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 .

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

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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 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 .

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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 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 .

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

(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 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

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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 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 .

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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 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

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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 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

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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 protein overexpression in a two - dimensional concentration gradient of TMP and 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 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

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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 ) , 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

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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 antibiotics 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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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

(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 the pathway of pluripotency of stem cells . The color bar showed the relative RNA - 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 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 ) .

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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 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 ) .

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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 and TUBULIN in 7 paired human normal lungs ( N ) and ADC samples ( T ) and 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 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 .

(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 ) The expression grade of IHC staining 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 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 ) .

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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 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 .

O O O 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 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

(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 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 .

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 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.


( 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 ) .

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

(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 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 ) .

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

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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 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 ) .

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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 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 .

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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 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 .

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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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- 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 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 ) .

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 and shaded area 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 and RPB1 - 25R cells 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 and RPB1 - 25R cells . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . 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

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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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- 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 and shaded area 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 and shaded area 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 and RPB1 - 25R cells . Solid lines represent median signal and shaded area 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 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 .

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

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 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 .

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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 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 .

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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 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 .

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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 and RPB1 - 25R cells .

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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 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 .

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

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 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 .

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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 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 .

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 cells ) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology . Scale bar : 1 μm .

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

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

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(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 and Mfn2 KO cells in basal conditions .

(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 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 .

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(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 , and XBP ‐ 1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated .

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(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 .

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


( 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 .

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(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 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 .

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(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 and Mfn2 KO cells stably expressing a scrambled shRNA ( Scr ) or a shRNA directed against PERK ( PERK KD ) .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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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 .

(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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 and p ‐ eIF2α in Mfn2 KO MEFs , Mfn2 knockdown 3T3 ‐ L1 fibroblasts , and Mfn2 knockdown C2C12 myoblasts .

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


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

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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 , and 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 and PERK in WT or Mfn2 KO cells 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 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 .

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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 images of mitochondrial morphology in scrambled ( Scr ) and PERK KD Mfn2 KO cells 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 morphology quantification . Data are mean ± s . e . m . ( n = 3 ; 100 cells were analyzed per experiment and group ) . * P0 . 05 versus Scr group .

(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 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 .

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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 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 .

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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 quantification of ROS levels in Scr and PERK KD WT and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 cells from B was analyzed by SDS - PAGE and 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 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 .

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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 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 .

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 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 .

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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 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 .

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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 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 .

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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.


C UNC - 51 phosphorylates PHIP - 1 at Ser - 112 . COS - 7 cells were co - transfected with Flag - PHIP - 1 ( WT or mutants ) and GFP - UNC - 51 [ WT or ∆ AIKAI ( KD ) ] , and cell lysates were analyzed using Phos - tag SDS - PAGE . Total lysates were immunoblotted ( IB ) with antibodies , as indicated . Filled and open arrowheads indicate unmodified and phosphorylated PHIP - 1 , respectively . Asterisk indicates non - specific band .

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C UNC-51 phosphorylates PHIP-1 at Ser-112. COS-7 cells were co-transfected with Flag-PHIP-1 (WT or mutants) and GFP-UNC-51 [WT or ∆AIKAI (KD)], and cell lysates were analyzed using Phos-tag SDS-PAGE. Total lysates were immunoblotted (IB) with antibodies, as indicated. Filled and open arrowheads indicate unmodified and phosphorylated PHIP-1, respectively. Asterisk indicates non-specific band.


A . Western blot analysis of Tace using lysates from WT and TaceFl / Fl ; P0 - Cre mousesciatic nerves at P10 ( 65ug of total protein lysate loaded ) . The arrowhead indicates the pro - protein and the arrow shows the cleaved active form of Tace . Representative of two independent experiments .

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A. Western blot analysis of Tace using lysates from WT and TaceFl/Fl;P0-Cre mousesciatic nerves at P10 (65ug of total protein lysate loaded). The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments.


B . Western blot analysis ( 50ug of total protein lysate loaded ) shows that Tace expression in axons is maximal at P5 - P10 and then progressively declines in the adult . Representative of two independent experiments .

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B. Western blot analysis (50ug of total protein lysate loaded) shows that Tace expression in axons is maximal at P5-P10 and then progressively declines in the adult. Representative of two independent experiments.


C . Western blot analysis of Tace in Mtmr2 - / - sciatic nerves at P5 , P10 , and P20 . Representative of two independent experiments .

C. Western blot analysis of Tace in Mtmr2-/- sciatic nerves at P5, P10, and P20. Representative of two independent experiments.


D . Western blot analysis of Tace using lysates from Mtmr2 - / - mouse sciatic nerves at 5 months , with quantification , p = 0 . 10 , two - tailed Mann Whitney U test . Representative of two independent experiments .

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D. Western blot analysis of Tace using lysates from Mtmr2-/- mouse sciatic nerves at 5 months, with quantification, p=0.10, two-tailed Mann Whitney U test. Representative of two independent experiments.


E . Phosphorylation of ErbB2 in Mtmr2 - / - sciatic nerves at P2 , P10 and P15 . At P2 , each lane is a pool of n = 7 animals per genotype , representative of six independent experiments using n = 6 different nerve pools per genotype , and quantification using WT values arbitrary set to1 , p = 0 . 0313 , Wilcoxon Rank Sum test . At P10 and P15 , mean values of two samples are shown . At P10 , each lane is a pool of n = 3 / 4 animals per genotype , representative of three independent experiments using n = 6 different pools per genotype . Nf - l is the neurofilament light chain and Clx is calnexin .

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E. Phosphorylation of ErbB2 in Mtmr2-/- sciatic nerves at P2, P10 and P15. At P2, each lane is a pool of n=7 animals per genotype, representative of six independent experiments using n=6 different nerve pools per genotype, and quantification using WT values arbitrary set to1, p=0.0313, Wilcoxon Rank Sum test. At P10 and P15, mean values of two samples are shown. At P10, each lane is a pool of n=3/4 animals per genotype, representative of three independent experiments using n=6 different pools per genotype. Nf-l is the neurofilament light chain and Clx is calnexin.


F . Phosphorylation of ErbB2 in Mtmr2 - / - Schwann cell / DRG neuron co - cultures after 4 days of ascorbic acid treatment . Each lane is a pool from at least 10 coverslips / DRG per genotype . Representative of two independent experiments using n = 4 different pools of coverslips / DRG per genotype , p = 0 . 20 , two - tailed Mann Whitney U test .

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F. Phosphorylation of ErbB2 in Mtmr2-/- Schwann cell/DRG neuron co-cultures after 4 days of ascorbic acid treatment. Each lane is a pool from at least 10 coverslips/DRG per genotype. Representative of two independent experiments using n=4 different pools of coverslips/DRG per genotype, p=0.20, two-tailed Mann Whitney U test.


A . ShRNA targeting Nrg1 type III were validated in rat purified neurons . Western blot analysis for Nrg1 shows reduction of its expression ( pro - protein at 150 KDa ) using two different harpins , representative of two independent experiments . Clx is calnexin .

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A. ShRNA targeting Nrg1 type III were validated in rat purified neurons. Western blot analysis for Nrg1 shows reduction of its expression (pro-protein at 150 KDa) using two different harpins, representative of two independent experiments. Clx is calnexin.