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 .

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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 ) .

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(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 .

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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 .

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(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 .

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(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 .

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(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

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(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 .

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(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

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(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

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(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 .

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(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

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(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

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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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 ) .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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.


( I , J ) Statistical of Ki67 ( I ) CC3 ( J ) activity in ADC IMA lung sections of vehicle phenformin ( Phen ) groups . numbers of tumors analyzed over vehicle group phenformin groups for ADC IMA were 17 , 8 , 9 for Ki67 , 10 , 10 , 7 for CC3 . Results were shown as mean ± . Significance was calculated by one - way ANOVA with Dunnett ' s multiple comparisons . * * p = 0 . 0010 ( I ) , * * * p = 0 . 0009 ( I ) , * p = 0 . 0427 ( J ) .

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(I, J) Statistical analysis of Ki67 (I) and CC3 (J) activity in ADC and IMA lung sections of vehicle and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17, 8, 9 for Ki67, 10, 10, 7 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0010 (I), ***p=0.0009 (I), *p=0.0427(J).


( E , F ) Statistical of Ki67 ( E ) CC3 ( F ) reactivity in lung sections of KLN mice following vehicle , LY - 411575 treatment or a combinational therapy of phenformin LY - 411575 . numbers of tumors analyzed over vehicle , phenformin combinational therapy groups were 20 , 30 , 13 for Ki67 , 14 , 11 , 9 for CC3 . Results were shown as mean ± . Significance was calculated by one - way ANOVA with Dunnett ' s multiple comparisons . * * p = 0 . 0014 ( E ) , * * * p = 0 . 0001 ( F ) .

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(E, F) Statistical analysis of Ki67 (E) and CC3 (F) reactivity in lung sections of KLN mice following vehicle, LY-411575 treatment or a combinational therapy of phenformin and LY-411575. The numbers of tumors analyzed over vehicle, phenformin and combinational therapy groups were 20, 30, 13 for Ki67, 14, 11, 9 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0014 (E), ***p=0.0001 (F).


B - Western blot verifying expression of CTD variants RPB1 - 52R RPB1 - 25R in U2OS in whole extracts . U1 snRNP was used as a loading control .

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B- Western blot verifying the expression of the CTD variants RPB1-52R and RPB1-25R in U2OS cells in whole cell extracts. U1 snRNP was used as a loading control.


C - Changes in synthesis upon CTD shortening . MA plot showing synthesis changes in TT - seq datasets upon CTD shortening in U2OS in steady state conditions . RPB1 - 52R are used as a control data was normalized using spike - in . 16214 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red . 473 genes were significantly upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) 704 genes were significantly downregulated ( adjusted p value < 0 . 05 , log2 fold change ≤ 1 ) .

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C- Changes in RNA synthesis upon CTD shortening. MA plot showing RNA synthesis changes in TT-seq datasets upon CTD shortening in U2OS cells in steady state conditions. RPB1-52R cells are used as a control and the data was normalized using spike-in counts. 16214 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red. 473 genes were significantly upregulated (adjusted p value < 0.05, log2 fold change >=1) and 704 genes were significantly downregulated (adjusted p value < 0.05, log2 fold change ≤1).


D - Box plot of estimated synthesis rates of expressed RefSeq transcripts ( 12014 transcripts ) on TT - seq - seq datasets in RPB1 - 52R RPB1 - 25R . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

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D- Box plot of estimated RNA synthesis rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


E - Box plot of estimated degradation rates of expressed RefSeq transcripts ( 12014 transcripts ) on TT - seq - seq datasets in RPB1 - 52R RPB1 - 25R . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

E- Box plot of estimated RNA degradation rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


F - Box plot of estimated half - lives of expressed RefSeq transcripts ( 12014 transcripts ) on TT - seq - seq datasets in RPB1 - 52R RPB1 - 25R . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

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F- Box plot of estimated RNA half-lives of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


A - Box plots showing ratios of spliced TT - seq reads over total unspliced spliced TT - seq reads for constitutive 5 ' SSs 3 ' SSs detected in major isoforms in RPB1 - 52R RPB1 - 25R . p value was calculated using Mann - Whitney U . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed . B - D - Box plots showing ratios of spliced TT - seq reads over total unspliced spliced TT - seq reads in RPB1 - 52R RPB1 - 25R for constitutive 5 ' SSs 3 ' SSs detected in major isoforms on intron position in transcript : ( B ) first intron junction , ( C ) intermediate intron junctions ( D ) last intron junctions . p values were calculated using Mann - Whitney U . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

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A- Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads for all constitutive 5'SSs and 3'SSs detected in major RNA isoforms in RPB1-52R and RPB1-25R cells. p value was calculated using Mann-Whitney U test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. B-D- Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads in RPB1-52R and RPB1-25R cells for all constitutive 5'SSs and 3'SSs detected in major RNA isoforms based on intron position in the transcript: (B) first intron junction, (C) intermediate intron junctions and (D) last intron junctions. p values were calculated using Mann-Whitney U test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


E - Differential mRNA isoform usage upon CTD shortening . Scatter plot showing read for mRNA isoforms detected in fraction of RPB1 - 52R RPB1 - 25R using long read sequencing . 47416 isoforms were detected , RPB1 - 52R condition was used as a control . 335 isoforms show differential expression ( p value < 0 . 05 , Fischer ' s exact ) .

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E- Differential mRNA isoform usage upon CTD shortening. Scatter plot showing read counts for mRNA isoforms detected in the chromatin fraction of RPB1-52R and RPB1-25R cells using long read sequencing. 47416 isoforms were detected, RPB1-52R condition was used as a control. 335 isoforms show differential expression (p value < 0.05, Fischer's exact test).


A - CTD shortening leads to a decreased Pol II occupancy in promoter - proximal region . Median coverage plot of PRO - seq signal at genes encoding major isoforms . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

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A- CTD shortening leads to a decreased Pol II occupancy in promoter-proximal region. Median coverage plot of PRO-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


B , C - Pol II pause position remains unchanged upon CTD shortening . Histogram of estimated positions of paused Pol II in RPB1 - 52R RPB1 - 25R at genes encoding major isoforms with constitutive first exon longer than 100 bp .

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B,C- Pol II pause position remains unchanged upon CTD shortening. Histogram of estimated positions of paused Pol II in RPB1-52R and RPB1-25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp.


D - Median coverage plot of TT - seq signal at genes encoding major isoforms in RPB1 - 52R RPB1 - 25R . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

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D- Median coverage plot of TT-seq signal at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


E - Box plot showing productive transcription initiation rate in RPB1 - 52R RPB1 - 25R for genes encoding major isoforms . p value = 0 . 13 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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E- Box plot showing productive transcription initiation rate in RPB1-52R and RPB1-25R cells for genes encoding major isoforms. p value=0.13 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


F - Box plot showing Pol II pausing duration in RPB1 - 52R RPB1 - 25R for genes encoding major isoforms with constitutive first exon longer than 100 bp . p value < 2 . 2e - 16 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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F- Box plot showing Pol II pausing duration in RPB1-52R and RPB1-25R cells for genes encoding major isoforms with constitutive first exon longer than 100 bp. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


G - Median scaled coverage plot of TT - seq signal at genes encoding major isoforms . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

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G- Median scaled coverage plot of TT-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


H - Pol II velocity remains unchanged upon CTD shortening . Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals .

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H- Pol II velocity remains unchanged upon CTD shortening. Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals.


I - Median coverage plot of TT - seq signal centered at PAS at genes encoding major isoforms in RPB1 - 52R RPB1 - 25R . Solid lines represent median signal shaded refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed .

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I- Median coverage plot of TT-seq signal centered at PAS at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.


J - Box plot showing of poly ( A ) tail in RPB1 - 52R RPB1 - 25R measured using Nanopore sequening data . p value < 2 . 2e - 16 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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J - Box plot showing length of the poly(A) tail in RPB1-52R and RPB1-25R cells measured using Nanopore sequening data. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


K - Box plot showing from polyadenylation site ( PAS ) to transcription termination site ( TTS ) in RPB1 - 52R RPB1 - 25R for 1958 genes encoding major isoforms . p value = 0 . 8 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range .

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K- Box plot showing distance from the polyadenylation site (PAS) to the transcription termination site (TTS) in RPB1-52R and RPB1-25R cells for 1958 genes encoding major isoforms. p value= 0.8 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


B - Box plots showing synthesis ( RPKs ) of putative eRNAs annotated using TT - seq data in RPB1 - 52R RPB1 - 25R . p value = 9 . 27e - 103 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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B- Box plots showing RNA synthesis levels (RPKs) of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. p value =9.27e-103 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


C - Box plots showing lengths of putative eRNAs annotated using TT - seq data in RPB1 - 52R RPB1 - 25R . p value = 0 . 000519 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

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C- Box plots showing lengths of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. p value =0.000519 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


D - Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1 - 52R RPB1 - 25R .

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D - Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells.


A - Changes in synthesis in response to 15 min of TPA treatment . MA plot showing synthesis changes in TT - seq datasets upon 15 min treatment with TPA ( 200 nM ) in RPB1 - 52R RPB1 - 25R . DMSO treatment of respective was used as a control data was normalized using library normalization . 14636 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red ( adjusted p value < 0 . 05 , log2 fold change > = 1 for upregulated genes adjusted p value < 0 . 05 , log2 fold change ≤ 1 for downregulated genes ) . B - Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1 - 52R RPB1 - 25R ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) . P value = 0 . 02257 ( Mann - Whitney ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed . C - Changes in synthesis in response to 30 min of TPA treatment . MA plot showing synthesis changes in TT - seq datasets upon 30 min treatment with TPA ( 200 nM ) in RPB1 - 52R RPB1 - 25R . DMSO treatment of respective was used as a control data was normalized using library normalization . 14636 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red ( adjusted p value < 0 . 05 , log2 fold change > = 1 for upregulated genes adjusted p value < 0 . 05 , log2 fold change ≤ 1 for downregulated genes ) D - Box plots showing log2 fold change in synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1 - 52R RPB1 - 25R ( adjusted p value < 0 . 05 , log2 fold chage > = 1 ) . P value = 0 . 001749 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNA synthesis in response to 15 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 15 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes ). B- Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold change >=1). P value =0.02257 (Mann-Whitney test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Changes in RNA synthesis in response to 30 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 30 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes) D- Box plots showing log2 fold change in RNA synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold chage >=1). P value =0.001749 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


A - Histogram showing distribution of a number of putative eRNAs paired to genes in RPB1 - 52R RPB1 - 25R upon 15 30 min TPA treatment ( 200 nM ) as well as in DMSO controls .

A- Histogram showing the distribution of a number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells upon 15 and 30 min TPA treatment (200 nM) as well as in the DMSO controls.


B - Box plots showing synthesis ( RPK ) of putative eRNAs paired with genes upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) in RPB1 - 52R RPB1 - 25R upon 15 min of TPA treatment ( 200 nM ) . p value = 0 . 000732 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed . C - Box plots showing synthesis ( RPK ) of putative enhancer eRNAs paired with genes upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) in RPB1 - 52R RPB1 - 25R upon 30 min of TPA treatment ( 200 nM ) . p value = 2 . 77e - 5 ( Mann - Whitney U ) Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Two independent biological replicates were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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- Box plots showing RNA synthesis levels (RPK) of putative eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 15 min of TPA treatment (200 nM). p value = 0.000732 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Box plots showing RNA synthesis levels (RPK) of putative enhancer eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 30 min of TPA treatment (200 nM). p value = 2.77e-5 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


( A ) WT or Mfn2 KO MEFs ( Mfn2 KO ) were treated with 1 μM Tg for 12 h then processed for EM visualization of ER . Scale bar : 1 μm .

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

(A) WT or Mfn2 KO MEFs (Mfn2 KO cells) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology. Scale bar: 1 μm.


( B ) EM of Tg ‐ treated Mfn2 KO show accumulation of ER stacking . Scale bar : 1 μm .

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

(B) EM images of Tg‐treated Mfn2 KO cells show accumulation of ER membrane stacking. Scale bar: 1 μm.


( C ) WT , Mfn2 KO or Mfn1 KO were transfected with Sec61β ‐ GFP plasmid treated with 1 μM Tg for 24 h . Confocal microscopy show ER vacuolization in Mfn2 KO treated with Tg . Scale bar : 10 μm . Insets show × 10 zoomed . Scale bar : 5 μm .

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 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) WT, Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β‐GFP plasmid and treated with 1 μM Tg for 24 h. Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg. Scale bar: 10 μm. Insets show × 10 zoomed images. Scale bar: 5 μm.


( D ) WT Mfn2 KO were treated with Tg 1 μM for 3 h then incubated with brefeldin A ‐ bodipy to stain ER Golgi . Representative flow ‐ cytometry histograms ( upper panel ) . Mean was used to quantify ER expansion ( n = 5 ) ( lower panel ) . Data are mean ± s . e . m . * P0 . 05 versus WT group .

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

(D) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A‐bodipy to stain ER and Golgi. Representative flow‐cytometry histograms (upper panel). Mean fluorescence intensity was used to quantify ER expansion (n=5) (lower panel). Data are mean±s.e.m. *P0.05 versus WT group.


( A ) WT Mfn2 KO were treated with 1 μM Tg for 12 or 24 h . Total caspase 3 were detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( B , C ) WT Mfn2 KO were treated with 0 . 5 μg / ml tunicamycin ( Tm ) , 100 ng / ml brefeldin A ( Bref ) , or 1 μM Tg for 24 h . Total caspase 3 were detected by western blot ( B ) caspase activity ( C ) by measurement of DEVD ‐ AFC substrate processing . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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 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

(B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( D ) Flow ‐ cytometry of Sub G1 fragmentation in methanol ‐ fixed WT Mfn2 KO after incubation with or without 1 μM Tg for 24 h . Data are given as mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(D) Flow‐cytometry analysis of the Sub G1 DNA fragmentation in methanol‐fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.


( E ) Scr ( stably expressing scrambled shRNA ) Mfn2 knockdown ( KD ) ( stably expressing shRNA directed against Mfn2 ) 3T3 ‐ L1 fibroblasts were incubated in presence or absence of 1 μM Tg for 24 h . Total caspase 3 were detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3‐L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( F ) WT Mfn2 KO were treated with 1 μM Tg for 24 h . Lactate dehydrogenase ( LDH ) release was analyzed by flow cytometry to assess necrotic . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( G ) WT Mfn2 KO were incubated for 24 h with or without 1 μM Tg in presence or absence of z ‐ VAD ‐ fmk stained for annexin V / PI . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT + Tg group .

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

(G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z‐VAD‐fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P0.05 versus WT+Tg group.


( H ) Mfn2 KO transfected with pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h ( 3 h of pre ‐ incubation with CHX ) . Florescence microscopy show that CHX prevents vacuolization . Scale bar : 10 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

(H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre‐incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm.


( I ) WT ( black circles ) Mfn2 KO ( circles ) were incubated with 1 μM Tg for varying times , ALIX was detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . Source data for this figure is available on online supplementary information page .

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

(I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( A , upper panel ) WT ( black circles ) or Mfn2 KO ( circles ) were treated with 1 μM Tg for a range of times . ( A , lower ) Densitometric . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group ; # P0 . 05 versus WT non ‐ Tg ‐ treated group .

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

(A, upper panel) WT (black circles) or Mfn2 KO cells (white circles) were treated with 1 μM Tg for a range of times. (A, lower) Densitometric quantification. Data are mean±s.e.m. (n=3). *P0.05 versus WT group; #P0.05 versus WT non‐Tg‐treated group.


( B , upper panel ) WT or Mfn2 KO were treated with Tg for 0 , 6 or 12 h , in presence or absence of Bafilomycin ( Baf , 100 nM ) . LC3b ‐ I LC3b ‐ II expression was measured by western blot . ( B , lower panel ) Densitometric of LC3b ‐ II ( relative to tubulin ) . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT + Baf + Tg group .

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 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 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, upper panel) WT or Mfn2 KO cells were treated with Tg for 0, 6 or 12 h, in the presence or absence of Bafilomycin (Baf, 100 nM). LC3b‐I and LC3b‐II expression was measured by western blot. (B, lower panel) Densitometric quantification of LC3b‐II levels (relative to tubulin). Data are mean±s.e.m. (n=3). *P0.05 versus WT+Baf+Tg group.


( C ) WT or Mfn2 KO stably expressing mCherry ‐ GFP ‐ LC3b were treated with 1 μM Tg , 100 ng / ml Brefeldin ( Bref ) , or 0 . 5 μg / ml tunicamycin ( Tm ) for 24 h examined by confocal microscopy . Scale bar : 10 μm .

O O O O O B-CONTROLLED_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

(C) WT or Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b were treated with 1 μM Tg, 100 ng/ml Brefeldin (Bref), or 0.5 μg/ml tunicamycin (Tm) for 24 h and examined by confocal microscopy. Scale bar: 10 μm.


( D ) of mCherry + / GFP ‐ ( mCherry ‐ LC3 ) red per ( a measure of acidic autophagosomes ) is shown . Data are mean ± s . e . m . ( n = 3 ; 100 were analyzed per experiment group ) . * P0 . 05 versus WT group .

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

(D) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus WT group.


( E ) Acidic compartments of WT or Mfn2 KO were stained with Lysotracker Green analyzed by flow cytometry . Data are given as mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(E) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.


( F ) LAMP1 expression was immunodetected in WT Mfn2 KO in basal conditions .

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

(F) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions.


( G ) Expression of Beclin ‐ 1 or LC3b transcripts in WT or Mfn2 KO treated with or without 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . Source data for this figure is available on online supplementary information page .

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

(G) Expression of Beclin‐1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( B ) Immunodetection of p ‐ PERK , PERK , GADD34 , p ‐ eIF2α , eIF2α , ATF4 , CHOP , XBP ‐ 1s in WT Mfn2 KO treated with 1 μM Tg for times indicated .

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

(B) Immunodetection of p‐PERK,PERK, GADD34, p‐eIF2α, eIF2α, ATF4, CHOP, and XBP‐1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated.


( C ) Densitometric quantifications . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

O O O O O O 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) Densitometric quantifications. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( D ) Immunodetection of p ‐ eIF2α , eIF2α , ATF4 in WT Mfn2 KO treated with 0 . 5 μg / ml Tunicamycin ( Tm ) for times indicated .

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

(D) Immunodetection of p‐eIF2α, eIF2α, and ATF4 in WT and Mfn2 KO cells treated with 0.5 μg/ml Tunicamycin (Tm) for the times indicated.


( E ) Transcriptional activity driven by ATF6 . WT Mfn2 KO were co ‐ transfected with 5xATF6 ‐ GL3 TK ‐ Renilla plasmids treated with 1 μM Tg for 24 h after transfection . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT group . Source data for this figure is available on online supplementary information page .

O O O O O O O B-MEASURED_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 O O O O O O 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) Transcriptional activity driven by ATF6. WT and Mfn2 KO cells were co‐transfected with the 5xATF6‐GL3 and TK‐Renilla plasmids and treated with 1 μM Tg for 24 h after transfection. Data are mean±s.e.m. (n=4). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of PERK in WT Mfn2 KO stably expressing a scrambled shRNA ( Scr ) or a shRNA directed against PERK ( PERK KD ) .

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

(A) Immunodetection of PERK in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed against PERK (PERK KD).


( B ) Caspase activity was detected by measurement of DEVD - AFC substrate processing in WT or Mfn2 KO subjected to PERK silencing treated with 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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) Caspase activity was detected by measurement of DEVD-AFC substrate processing in WT or Mfn2 KO cells subjected to PERK silencing and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.


( C , D ) Total caspase 3 were detected by western blot . Densitometric is shown in D . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group .

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

(C, D) Total and cleaved caspase 3 levels were detected by western blot. Densitometric quantification is shown in D. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.


( E , F ) Immunodetection of LC3b ‐ I ‐ II in WT Mfn2 KO subjected to PERK silencing treated as indicated with Tg or Baf ( 1 μM Tg ; 100 nM Baf ) for 6 h . Densitometric is shown in F . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group . Source data for this figure is available on online supplementary information page .

O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunodetection of LC3b‐I and ‐II in WT and Mfn2 KO cells subjected to PERK silencing and treated as indicated with Tg or Baf (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of XBP ‐ 1s in WT Mfn2 KO stably expressing a scrambled shRNA ( Scr ) or a shRNA directed to XBP ‐ 1 ( XBP ‐ 1 KD ) , treated with Tg for 6 h .

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(A) Immunodetection of XBP‐1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed to XBP‐1 (XBP‐1 KD), and treated with Tg for 6 h.


( B ) Caspase activity was detected by measurement of DEVD ‐ AFC substrate processing . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . # P0 . 05 versus Scr group .

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(B) Caspase activity was detected by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. #P0.05 versus Scr group.


( C , D ) Total caspase 3 were detected by western blot . WT Mfn2 KO subjected or not to XBP ‐ 1 silencing ( Scr , KD ) treated with 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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(C, D) Total and cleaved caspase 3 levels were detected by western blot. WT and Mfn2 KO cells subjected or not to XBP‐1 silencing (Scr, and KD) and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( E , F ) Immunodetection of LC3b ‐ I ‐ II in Scr XBP ‐ 1 KD WT or Mfn2 KO treated as indicated ( 1 μM Tg ; 100 nM Baf ) for 6 h . Densitometric is shown in F . Data are mean ± s . e . m . ( n = 3 ) . # P0 . 05 versus Scr group .

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(E, F) Immunodetection of LC3b‐I and ‐II in Scr and XBP‐1 KD WT or Mfn2 KO cells treated as indicated (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). #P0.05 versus Scr group.


( G ) Mfn2 KO stably expressing mCherry ‐ GFP ‐ LC3b subjected or not to XBP ‐ 1 silencing ( Scr or KD ) were treated with 1 μM Tg , 100 ng / ml Bref , or 0 . 5 μg / ml Tm for 24 h examined by confocal microscopy . Scale bar : 10 μm .

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(G) Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b and subjected or not to XBP‐1 silencing (Scr or KD) were treated with 1 μM Tg, 100 ng/ml Bref, or 0.5 μg/ml Tm for 24 h and examined by confocal microscopy. Scale bar: 10 μm.


( H ) of mCherry + / GFP ‐ ( mCherry ‐ LC3 ) red per ( a measure of acidic autophagosomes ) is shown . Data are mean ± s . e . m . ( n = 3 ; 100 were analyzed per experiment group ) . # P0 . 05 versus Scr group . Source data for this figure is available on online supplementary information page .

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(H) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of p ‐ PERK p ‐ eIF2α in Mfn2 KO MEFs , Mfn2 knockdown 3T3 ‐ L1 fibroblasts , Mfn2 knockdown C2C12 myoblasts .

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(A) Immunodetection of p‐PERK and p‐eIF2α in Mfn2 KO MEFs, Mfn2 knockdown 3T3‐L1 fibroblasts, and Mfn2 knockdown C2C12 myoblasts.


( B ) Densitometric of p ‐ PERK p ‐ eIF2α in MEFs . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT .

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(B) Densitometric quantification of p‐PERK and p‐eIF2α in MEFs. Data are mean±s.e.m. (n=3). *P0.05 versus WT cells.


( C , D ) Immunodetection of p ‐ eIF2α , eIF2α , CHOP , XBP ‐ 1s , LC3b , p62 in Mfn2 ‐ deficient liver ( C ) or skeletal muscle ( D ) from tissue ‐ specific KO mice .

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(C, D) Immunodetection of p‐eIF2α, eIF2α, CHOP, XBP‐1s, LC3b, and p62 in Mfn2‐deficient liver (C) or skeletal muscle (D) from tissue‐specific KO mice.


( E ) Co ‐ immunoprecipitation of Mfn2 PERK in WT or Mfn2 KO stably expressing PERK ‐ myc or an empty vector ( pBABE ‐ myc ; negative control ) .

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(E) Co‐immunoprecipitation of Mfn2 and PERK in WT or Mfn2 KO cells stably expressing PERK‐myc or an empty vector (pBABE‐myc; negative control).


( F ) Co ‐ immunoprecipitation of endogenous Mfn2 PERK in WT MEF lysates . PERK was immunoprecipitated with a C ‐ terminal antibody co ‐ immunoprecipitation of Mfn2 was detected by western blot . Source data for this figure is available on online supplementary information page .

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(F) Co‐immunoprecipitation of endogenous Mfn2 and PERK in WT MEF lysates. PERK was immunoprecipitated with a C‐terminal antibody and co‐immunoprecipitation of Mfn2 was detected by western blot.Source data for this figure is available on the online supplementary information page.


( A ) Representative confocal of mitochondrial in scrambled ( Scr ) PERK KD Mfn2 KO stained with MitoTracker Green .

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(A) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD Mfn2 KO cells stained with MitoTracker Green.


( B ) Mitochondrial . Data are mean ± s . e . m . ( n = 3 ; 100 were analyzed per experiment group ) . * P0 . 05 versus Scr group .

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(B) Mitochondrial morphology quantification. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus Scr group.


( C , D ) Representative confocal of mitochondrial in scrambled ( Scr ) PERK KD WT ( C ) or scrambled ( Scr ) PERK KD Mfn1 KO ( D ) stained with MitoTracker Green .

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(C, D) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD WT cells (C) or scrambled (Scr) and PERK KD Mfn1 KO cells (D) stained with MitoTracker Green.


( E ) Representative confocal of mitochondrial in Mfn2 KO treated with 1 μM TUDCA or 10 mM 4 ‐ phenyl butyric acid for 6 h stained with MitoTracker Green .

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(E) Representative confocal images of mitochondrial morphology in Mfn2 KO cells treated with 1 μM TUDCA or 10 mM 4‐phenyl butyric acid for 6 h and stained with MitoTracker Green.


( F ) Flow ‐ cytometry of ROS in Scr PERK KD WT Mfn2 KO

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(F) Flow‐cytometry quantification of ROS levels in Scr and PERK KD WT and Mfn2 KO cells


( G ) or Scr PERK KD WT Mfn1 using DHR1 , 2 , 3 . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT group , # P0 . 05 versus Scr group . Scale bars : 10 μm .

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(G) or Scr and PERK KD WT and Mfn1 cells using DHR1,2,3. Data are mean±s.e.m. (n=4). *P0.05 versus WT group, #P0.05 versus Scr group. Scale bars: 10 μm.


( A ) Mitochondrial calcium overload in Scr PERK KD Mfn2 KO . were loaded with Rhod ‐ 2 then treated with 2 . 5 mM CaCl2 ( left ) . Calcium uptake was monitored by confocal microscopy . Representative confocal of mitochondrial in WT Mfn2 KO stained with Rhod ‐ 2 incubated with 2 . 5 mM CaCl2 for 5 min ( right ) . Scale bars : 10 μm .

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(A) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells. Cells were loaded with Rhod‐2 and then treated with 2.5 mM CaCl2 (left). Calcium uptake was monitored by confocal microscopy. Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod‐2 and incubated with 2.5 mM CaCl2 for 5 min (right). Scale bars: 10 μm.


( B - E ) Mitochondrial oxygen consumption ( OCR ) was measured in Scr PERK KD Mfn2 KO ( B ) , Scr PERK KD WT ( C ) , Scr PERK KD Mfn1 KO ( D ) , WT stably expressing PERK ‐ myc ( E ) . Data are mean of three independent experiments . following parameters were measured : oxygen consumption under routine conditions ( DMEM with 5 . 5 mM glucose ) , maximal respiratory capacity reached after uncoupling with FCCP , respiratory leak , measured after inhibition of ATP synthase with oligomycin . Data are mean ± s . e . m . * P0 . 05 versus Scr group or versus empty plasmid group .

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(B-E) Mitochondrial oxygen consumption (OCR) was measured in Scr and PERK KD Mfn2 KO cells (B), Scr and PERK KD WT cells (C), Scr and PERK KD Mfn1 KO cells (D), and WT cells stably expressing PERK‐myc (E). Data are mean of three independent experiments. The following parameters were measured: oxygen consumption under routine conditions (DMEM with 5.5 mM glucose), maximal respiratory capacity reached after uncoupling with FCCP, and respiratory leak, measured after inhibition of ATP synthase with oligomycin. Data are mean±s.e.m. *P0.05 versus Scr group or versus the empty plasmid group.


( F , G ) Representative confocal of mitochondrial in WT stably expressing PERK ‐ myc stained with MitoTracker Green . Scale bars : 10 μm . Quantitative of mitochondrial is shown in G . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus empty plasmid group .

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(F, G) Representative confocal images of mitochondrial morphology in WT cells stably expressing PERK‐myc stained with MitoTracker Green. Scale bars: 10 μm. Quantitative analysis of mitochondrial morphology is shown in G. Data are mean±s.e.m. (n=3). *P0.05 versus the empty plasmid group.


A . Vps10yEGFP localization . Yeast carrying Vps10yEGFP expressing indicated vps35 alleles as sole source of Vps35 were logarithmically grown in SC medium . Their vacuoles were labelled with FM4 - 64 . were harvested by brief centrifugation immediately imaged by confocal microscopy . Single confocal planes are shown . A brightfield image was used to outline boundaries ( shown in merged ) . B . Co - localization of Vps10yEGFP FM4 - 64 in from a was measured using Pearson ' s coefficient . 15 Confocal planes with 20 to 30 each from 3 independent experiments were analyzed .

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A. Vps10yEGFP localization. Yeast cells carrying Vps10yEGFP and expressing the indicated vps35 alleles as the sole source of Vps35 were logarithmically grown in SC medium. Their vacuoles were labelled with FM4-64. Cells were harvested by brief centrifugation and immediately imaged by confocal microscopy. Single confocal planes are shown. A brightfield image was used to outline the cell boundaries (shown in the merged images). B. Co-localization of Vps10yEGFP and FM4-64 in cells from a was measured using Pearson's coefficient. 15 Confocal planes with 20 to 30 cells each from 3 independent experiments were analyzed. Means


B . Influence of VPS35 variants on GLUT1 . HK2 silenced for VPS35 were transfected with a plasmid carrying siRNA resistant wildtype or mutant forms of GFP - VPS35 . GLUT1 was detected by fixation immunofluorescence as in a . Maximum projections of image stacks ( step in z of 300 nm ) are shown .

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B. Influence of VPS35 variants on GLUT1. HK2 cells silenced for VPS35 were transfected with a plasmid carrying siRNA resistant wildtype or mutant forms of GFP-VPS35. GLUT1 was detected by fixation and immunofluorescence staining as in a. Maximum projections of image stacks (step size in z of 300 nm) are shown.


C . of GLUT1 immunofluorescence in from B . Regions of interest ( ROIs ) corresponding to expressing indicated VPS35 variants , some regions outside ( background ) , were manually defined using ImageJ software . Total was integrated corrected for background . 105 per condition stemming from 3 independent experiments were analyzed . was performed with 99 % confidence : * * * p < 0 . 001 .

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C. Quantification of GLUT1 immunofluorescence in cells from B. Regions of interest (ROIs) corresponding to cells expressing the indicated VPS35 variants, and some regions outside the cells (background), were manually defined using ImageJ software. Total cell fluorescence was integrated and corrected for background fluorescence. 105 cells per condition stemming from 3 independent experiments were analyzed. The analysis was performed with 99% confidence: *** p < 0.001.


D . Expression of Vps35 variants in from B was analyzed by SDS - PAGE Western blot against Vps35 . Tubulin served as loading control .

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D. Expression of Vps35 variants in cells from B was analyzed by SDS-PAGE and Western blot against Vps35. Tubulin served as loading control.


A PHIP - 1 interaction with GPB - 1 by yeast two - hybrid . reporter PJ69 - 4A was co - transformed with expression vectors encoding GAL4 DBD - PHIP - 1 ( H45A ) GAL4 AD - GPB - 1 , as indicated . Yeast strains carrying indicated plasmids were cultured on a selective plate lacking histidine containing 5 mM 5 - aminotriazole for 4 days .

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A PHIP-1 interaction with GPB-1 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-GPB-1, as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.


A His - phosphorylation of GPB - 1 in . phip - 1 ( km96 ) mutant carrying 3XFLAG : : gpb - 1 or 3XFLAG : : gpb - 1 ( H266F ) knock - in allele were lysed . lysates were treated with or without heating ( 95 ° C ) immunoblotted ( IB ) with anti - 3 - pHis anti - FLAG antibodies .

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A His-phosphorylation of GPB-1 in animals. The phip-1(km96) mutant animals carrying the 3XFLAG::gpb-1 or 3XFLAG::gpb-1(H266F) knock-in allele were lysed. The lysates were treated with or without heating (95°C) and immunoblotted (IB) with anti-3-pHis and anti-FLAG antibodies.


B NDK - 1 phosphorylates GPB - 1 in vitro . COS - 7 were co - transfected with HA - GPB - 1 or HA - GPB - 1 ( H266F ) T7 - GPC - 2 , lysates were immunoprecipitated ( IP ) with anti - HA antibodies . Immunopurified GPB - 1 was subjected to in vitro kinase with recombinant GST - NDK - 1 . Phosphorylated GPB - 1 was detected by immunoblotting ( IB ) with anti - 3 - pHis antibodies .

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B NDK-1 phosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 or HA-GPB-1(H266F) and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. Immunopurified GPB-1 was subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.


C PHIP - 1 dephosphorylates GPB - 1 in vitro . COS - 7 were co - transfected with HA - GPB - 1 T7 - GPC - 2 , lysates were immunoprecipitated ( IP ) with anti - HA antibodies . immunopurified HA - GPB - 1 was first subjected to in vitro kinase with recombinant GST - NDK - 1 . Phosphorylated GPB - 1 was then equally aliquoted subjected to in vitro phosphatase with recombinant GST - PHIP - 1 or its variants . Phosphorylated GPB - 1 was detected by immunoblotting ( IB ) with anti - 3 - pHis antibodies .

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C PHIP-1 dephosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. The immunopurified HA-GPB-1 was first subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was then equally aliquoted and subjected to the in vitro phosphatase assay with recombinant GST-PHIP-1 or its variants. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.


A PHIP - 1 interaction with UNC - 51 by yeast two - hybrid . reporter PJ69 - 4A was co - transformed with expression vectors encoding GAL4 DBD - PHIP - 1 ( H45A ) GAL4 AD - UNC - 51 ( 274 - 856 ) , as indicated . Yeast strains carrying indicated plasmids were cultured on a selective plate lacking histidine containing 5 mM 5 - aminotriazole for 4 days .

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A PHIP-1 interaction with UNC-51 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-UNC-51(274-856), as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.