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

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


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

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


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

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


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

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B FOXL2 protein-expression levels were determined by western blotting after transfecting KGN cells with control, DHX9, or GW182 siRNAs for 48 h. Quantification of FOXL2 protein expression is presented in the bottom panel. The data are presented as the mean ± SEM from three independent experiments. The p values were analyzed by unpaired, two-tailed Student's t-test (**p < 0.01, ***p < 0.001).


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

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


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

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

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


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

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


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

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


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

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


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

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


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 .

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KGN cells were transfected with 200 nM of scrambled control or FOXL2-specific siRNAs for 24 h. Then, KGN cells were further transfected with 20 nM of control miRNA, miR-1236, control anti-miRNA, or anti-miR-1236. The population at S phase (C and D) were analyzed by flow cytometry. The data are presented as the mean ± SEM of three independent experiments. Different letters denote statistically significant differences (p < 0.0001; Student-Newman-Keuls test). Efficient silencing of FOXL2 using specific siRNAs was confirmed by western blotting.


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

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E, F KGN cells were transfected with control siRNA or siFOXL2 for 24 h. Then, KGN cells were further transfected with control miRNA or miR-1236 (E) or with control anti-miRNA or anti-miR-1236 (F) for 48 h, and Transwell-migration assays were performed. The migrated cells were imaged under a bright-field microscope (100 × magnification, scale bar = 100 µm). The results are from three independent experiments and represent fold-changes in the average number of cells/field (mean ± SEM). Different letters denote statistically significant differences (p < 0.01; Student-Newman-Keuls test).


G , H COV434 cells were transfected with miR - control or miR - 1236 for 48 h , after which cell viabilities ( G ) and migration abilities ( H ) were measured . The data are presented as the mean ± SEM of three independent experiments . Immunoblots showing no change in FOXL2 protein are presented in the top panel , and images of migrated cells are presented in the right panel . The migrated cells were imaged under a bright - field microscope ( 100 × magnification , scale bar = 100 µm ) .

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G, H COV434 cells were transfected with miR-control or miR-1236 for 48 h, after which cell viabilities (G) and migration abilities (H) were measured. The data are presented as the mean ± SEM of three independent experiments. Immunoblots showing no change in FOXL2 protein are presented in the top panel, and images of migrated cells are presented in the right panel. The migrated cells were imaged under a bright-field microscope (100 × magnification, scale bar = 100 µm).


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

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


L No difference in the cell - migration activities of control and two independent miR - 1236 - / - ( # 1 and # 2 ) COV434 cell lines . The migrated cells were imaged under a bright - field microscope ( 100 × magnification , scale bar = 100 µm ; top ) and the results ( bottom ) represent fold - changes in the average number of cells / field . The data are presented as the mean ± SEM of three independent experiments .

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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.


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 .

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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 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 ) .

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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 - 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 .

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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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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(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.