .

O O O O O O O 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 B-CONTROLLED_VAR 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

(A) TMP dose-response curve in minimal medium with glucose as carbon source and at lower drug-free growth rate due to glucose limitation, achieved by increasing the αMG /glucose ratio from 0 (black) to 5 (gray). Glucose limitation results in a steeper dose-response curve. Lines show Hill function fits (B) Steepness of TMP dose-response curves (dose-sensitivity n) versus drug-free growth rate at different αMG concentrations Numbers next to data points show αMG/glucose ratio. Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in B show standard deviation of parameter estimates from Hill function fit.


( C , D ) As A , B but for growth limitation by gratuitous overexpression in rich growth medium Inducing overexpression with IPTG at 10 mM ( gray ) steepens dose - response curve compared to no induction ( black ) . Numbers next to data points in D show IPTG in mM . Data information : Growth rate error bars show standard deviation of three replicates ; vertical error bars in D , show standard deviation of parameter estimates from Hill function fit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) As A,B but for growth limitation by gratuitous protein overexpression in rich growth medium Inducing overexpression with IPTG at 10 mM (light gray) steepens the dose-response curve compared to no induction (black). Numbers next to data points in D show IPTG concentration in mM. Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in D, show standard deviation of parameter estimates from Hill function fit.


( E , F ) As A , B but for growth limitation by varying carbon source in a minimal medium Data information : Growth rate error bars show standard deviation of three replicates ; vertical error bars in F show standard deviation of parameter estimates from Hill function fit .

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

(E,F) As A,B but for growth limitation by varying the carbon source in a minimal medium Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in F show standard deviation of parameter estimates from Hill function fit.


( A ) Dependence of growth rate ( black ) folA expression ( green ) on TMP . Schematic : FolA expression was measured using a promoter - GFP reporter inserted at a neutral site in genome Data information : Black in A shows Hill function fit other lines show polynomial fits of second order ( A to guide eye . Horizontal dotted green shows folA expression level in absence of TMP . Error bars show standard deviation of three biological replicates .

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

(A) Dependence of growth rate (black) and folA expression (green) on TMP concentration. Schematic: FolA expression was measured using a promoter-GFP reporter inserted at a neutral site in the genome Data information: Black line in A shows Hill function fit other lines show polynomial fits of second order (A to guide the eye. Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.


( B ) Growth rate ( black ) folA expression ( green ) in absence of TMP at different growth rates achieved by different ratios of αMG / glucose . Data information : other lines show polynomial fits of second order B ) to guide eye . Horizontal dotted green shows folA expression level in absence of TMP . Error bars show standard deviation of three biological replicates .

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

(B) Growth rate (black) and folA expression (green) in the absence of TMP at different growth rates achieved by different ratios of αMG/glucose. Data information: other lines show polynomial fits of second order B) to guide the eye. Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.


( C ) Scatterplot of folA expression level with growth rate across combinations of TMP concentrations αMG / glucose ratios Pearson ' s correlation coefficient ρ p - value from two - sided t - are shown . FolA expression is largely determined by growth rate , suggesting that this regulation is growth rate - dependent not mediated by a specific molecular mechanism . Data information : ; other lines show polynomial fits of first ( C ) order to guide eye . Horizontal dotted green shows folA expression level in absence of TMP . Error bars show standard deviation of three biological replicates .

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

(C) Scatterplot of folA expression level with growth rate across all combinations of TMP concentrations and αMG/glucose ratios Pearson's correlation coefficient ρ and p-value from two-sided t-test are shown. FolA expression is largely determined by growth rate, suggesting that this regulation is growth rate-dependent and not mediated by a specific molecular mechanism. Data information: ; other lines show polynomial fits of first (C) order to guide the eye. Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.


( D ) Dependence of folA expression on TMP at four different αMG / glucose ratios ( 0 , 2 . 5 , 5 , 10 as shown ) . Darker green indicates greater αMG / glucose . FolA expression converges approximately to same level at high TMP concentrations . Data information : Horizontal dotted green shows folA expression level in absence of TMP . Error bars show standard deviation of three biological replicates .

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

(D) Dependence of folA expression on TMP concentration at four different αMG/glucose ratios (0, 2.5, 5, and 10 as shown). Darker green indicates greater αMG/glucose ratio. FolA expression converges approximately to the same level at high TMP concentrations. Data information: Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.


( B ) Growth rate as a function of TMP for different paths through IPTG - TMP space Constant FolA is shown in black inverted FolA regulation in red . Wild - type dose - response curve ( blue ; is shown for comparison . ( C ) Steepness of dose response curve ( quantified as dose - sensitivity n ) for three cases in B . Inset : Normalized FolA expression level as a function of TMP for three cases in B ; WT ( blue ) colors as in bar chart in B . Data information : Error bars in B show standard deviation of measured growth rates used for interpolating values shown ; entire experiment was replicated once Error bars in C show standard deviation of parameter estimates from Hill function fit .

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

(B) Growth rate as a function of TMP concentration for different paths through IPTG-TMP concentration space Constant FolA is shown in black and inverted FolA regulation in red. Wild-type dose-response curve (blue line; is shown for comparison. (C) Steepness of the dose response curve (quantified as dose-sensitivity n) for the three cases in B. Inset: Normalized FolA expression level as a function of TMP concentration for the three cases in B; WT (blue) colors as in the bar chart and in B. Data information: Error bars in B show standard deviation of the measured growth rates used for interpolating the values shown; the entire experiment was replicated once Error bars in C show standard deviation of parameter estimates from Hill function fit.


( C ) Heat map demonstration of genes that were mostly enriched in pathway of pluripotency of . color bar showed relative - seq signal ( Z - score of normalized FPKM ) .

O O O O O O O O O O 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) Heat map demonstration of genes that were mostly enriched in the pathway of pluripotency of stem cells. The color bar showed the relative RNA-seq signal (Z-score of normalized FPKM).


Histological examinations of types in ( F ) K versus KrasG12D ; NanogL / L ( KN ) tissues collected form indicated mice analyzed using Haematoxylin Eosin ( H & E ) . Representative H & E are shown . Scale bar , 500 μm ( top ) , 50 μm ( bottom ) .

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

Histological examinations of tumor types in (F) K versus KrasG12D; NanogL/L (KN) Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom).


( G ) qPCR of stomach - restricted gene set between KL ADC KLN mucinous sections ( n = 3 or 4 per group ) . Results were shown in a box whisker plot with minimum value , first quartile , median , third quartile maximum value . box represented 50 % of central data , with a inside represented median . significance was calculated by two - unpaired Student ' s t - with Welch ' s correction . * p = 0 . 0260 ( Hnf4α ) , * p = 0 . 0275 ( Vsig1 ) , * p = 0 . 0122 ( Gkn3 ) , * p = 0 . 0324 ( Ctse ) , * * p = 0 . 0084 ( Onecut2 ) .

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

(G) qPCR analysis of stomach-restricted gene set between KL ADC and KLN mucinous tumor sections (n=3 or 4 per group). Results were shown in a box and whisker plot with the minimum value, first quartile, median, third quartile and maximum value. The box represented the 50% of the central data, with a line inside represented the median. The significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p=0.0260 (Hnf4α), *p=0.0275 (Vsig1), *p=0.0122 (Gkn3), *p=0.0324 (Ctse), **p=0.0084 (Onecut2).


( D ) Western blot for NANOG TUBULIN in 7 paired human normal lungs ( N ) ADC samples ( T ) human ESCs . Mr , relative molecular mass . kDa , killoDalton .

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

(D) Western blot for NANOG and TUBULIN in 7 paired human normal lungs (N) and ADC samples (T) and human ESCs. Mr, relative molecular mass. kDa, killoDalton.


( H ) Statistic of LKB1 expression in ADC IMA . tissues were analyzed by graded as low , medium , high expression . Significance was calculated by χ2 for trend .

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

(H) Statistic analysis of LKB1 expression in ADC and IMA. The tumor tissues were analyzed by IHC staining and graded as low, medium, high expression. Significance was calculated by χ2 test for trend.


( I ) expression grade of of NANOG , NKX2 - 1 , LKB1 in 76 human IMA ( 1 sample / row ) .

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

(I) The expression grade of IHC staining of NANOG, NKX2-1, LKB1 in 76 human IMA (1 sample/row).


( B - D ) H & E of tumors , ( B ) of NICD ( C ) HES1 ( D ) in KL ADC KLN IMA . Representative were shown . Scale bar , 50 μm . Results were shown as mean ± SD . numbers of tumors analyzed over KL ADC KLN IMA were 15 , 18 for NICD , 16 , 15 for HES1 . Significance was calculated by two - unpaired Student ' s t - with Welch ' s correction . * p = 0 . 0188 ( C ) , * * p = 0 . 0040 ( D ) .

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

(B-D) H&E staining of tumors, IHC staining (B) and quantification of NICD (C) and HES1 (D) in KL ADC and KLN IMA. Representative images were shown. Scale bar, 50 μm. Results were shown as mean ± SD. The numbers of tumors analyzed over KL ADC and KLN IMA were 15, 18 for NICD, 16, 15 for HES1. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p=0.0188 (C), **p=0.0040 (D).


( G , H ) Statistical of NICD ( G ) HES1 ( H ) expression in lung sections of KLN mice following vehicle or LY - 411575 treatment . numbers of tumors analyzed over vehicle LY - 411575 groups were 22 , 19 for NICD , 22 , 22 for HES1 . Results were shown as mean ± SD . Significance was calculated by two - unpaired Student ' s t - with Welch ' s correction . * * * * p < 0 . 0001 .

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

(G, H) Statistical analysis of NICD (G) and HES1 (H) expression in lung sections of KLN mice following vehicle or LY-411575 treatment. The numbers of tumors analyzed over vehicle and LY-411575 groups were 22, 19 for NICD, 22, 22 for HES1. Results were shown as mean ± SD. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. ****p<0.0001.


( I ) H & E of NICD HES1 in lung tissues of vehicle LY - 411575 groups . Scale bar , 50 μm . ( J ) H & E , AB PAS in lung tumors of vehicle LY - 411575 groups . Scale bar , 50 μm .

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

(I) H&E and IHC staining of NICD and HES1 in lung tumor tissues of vehicle and LY-411575 groups. Scale bar, 50 μm. (J) H&E, AB and PAS staining in lung tumors of vehicle and LY-411575 groups. Scale bar, 50 μm.