( A ) Growth measured by optical density ( OD ) at 600nm for 3 biological replicates each of the T - and A - strains over 6 hrs of exponential growth in YES media . The x - axis refers to the time since inoculation of the culture . ( B ) The same data as in ( A ) plotted on a log2 scale ( solid lines ) with lines fitted ( dotted lines ) . The doubling time is the inverse of the slope .

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(A) Growth measured by optical density (OD) at 600nm for 3 biological replicates each of the T- and A-strains over 6 hrs of exponential growth in YES media. The x-axis refers to the time since inoculation of the culture. (B) The same data as in (A) plotted on a log2 scale (solid lines) with lines fitted (dotted lines). The doubling time is the inverse of the slope.


( C ) Media samples were taken from the same cultures ( n = 3 for both strains ) at the 8 hr timepoint and the remaining glucose was quantified . The consumed glucose was calculated based on the amount of glucose measured in the same , fresh media and normalised to the OD of each culture at the time of sampling .

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(C) Media samples were taken from the same cultures (n=3 for both strains) at the 8 hr timepoint and the remaining glucose was quantified. The consumed glucose was calculated based on the amount of glucose measured in the same, fresh media and normalised to the OD of each culture at the time of sampling.


( D ) Oxygen consumption rates in A - and T - strains ( ratio of means = 0 . 65 , p = 0 . 0002 , Welch ' s T - test , 3 biological replicates , each measured in technical duplicates ) . Data information : Vertical bars show the mean of the data . Error bars and shaded areas in all cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - test ) .

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(D) Oxygen consumption rates in A- and T-strains (ratio of means=0.65, p=0.0002, Welch's T-test, 3 biological replicates, each measured in technical duplicates). Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( E ) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain . Data information : Vertical bars show the mean of the data . Error bars and shaded areas in all cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - test ) .

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(E) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain. Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( A ) Spot assays on solid media from a three - fold dilution series of exponential cultures at the same cell density in 96 - well plates ( 3 biological replicates of each strain ) and spotted in 16 technical replicates ( each dilution in 4x4 square ) . The A - strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide . A control without toxin ( left ) was included in each batch of spot assays performed and a representative image is shown here .

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(A) Spot assays on solid media from a three-fold dilution series of exponential cultures at the same cell density in 96-well plates (3 biological replicates of each strain) and spotted in 16 technical replicates (each dilution in 4x4 square). The A-strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide. A control without toxin (left) was included in each batch of spot assays performed and a representative image is shown here.


( B ) Fitness ( approximated by maximum slope of smoothed growth curves ) of A - and T - strains on 12 carbon sources , with either yeast extract ( YE ) or ammonium ( NH4 ) , with or without 0 . 1 % priming glucose to support initial growth . For all 48 conditions , two biological replicates of A - and T - strains were grown in technical quadruplicates each . Dotted lines in panels mark a fitness ratio of 1 ( i . e . , same fitness ) .

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(B) Fitness (approximated by maximum slope of smoothed growth curves) of A- and T-strains on 12 carbon sources, with either yeast extract (YE) or ammonium (NH4), with or without 0.1% priming glucose to support initial growth. For all 48 conditions, two biological replicates of A- and T-strains were grown in technical quadruplicates each. Dotted lines in panels mark a fitness ratio of 1 (i.e., same fitness).


( C ) Fitness of A - and T - strains on 95 nitrogen sources on Biolog Phenotype MicroArrays . Conditions with no substantial growth were excluded ( black circles , maximum slope < 0 . 015 ) . Red lines show arbitrary significance cut - off , put at | log2 ( A - strain / T - strain ) | > 0 . 75 .

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(C) Fitness of A- and T-strains on 95 nitrogen sources on Biolog Phenotype MicroArrays. Conditions with no substantial growth were excluded (black circles, maximum slope <0.015). Red lines show arbitrary significance cut-off, put at |log2(A-strain/T-strain)|>0.75.


( D ) Fitness of A - and T - strains on 72 different drugs and toxins , at 4 concentrations each , on Biolog Phenotype MicroArrays . ( Results for benzamidine were inconclusive , with both strains comparatively resistant in one concentration each . )

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(D) Fitness of A- and T-strains on 72 different drugs and toxins, at 4 concentrations each, on Biolog Phenotype MicroArrays. (Results for benzamidine were inconclusive, with both strains comparatively resistant in one concentration each.)


( E ) Spot assays to validate selected results Several assays were performed with both h - and h90 strains , and no mating - type specific differences were evident between the two sets of allele - swap strains .

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(E) Spot assays to validate selected results Several assays were performed with both h- and h90 strains, and no mating-type specific differences were evident between the two sets of allele-swap strains.


( F ) Chronological lifespan of A - and T - strains , i . e . the proportion of non - dividing cells in stationary phase that maintain proliferative potential after refeeding . The data show colony forming units ( CFUs ) per ml of culture over 7 days of stationary phase in glucose - depleted rich media . Three biological repeats were carried out for both strains , with each repeat measured as technical triplicates . Error bars represent standard error of the biological replicates .

(F) Chronological lifespan of A- and T-strains, i.e. the proportion of non-dividing cells in stationary phase that maintain proliferative potential after refeeding. The data show colony forming units (CFUs) per ml of culture over 7 days of stationary phase in glucose-depleted rich media. Three biological repeats were carried out for both strains, with each repeat measured as technical triplicates. Error bars represent standard error of the biological replicates.


( G ) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection . The T - strains had a higher mean fitness in H2O2 than the A - strains ( 1 . 01 ± 0 . 06 vs 0 . 95 ± 0 . 11 ; p = 0 . 0021 , Welch ' s t - test ) . The resistance score was obtained as for antimycin A .

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(G) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection. The T-strains had a higher mean fitness in H2O2 than the A-strains (1.01±0.06 vs 0.95±0.11; p=0.0021, Welch's t-test). The resistance score was obtained as for antimycin A.


( H ) Boxplot showing growth fitness on rich media , grouped by pyk1 allele for 158 S . pombe strains ( 0 . 88 ± 0 . 16 vs 0 . 94 ± 0 . 24 ; p = 0 . 18 , Welch ' s t - test ) .

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(H) Boxplot showing growth fitness on rich media, grouped by pyk1 allele for 158 S. pombe strains (0.88±0.16 vs 0.94±0.24; p=0.18, Welch's t-test).


B ) Log ( odds enrichment ) scores for charged GKs in the flanks of the 30 % strongest versus the 30 % weakest APRs in the E . coli cytoplasmic proteome . Whiskers indicate 95 % confidence intervals for no enrichment as determined through bootstrapping analysis .

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B) Log(odds enrichment) scores for charged GKs in the flanks of the 30 % strongest versus the 30 % weakest APRs in the E. coli cytoplasmic proteome. Whiskers indicate 95 % confidence intervals for no enrichment as determined through bootstrapping analysis.


C ) Percentual difference in non - synonymous mutation rate in E . coli for GK versus background i . e . versus the corresponding residue in non - gatekeeper positions .

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C) Percentual difference in non-synonymous mutation rate in E. coli for GK versus background i.e. versus the corresponding residue in non-gatekeeper positions.


D ) Percentual difference in non - synonymous mutation rate in mammalian species for GK versus background i . e . versus the corresponding residue in non - gatekeeper positions .

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D) Percentual difference in non-synonymous mutation rate in mammalian species for GK versus background i.e. versus the corresponding residue in non-gatekeeper positions.


B ) Time - resolved Dynamic Light Scattering ( DLS ) analysis indicating average particle size over time for each of the peptides in the β - Gal peptide set ( aggregating core sequence SVIIWSLGN ) .

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B) Time-resolved Dynamic Light Scattering (DLS) analysis indicating average particle size over time for each of the peptides in the β-Gal peptide set (aggregating core sequence SVIIWSLGN).


C ) ThT binding kinetics of the β - Gal peptide set . Background - corrected fluorescence over time is shown for 3 independent repeats .

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C) ThT binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.


D ) pFTAA binding kinetics of the β - Gal peptide set . Background - corrected fluorescence over time is shown for 3 independent repeats .

D) pFTAA binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.


E ) End - point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β - Gal peptide set . Bars depict mean concentration , whiskers indicate standard deviation ( n = 4 ) . Statistical significance was determined through two - way ANOVA with Bonferroni ' s correction for multiple comparisons . P - values for significant differences are indicated .

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E) End-point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β-Gal peptide set. Bars depict mean concentration, whiskers indicate standard deviation (n=4). Statistical significance was determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.


F ) TEM images of the β - Gal peptide set after 3 days incubation . Top panels give an overview image of one square in a TEM grid , lower panel shows enlarged image ( magnification 12000X ) . Scale bars indicate 12 µm ( top row ) or 1 µm ( bottom row ) .

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F) TEM images of the β-Gal peptide set after 3 days incubation. Top panels give an overview image of one square in a TEM grid, lower panel shows enlarged image (magnification 12000X). Scale bars indicate 12 µm (top row) or 1 µm (bottom row).


H ) Violin and boxplots showing the distribution of end - point solubilities across 11 aggregating cores , grouped by GK . The dotted line indicates a concentration of µM , corresponding to completely insoluble peptides .

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H) Violin and boxplots showing the distribution of end-point solubilities across 11 aggregating cores, grouped by GK. The dotted line indicates a concentration of µM, corresponding to completely insoluble peptides.


I ) Solubility analysis of the in vitro solubility screen performed by Niwa et al ( Niwa et al , 2009 ) . Left panel shows combined box - and violin plots depicting the solubility distributions of proteins containing at least one strong APR ( TANGO score > 70 ) not protected by any negative GKs ( red ) versus proteins where all strong APRs are flanked by at least one negative charge ( blue ) . The right panel shows a control analysis , in which proteins were classified by the occurrence of at least one strong APR ( TANGO score > 70 ) not flanked by any positive GKs ( red ) versus proteins where all strong APRs are flanked by at least one positive charge ( blue ) . The upper boxplot whiskers indicate the largest value no further than 1 . 5 times the Inter - Quartile Range ( IQR ) from the upper hinge , the lower whiskers show the smallest value at least 1 . 5 * IQR from the lower hinge . Notches extend 1 . 58 * IQR / sqrt ( n ) from either side of the median and represent a 95 % confidence interval for the median value . Statistical significance was determined through Kruskal - Wallis test with a post - hoc Wilcoxon analysis for pairwise testing . P - values are indicated .

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I) Solubility analysis of the in vitro solubility screen performed by Niwa et al (Niwa et al, 2009). Left panel shows combined box- and violin plots depicting the solubility distributions of proteins containing at least one strong APR (TANGO score > 70) not protected by any negative GKs (red) versus proteins where all strong APRs are flanked by at least one negative charge (blue). The right panel shows a control analysis, in which proteins were classified by the occurrence of at least one strong APR (TANGO score > 70) not flanked by any positive GKs (red) versus proteins where all strong APRs are flanked by at least one positive charge (blue). The upper boxplot whiskers indicate the largest value no further than 1.5 times the Inter-Quartile Range (IQR) from the upper hinge, the lower whiskers show the smallest value at least 1.5 * IQR from the lower hinge. Notches extend 1.58 * IQR / sqrt(n) from either side of the median and represent a 95% confidence interval for the median value. Statistical significance was determined through Kruskal-Wallis test with a post-hoc Wilcoxon analysis for pairwise testing. P-values are indicated.


D ) Time - resolved Dynamic Light Scattering ( DLS ) analysis showing average particle radius over time for the β - Gal peptides flanked by both natural and non - natural GKs , at increasing NaCl concentrations ( indicated in µM ) . Structures of the amino acids flanking the aggregating core are indicated on the right - hand side , the sequence of the aggregating core is indicated below .

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D) Time-resolved Dynamic Light Scattering (DLS) analysis showing average particle radius over time for the β-Gal peptides flanked by both natural and non-natural GKs, at increasing NaCl concentrations (indicated in µM). Structures of the amino acids flanking the aggregating core are indicated on the right-hand side, the sequence of the aggregating core is indicated below.


A ) DnaK binding to cellulose - bound peptide array . Sequences of the aggregating cores are indicated in the top of the panel , the GKs flanking them in each instance are indicated above the blots .

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A) DnaK binding to cellulose-bound peptide array. Sequences of the aggregating cores are indicated in the top of the panel, the GKs flanking them in each instance are indicated above the blots.


C ) Representative Western blot of in vitro translation analysis of folding - incompetent model constructs . Bands show Total ( T ) and Soluble ( S ) fractions after centrifugation at 21 , 000 g for 30 mins . " + Dnak " indicates addition of either DnaK mix - containing DnaK , DnaJ and GrpE .

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C) Representative Western blot of in vitro translation analysis of folding-incompetent model constructs. Bands show Total (T) and Soluble (S) fractions after centrifugation at 21,000 g for 30 mins. "+ Dnak" indicates addition of either DnaK mix - containing DnaK, DnaJ and GrpE.


D ) Bar plots showing mean solubility as determined through quantification of western blot in ( C ) , whiskers indicate standard deviation ( n = 4 ) . Statistically significant differences between the - DnaK and + DnaK conditions were determined through two - way ANOVA with Bonferroni ' s correction for multiple comparisons . P - values for significant differences are indicated .

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D) Bar plots showing mean solubility as determined through quantification of western blot in (C), whiskers indicate standard deviation (n=4). Statistically significant differences between the -DnaK and +DnaK conditions were determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.


E ) Representative western blot of the solubility analysis of GK variants upon overexpression in E . coli K12 . Bands show Soluble ( Sol ) and Pellet fractions after centrifugation at 17 , 100 g for 15 mins . " - DnaK " indicates expression in E . coli K12 DnaK knockout line , " + Dnak " indicates co - expression of pKJE7 , encoding DnaK , DnaJ and GrpE .

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E) Representative western blot of the solubility analysis of GK variants upon overexpression in E. coli K12. Bands show Soluble (Sol) and Pellet fractions after centrifugation at 17,100 g for 15 mins. "-DnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.


F ) Barplots showing mean solubility as determined through quantification of western blot bands in ( E ) , whiskers indicate standard deviation ( n = 7 , except for the AA construct , where n = 4 ) . Statistical significance was determined through two - way ANOVA followed by Tukey ' s multiple comparisons test . P - values are indicated .

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F) Barplots showing mean solubility as determined through quantification of western blot bands in (E), whiskers indicate standard deviation (n=7, except for the AA construct, where n=4). Statistical significance was determined through two-way ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.


G ) Structured Illumination Microsopy ( SIM ) images of E . coli after three hours of expression of one of the GK variants . GFP fluorescence is shown in green . " ΔDnaK " indicates expression in E . coli K12 DnaK knockout line , " + Dnak " indicates co - expression of pKJE7 , encoding DnaK , DnaJ and GrpE .

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G) Structured Illumination Microsopy (SIM) images of E. coli after three hours of expression of one of the GK variants. GFP fluorescence is shown in green. "ΔDnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.


Plasma corticosterone levels at baseline , 30 minutes after induction of stress by placing mice in a clean cage , and after 90 minutes upon return in the home cage . DEX mice received an injection of dexamethasone at 0 . 1 mg / kg body weight 150 minutes before collecting the first blood sample ( n = 5 - 6 animals per group ) . Flox and GRBATKO mice were injected with 0 . 02 mg / kg dexamethasone and the procedure described in ( A ) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in ( A ) ( n = 6 - 7 animals per group ) .

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Plasma corticosterone levels at baseline, 30 minutes after induction of stress by placing mice in a clean cage, and after 90 minutes upon return in the home cage. DEX mice received an injection of dexamethasone at 0.1 mg/kg body weight 150 minutes before collecting the first blood sample (n = 5-6 animals per group). Flox and GRBATKO mice were injected with 0.02 mg/kg dexamethasone and the procedure described in (A) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in (A) (n = 6-7 animals per group).


GR mRNA levels in BAT of Flox and GRBATKO mice . Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage . Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure , controls received an injection with saline ( n = 5 - 6 animals per group ) .

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GR mRNA levels in BAT of Flox and GRBATKO mice. Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage. Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure, controls received an injection with saline (n = 5-6 animals per group).


Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice ( n = 5 - 6 animals per group ) . Blood glucose levels ( n = 5 - 6 animals per group ) .

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Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice (n = 5-6 animals per group). Blood glucose levels (n = 5-6 animals per group).


Knockdown of GR in BAT assessed by immunoblotting . Β - Tubulin was used as loading control . Quantification of immunoblot in ( A ) using Image Lab ( n = 6 animals per group ) .

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Knockdown of GR in BAT assessed by immunoblotting. Β-Tubulin was used as loading control. Quantification of immunoblot in (A) using Image Lab (n = 6 animals per group).


Body temperature over the course of the cold exposure , determined daily ( n = 6 animals per group ) .

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Body temperature over the course of the cold exposure, determined daily (n = 6 animals per group).


Body weight at the end of the cold exposure ( n = 6 animals per group ) .

Body weight at the end of the cold exposure (n = 6 animals per group).


BAT weight at the end of the cold exposure ( n = 6 animals per group ) .

BAT weight at the end of the cold exposure (n = 6 animals per group).


Representative Hematoxilin and eosin staining image of BAT of cold - exposed Flox and GRBATKO mice . Scale bar 50 µm .

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Representative Hematoxilin and eosin staining image of BAT of cold-exposed Flox and GRBATKO mice. Scale bar 50 µm.


Triglyceride content in BAT from Flox and GRBATKO animals ( n = 5 - 6 animals per group ) .

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Triglyceride content in BAT from Flox and GRBATKO animals (n = 5-6 animals per group).


Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals , indicating unchanged thermogenic marker gene expression . Color gradient ranges from 2 - fold higher expression in GRBATKO over Flox controls ( red ) to 0 . 1 - fold ( lower ) expression ( blue ) ( n = 4 animals per group ) .

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Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals, indicating unchanged thermogenic marker gene expression. Color gradient ranges from 2-fold higher expression in GRBATKO over Flox controls (red) to 0.1-fold (lower) expression (blue) (n = 4 animals per group).


Volcano plot from microarray gene expression data as in ( I ) , with cut - off values for the adjusted p - value < 0 . 1 and fold change > 1 . 5 .

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Volcano plot from microarray gene expression data as in (I), with cut-off values for the adjusted p-value <0.1 and fold change >1.5.


Serum corticosterone levels ( n = 6 animals per group ) .

Serum corticosterone levels (n = 6 animals per group).


Body weight after 16 h fasting and 6 h chow refeeding at 22 ° C ( n = 6 animals per group )

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Body weight after 16 h fasting and 6 h chow refeeding at 22°C (n = 6 animals per group)


Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h ( n = 6 animals per group ) .

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Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h (n = 6 animals per group).


Serum insulin and corticosterone levels determined by ELISA ( n = 6 animals per group ) .

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Serum insulin and corticosterone levels determined by ELISA (n = 6 animals per group).


Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding ( n = 6 animals per group ) .

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Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding (n = 6 animals per group).


Body weight gain over a 24 - week period of high - fat feeding ( n = 13 - 14 animals per group ) . Panel shows experimental design for Figures 4 - 6 .

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Body weight gain over a 24-week period of high-fat feeding (n = 13-14 animals per group). Panel shows experimental design for Figures 4-6.


Oxygen consumption over a 90 - hour period . Grays columns indicate dark phase ( n = 6 - 8 mice per group ) . Average oxygen consumption specified for light phase , dark phase , and total ( n = 6 - 8 animals per genotype ) .

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Oxygen consumption over a 90-hour period. Grays columns indicate dark phase (n = 6-8 mice per group). Average oxygen consumption specified for light phase, dark phase, and total (n = 6-8 animals per genotype).


Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight ( n = 6 - 8 animals per group ) .

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Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight (n = 6-8 animals per group).


Locomotor activity over a 90 - hour period ( n = 6 - 8 animals per group ) .

Locomotor activity over a 90-hour period (n = 6-8 animals per group).


Intraperitoneal GTT after 19 weeks of HFD - feeding , glucose injections at 1 . 5 mg per g of body weight ( n = 11 - 13 animals per group ) . Area under the curve calculated from ( A ) ( n = 11 - 13 animals per group ) .

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Intraperitoneal GTT after 19 weeks of HFD-feeding, glucose injections at 1.5 mg per g of body weight (n = 11-13 animals per group). Area under the curve calculated from (A) (n = 11-13 animals per group).


Plasma insulin levels during GTT ( n = 11 - 13 animals per group ) .

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Plasma insulin levels during GTT (n = 11-13 animals per group).


Intraperitoneal ITT after 20 weeks of high - fat diet intervention . Insulin was adjusted to 1 mU per g of body weight ( n = 11 - 13 animals per group ) . Area under the curve determined from ( D ) ( n = 11 - 13 animals per group ) .

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Intraperitoneal ITT after 20 weeks of high-fat diet intervention. Insulin was adjusted to 1 mU per g of body weight (n = 11-13 animals per group). Area under the curve determined from (D) (n = 11-13 animals per group).


Plasma insulin levels after a 5 - hour fast , before injecting insulin ( n = 11 - 13 animals per group ) .

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Plasma insulin levels after a 5-hour fast, before injecting insulin (n = 11-13 animals per group).


Body weight in Flox and GRBATKO after 24 weeks of high - fat feeding ( n = 10 - 13 animals per group ) .

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Body weight in Flox and GRBATKO after 24 weeks of high-fat feeding (n = 10-13 animals per group).


BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks ( n = 10 - 13 animals per group ) .

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BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks (n = 10-13 animals per group).


GR levels in BAT after 24 weeks high - fat feeding ( n = 10 - 13 mice per group ) .

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GR levels in BAT after 24 weeks high-fat feeding (n=10-13 mice per group).


Representative H & E stained sections of BAT from Flox and GRBATKO animals . Scale bar 100 µm .

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Representative H&E stained sections of BAT from Flox and GRBATKO animals. Scale bar 100 µm.


Triglyceride content in BAT per gram of wet tissue ( n = 10 - 13 animals per group ) .

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Triglyceride content in BAT per gram of wet tissue (n = 10-13 animals per group).


qRT - PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high - fat feeding ( n = 9 - 13 animals per group ) .

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qRT-PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high-fat feeding (n = 9-13 animals per group).


Representative immunohistochemistry for UCP1 in BAT . Scale bar 100 µm .

Representative immunohistochemistry for UCP1 in BAT. Scale bar 100 µm.


Serum insulin levels determined by ELISA ( n = 10 - 13 animals per group ) .

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Serum insulin levels determined by ELISA (n = 10-13 animals per group).


Blood glucose in Flox and GRBATKO animals after 24 weeks HFD ( n = 10 - 13 animals per group ) .

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Blood glucose in Flox and GRBATKO animals after 24 weeks HFD (n = 10-13 animals per group).


( A ) Heat map representation of the TNF - α - up - regulated genes . Genes were classified based on the TNF - α - treated time points , using the algorithm " HOPACH " .

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(A) Heat map representation of the TNF-α-up-regulated genes. Genes were classified based on the TNF-α-treated time points, using the algorithm "HOPACH".


( B ) Heat map showing down - regulated miRNAs at 4 hrs , but not 24 hrs , after TNF - α treatment . The middle column indicates the fold - change values ( control / TNF ) at 4 hrs . The right column indicates the relative expression level of each miRNA from the FANTOM5 database ( Consortium et al , 2014 ) . Asterisks indicate the top three most down - regulated miRNAs which were reported to be expressed in human endothelial cells ( ECs ) according to the FANTOM5 database .

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

(B) Heat map showing down-regulated miRNAs at 4 hrs, but not 24 hrs, after TNF-α treatment. The middle column indicates the fold-change values (control/TNF) at 4 hrs. The right column indicates the relative expression level of each miRNA from the FANTOM5 database (Consortium et al, 2014). Asterisks indicate the top three most down-regulated miRNAs which were reported to be expressed in human endothelial cells (ECs) according to the FANTOM5 database.


( C ) Bar plots showing mean mRNA levels of vascular cell adhesion molecule - 1 ( VCAM1 ) , intracellular adhesion molecule - 1 ( ICAM1 ) , and E - selectin ( SELE ) , measured 4 hrs after stimulation of human ECs with or without TNF - α ± miR - 3679 - 5p . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to TNF - α ( - ) mimics ( - ) . † P < 0 . 05 and ‡ P = 0 . 203 compared to TNF - α ( + ) mimics ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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

(C) Bar plots showing mean mRNA levels of vascular cell adhesion molecule-1 (VCAM1), intracellular adhesion molecule-1 (ICAM1), and E-selectin (SELE), measured 4 hrs after stimulation of human ECs with or without TNF-α ± miR-3679-5p. Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 and ‡ P = 0.203 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.


( D ) Western blots for VCAM1 , ICAM , SELE , and β - actin ( ACTB ) in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± miR - 3679 - 5p .

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(D) Western blots for VCAM1, ICAM, SELE, and β-actin (ACTB) in lysates from ECs treated with or without TNF-α (4 hrs) ± miR-3679-5p.


( E ) Representative images ( top ) and bar plot quantification ( bottom ) showing the adhesion of calcein - labeled U937 monocytes to ECs treated with or without TNF - α ± miR - 3679 - 5p . Scale bar represents 250 μm . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) mimics ( - ) . † P < 0 . 05 compared to TNF - α ( + ) mimics ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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

(E) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± miR-3679-5p. Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.


( F ) The scatter plot shows the correlation of RNA immunoprecipitation ( RIP ) followed by microarray data from two replicate screens for the identification of miR - 3679 - 5p target genes . Genes up - and down - regulated by miR - 3679 - 5p treatment ( | average Log2FC | > 1 ) are indicated in red and blue , respectively .

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(F) The scatter plot shows the correlation of RNA immunoprecipitation (RIP)　followed by microarray data from two replicate screens for the identification of miR-3679-5p target genes. Genes up- and down-regulated by miR-3679-5p treatment (|average Log2FC|>1) are indicated in red and blue, respectively.


( J and K ) Effect of miR - 3679 - 5p mimics ( J ) or miR - 3679 - 5p inhibitor ( K ) on luciferase activity in pmirGLO - transfected ECs expressing the 3 ′ UTR of KDM7A . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to control . Statistical differences were analyzed by the Student ' s t test .

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(J and K) Effect of miR-3679-5p mimics (J) or miR-3679-5p inhibitor (K) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of KDM7A. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.


( M and N ) The effect of miR - 3679 - 5p mimics ( M ) or miR - 3679 - 5p inhibitor ( N ) on luciferase activity in pmirGLO - transfected ECs expressing the 3 ′ UTR of UTX was measured . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to control . Statistical differences were analyzed by the Student ' s t test .

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(M and N) The effect of miR-3679-5p mimics (M) or miR-3679-5p inhibitor (N) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of UTX was measured. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.


( A ) Heat map representation of RNA - seq results . The p65 - dependent genes ( total 123 ; defined as genes up - regulated more than 2 - fold via TNF - α treatment and down - regulated more than 0 . 75 - fold via BAY 11 - 7082 treatment ) were classified based on the alteration patterns by the siRNA knockdown of KDM7A , UTX , and both , using the algorithm " HOPACH " .

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(A) Heat map representation of RNA-seq results. The p65-dependent genes (total 123; defined as genes up-regulated more than 2-fold via TNF-α treatment and down-regulated more than 0.75-fold via BAY 11-7082 treatment) were classified based on the alteration patterns by the siRNA knockdown of KDM7A, UTX, and both, using the algorithm "HOPACH".


( C - E ) Bar plot showing mean mRNA levels of VCAM1 , ICAM1 , and SELE measured 4 hrs after stimulation of ECs with or without TNF - α ± ( siKDM7A + siUTX ) . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to TNF - α ( - ) siKDM7A + siUTX ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A + siUTX ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(C-E) Bar plot showing mean mRNA levels of VCAM1, ICAM1, and SELE measured 4 hrs after stimulation of ECs with or without TNF-α ± (siKDM7A+siUTX). Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( F ) Western blots for VCAM1 , ICAM , SELE , and ACTB in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± ( siKDM7A + siUTX ) .

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(F) Western blots for VCAM1, ICAM, SELE, and ACTB in lysates from ECs treated with or without TNF-α (4 hrs) ± (siKDM7A+siUTX).


( G ) Representative images ( top ) and bar plot quantification ( bottom ) showing the adhesion of calcein - labeled U937 monocytes to ECs treated with or without TNF - α ± ( siKDM7A + siUTX ) . Scale bar represents 250 μm . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) siKDM7A + siUTX ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A + siUTX ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(G) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± (siKDM7A+siUTX). Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A and B ) Scatter plots showing the connection between KDM7A or UTX binding and RNA expression in TNF - α - stimulated ECs . The X and Y axes indicate the log2 ratio of fragments per kilobase of exon per million mapped sequence reads ( FPKM ) values and the ChIP - seq signals ( TNF - α 60min / 0min ) for KDM7A ( A ) and UTX ( B ) , respectively . Genes with more than a 1 . 5 - fold increase in FPKM values ( light red dots ) satisfying a 0 . 5 - fold increase in ChIP - seq signals are indicated as dark red dots . Genes with a 1 . 5 - fold decrease in FPKM values ( light blue dots ) satisfying a 0 . 5 - fold decrease in ChIP - seq signals are indicated as dark blue dots . These genes are defined as the target genes for KDM7A ( A ) and UTX ( B ) , respectively . The names of representative genes are shown in the graphs .

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

(A and B) Scatter plots showing the connection between KDM7A or UTX binding and RNA expression in TNF-α-stimulated ECs. The X and Y axes indicate the log2 ratio of fragments per kilobase of exon per million mapped sequence reads (FPKM) values and the ChIP-seq signals (TNF-α 60min/0min) for KDM7A (A) and UTX (B), respectively. Genes with more than a 1.5-fold increase in FPKM values (light red dots) satisfying a 0.5-fold increase in ChIP-seq signals are indicated as dark red dots. Genes with a 1.5-fold decrease in FPKM values (light blue dots) satisfying a 0.5-fold decrease in ChIP-seq signals are indicated as dark blue dots. These genes are defined as the target genes for KDM7A (A) and UTX (B), respectively. The names of representative genes are shown in the graphs.


( C and D ) Metagene representations of KDM7A ( C ) and UTX ( D ) ChIP - seq signals in units of read count per million mapped reads at a meta composite of the genomic regions around the TNF - α - up - regulated genes .

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(C and D) Metagene representations of KDM7A (C) and UTX (D) ChIP-seq signals in units of read count per million mapped reads at a meta composite of the genomic regions around the TNF-α-up-regulated genes.


( F ) Heat map of H3K27ac ( green ) , p65 ( yellow ) , KDM7A ( red ) , and UTX ( purple ) levels in resting and TNF - α - stimulated ECs . Each row shows ± 10 kb centered on the KDM7A ( top ) or UTX ( bottom ) peak . Rows are ordered by max KDM7A or UTX signal in each region . The ChIP - seq signal is depicted by color scaled intensities .

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(F) Heat map of H3K27ac (green), p65 (yellow), KDM7A (red), and UTX (purple) levels in resting and TNF-α-stimulated ECs. Each row shows ± 10 kb centered on the KDM7A (top) or UTX (bottom) peak. Rows are ordered by max KDM7A or UTX signal in each region. The ChIP-seq signal is depicted by color scaled intensities.


( G and H ) Gene tracks of ChIP - seq signals for H3K27Ac , p65 , KDM7A , and UTX , and RNA - seq signals around the VCAM1 ( G ) and SELE ( H ) loci in TNF - α ( - ) and TNF - α ( + ) ECs . ChIP - Seq and RNA - seq signals are visualized by Integrated Genome Viewer ( http : / / software . broadinstitute . org / software / igv / ) .

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(G and H) Gene tracks of ChIP-seq signals for H3K27Ac, p65, KDM7A, and UTX, and RNA-seq signals around the VCAM1 (G) and SELE (H) loci in TNF-α (-) and TNF-α (+) ECs. ChIP-Seq and RNA-seq signals are visualized by Integrated Genome Viewer (http://software.broadinstitute.org/software/igv/).


( I and J ) ChIP - qPCR of p65 ( I ) and KDM7A ( J ) at the VCAM1 TSS , normalized to input . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(I and J) ChIP-qPCR of p65 (I) and KDM7A (J) at the VCAM1 TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). † P < 0.05 compared to TNF-α (+) siKDM7A (-). Statistical differences were analyzed by the Tukey-Kramer test.


( K and L ) ChIP - qPCR of p65 ( K ) and UTX ( L ) at the SELE TSS , normalized to input . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(K and L) ChIP-qPCR of p65 (K) and UTX (L) at the SELE TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A and B ) Boxplots of the log2 fold change of ChIP - seq enrichment of H3K9me2 in KDM7A target genes ( A ) and that of H3K27me3 in UTX target genes ( B ) with TNF - α treatment ( TNF + ) and control ( TNF - ) . The horizontal line within the box represents the median sample value . The ends of the box represent the 3rd and 1st quantiles . The whiskers extend from the ends of the box to the outermost data point that falls within the distances computed as follows : 3rd quartile + 1 . 5 * ( interquartile range ) and 1st quartile - 1 . 5 * ( interquartile range ) . Each dot in the box plot shows data for one single target gene . Genes colored red represent the top five downregulated target genes with TNF - α - treatment , as compared to control . Graphs are representative of two independent experiments .

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(A and B) Boxplots of the log2 fold change of ChIP-seq enrichment of H3K9me2 in KDM7A target genes (A) and that of H3K27me3 in UTX target genes (B) with TNF-α treatment (TNF+) and control (TNF-). The horizontal line within the box represents the median sample value. The ends of the box represent the 3rd and 1st quantiles. The whiskers extend from the ends of the box to the outermost data point that falls within the distances computed as follows: 3rd quartile + 1.5*(interquartile range) and 1st quartile - 1.5*(interquartile range). Each dot in the box plot shows data for one single target gene. Genes colored red represent the top five downregulated target genes with TNF-α-treatment, as compared to control. Graphs are representative of two independent experiments.


( C ) Gene tracks of ChIP - seq signals for H3K9me2 and H3K27me3 around the VCAM1 locus in TNF - α ( - ) and TNF - α ( + ) ECs .

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(C) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around the VCAM1 locus in TNF-α (-) and TNF-α (+) ECs.


( D and E ) ChIP - qPCR showing enrichment ( percent input ) of H3K9me2 ( D ) and H3K27me3 ( E ) around the VCAM1 locus . Primer pairs targeting distinct regions are listed on the X axis . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Student ' s t test .

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(D and E) ChIP-qPCR showing enrichment (percent input) of H3K9me2 (D) and H3K27me3 (E) around the VCAM1 locus. Primer pairs targeting distinct regions are listed on the X axis. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.


( F ) Gene tracks of ChIP - seq signals for H3K9me2 and H3K27me3 around the SELE locus in TNF - α ( - ) and TNF - α ( + ) ECs .

(F) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around the SELE locus in TNF-α (-) and TNF-α (+) ECs.


( G and H ) ChIP - qPCR showing enrichment ( percent input ) of H3K9me2 ( G ) and H3K27me3 ( H ) around the SELE locus . Primer pairs targeting distinct regions are listed on the X axis . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Student ' s t test .

(G and H) ChIP-qPCR showing enrichment (percent input) of H3K9me2 (G) and H3K27me3 (H) around the SELE locus. Primer pairs targeting distinct regions are listed on the X axis. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.


( I and J ) ChIP - qPCR showing enrichment ( percent input ) of H3K9me2 and H3K27me3 around the HOXA13 ( I ) and GATA4 ( J ) loci . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by the Student ' s t test .

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(I and J) ChIP-qPCR showing enrichment (percent input) of H3K9me2 and H3K27me3 around the HOXA13 (I) and GATA4 (J) loci. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.


( K and L ) ChIP - qPCR showing enrichment ( percent input ) of H3K9me2 around VCAM1 - 11kb ( K ) and H3K27me3 around SELE - 12kb ( L ) . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A ( - ) or TNF - α ( + ) siUTX ( - ) . Statistical differences were analyzed by the Tukey - Kramer test .

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(K and L) ChIP-qPCR showing enrichment (percent input) of H3K9me2 around VCAM1 -11kb (K) and H3K27me3 around SELE -12kb (L). Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). † P < 0.05 compared to TNF-α (+) siKDM7A (-) or TNF-α (+) siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A ) Plot of enhancers defined in TNF - α - treated ECs , ranked by increasing bromodomain - containing protein 4 ( BRD4 ) signal . Super enhancers ( SEs ) were defined by ROSE ( Loven et al , 2013 , Whyte et al , 2013 ) , based on the ChIP - Seq signals for BRD4 ( Brown et al , 2014 ) . SEs are indicated by dashed lines and are colored yellow . The numbers show the total SEs and their classification based on KDM7A and UTX binding . Representative SE - related genes are indicated in the graph .

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(A) Plot of enhancers defined in TNF-α-treated ECs, ranked by increasing bromodomain-containing protein 4 (BRD4) signal. Super enhancers (SEs) were defined by ROSE (Loven et al, 2013, Whyte et al, 2013), based on the ChIP-Seq signals for BRD4 (Brown et al, 2014). SEs are indicated by dashed lines and are colored yellow. The numbers show the total SEs and their classification based on KDM7A and UTX binding. Representative SE-related genes are indicated in the graph.


( B and C ) Metagene representations of KDM7A ( B ) and UTX ( C ) ChIP - seq signals in units of read count per million mapped reads , at a meta composite of SEs and typical enhancers ( TEs ) . TEs were obtained from the pre - compiled HUVEC ngsplotdb ( https : / / github . com / shenlab - sinai / ngsplot ) .

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(B and C) Metagene representations of KDM7A (B) and UTX (C) ChIP-seq signals in units of read count per million mapped reads, at a meta composite of SEs and typical enhancers (TEs). TEs were obtained from the pre-compiled HUVEC ngsplotdb (https://github.com/shenlab-sinai/ngsplot).


( D ) Pirate plots showing ChIA - PET interaction changes at control - specific , common , and TNF - α - specific SEs . The Y axis indicates the log2 ratio of ChIA - PET signals ( TNF - α 60 min / 0 min ) . The central bar indicates the average .

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(D) Pirate plots showing ChIA-PET interaction changes at control-specific, common, and TNF-α-specific SEs. The Y axis indicates the log2 ratio of ChIA-PET signals (TNF-α 60 min/0 min). The central bar indicates the average.


( E and F ) Bean plots showing ChIA - PET interaction changes at KDM7A ( E ) and UTX ( F ) binding sites . The Y axis indicates the log2 ratio of ChIA - PET signals ( TNF - α 60 min / 0 min ) . The central bar indicates the median .

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(E and F) Bean plots showing ChIA-PET interaction changes at KDM7A (E) and UTX (F) binding sites. The Y axis indicates the log2 ratio of ChIA-PET signals (TNF-α 60 min/0 min). The central bar indicates the median.


( G and H ) ChIA - PET interactions of active RNA pol II around the VCAM1 ( G ) and IL8 ( H ) loci integrated with the ChIP - seq profiles of KDM7A , UTX , and BRD4 , and RNA - seq profiles in TNF - α ( - ) and TNF - α ( + ) ECs . ChIA - PET interactions were visualized by the WashU Epigenome Browser ( http : / / epigenomegateway . wustl . edu / browser / ) . Interactions detected by ChIA - PET are depicted with purple lines . Red bars show TNF - α - specific SEs .

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(G and H) ChIA-PET interactions of active RNA pol II around the VCAM1 (G) and IL8 (H) loci integrated with the ChIP-seq profiles of KDM7A, UTX, and BRD4, and RNA-seq profiles in TNF-α (-) and TNF-α (+) ECs. ChIA-PET interactions were visualized by the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/browser/). Interactions detected by ChIA-PET are depicted with purple lines. Red bars show TNF-α-specific SEs.


( I - K ) Gene tracks of ChIP - seq signals for H3K9me2 and H3K27me3 around VCAM1 ( I ) , IL8 ( J ) , and SELE ( K ) in TNF - α ( - ) and TNF - α ( + ) ECs . Red bars show TNF - α - specific SEs .

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(I-K) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around VCAM1 (I), IL8 (J), and SELE (K) in TNF-α (-) and TNF-α (+) ECs. Red bars show TNF-α-specific SEs.


( B ) Representative snapshot from the IVM analysis of leukocyte adhesive interactions in the femoral arteries . Margins of vessels are indicated with dashed lines . White spots represent fluorescently labeled leukocytes visualized by the intravenous injection of rhodamine 6G .

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(B) Representative snapshot from the IVM analysis of leukocyte adhesive interactions in the femoral arteries. Margins of vessels are indicated with dashed lines. White spots represent fluorescently labeled leukocytes visualized by the intravenous injection of rhodamine 6G.


( C ) Quantitative analyses of leukocyte adhesive interactions in the femoral arteries . Data are shown as means ± SE ( n = 3 / group ) . * P < 0 . 05 compared to the vehicle + TNF - α group . Statistical differences were analyzed by the Student ' s t test . See also Movies EV1 and 2 .

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(C) Quantitative analyses of leukocyte adhesive interactions in the femoral arteries. Data are shown as means ± SE (n=3/group). *P < 0.05 compared to the vehicle + TNF-α group. Statistical differences were analyzed by the Student's t test. See also Movies EV1 and 2.


( D ) Representative immunoblots ( n = 3 ) showing phosphorylation state of Smad2 and JNK during TGF - β - induced EMT in NMuMG cells . Lamin B serves as loading control .

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(D) Representative immunoblots (n=3) showing phosphorylation state of Smad2 and JNK during TGF-β-induced EMT in NMuMG cells. Lamin B serves as loading control.


( E ) Quantification of one set of representative immunoblots ( in figure D ) and its graphical representation showing dynamic of Smad2 and JNK activation during TGF - β - induced EMT in NMuMG .

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(E) Quantification of one set of representative immunoblots (in figure D) and its graphical representation showing dynamic of Smad2 and JNK activation during TGF-β-induced EMT in NMuMG.


( F ) Representative immunohistochemistry image for pSmad2 and pJNK in non - invasive ( n = 34 ) and invasive ( n = 14 ) breast cancer samples . Scale bars for 10X and 40X images are 200 µm and 50 µm respectively .

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(F) Representative immunohistochemistry image for pSmad2 and pJNK in non-invasive (n=34) and invasive (n=14) breast cancer samples. Scale bars for 10X and 40X images are 200 µm and 50 µm respectively.


( H ) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelialNMuMG cells ( d0 ) and cells treated with TGF - β for seven days in the presence of JNK inhibitor ( d7 JNKi ) or control ( d7 DMSO ) . Staining was performed with antibodies against Fibronectin 1 , with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques . It is important to note that the untreated epithelial cells are highly proliferative and appear small in size , round in shape and adhere to surrounding cells whereas day 7 TGF - β - treated ( mesenchymal ) cells are much bigger in size , elongated in shape and detached from the nearby cells . Scale bar , 100 μm , 63X magnification .

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(H) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelialNMuMG cells (d0) and cells treated with TGF-β for seven days in the presence of JNK inhibitor (d7 JNKi) or control (d7 DMSO). Staining was performed with antibodies against Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. It is important to note that the untreated epithelial cells are highly proliferative and appear small in size, round in shape and adhere to surrounding cells whereas day 7 TGF- β-treated (mesenchymal) cells are much bigger in size, elongated in shape and detached from the nearby cells. Scale bar, 100 μm, 63X magnification.


( I ) mRNA levels for classical EMT markers in cells treated as in ( F ) were measured by qRT - PCRs relative to Ctcf and fold change as compare to untreated and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates .

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(I) mRNA levels for classical EMT markers in cells treated as in (F) were measured by qRT-PCRs relative to Ctcf and fold change as compare to untreated and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.


( J ) Transwell migration assay for NMuMG cells treated as in G . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(J) Transwell migration assay for NMuMG cells treated as in G. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


. ( E ) ChIP assay using H3K27me3 - specific antibody in epithelial cells ( day 0 ) ) or NMuMG cells treated for seven days with TGF - β ( mesenchymal ) and DMSO or JNKi . qRT - PCRs were performed for indicated gene promoters and enrichments are plotted on the right y - axis as ratio of precipitated DNA ( bound ) to total input DNA . On the left y - axis RNA levels of analyzed genes derived from RNA - seq data ( fold change to untreated normalized tag counts ) are plotted . Mean and SEM is plotted from two independent biological replicates .

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. (E) ChIP assay using H3K27me3-specific antibody in epithelial cells (day 0)) or NMuMG cells treated for seven days with TGF-β (mesenchymal) and DMSO or JNKi. qRT-PCRs were performed for indicated gene promoters and enrichments are plotted on the right y-axis as ratio of precipitated DNA (bound) to total input DNA. On the left y-axis RNA levels of analyzed genes derived from RNA-seq data (fold change to untreated normalized tag counts ) are plotted. Mean and SEM is plotted from two independent biological replicates.


( F ) FAIRE assay was performed in cells treated as in ( D ) and qRT - PCRs were performed for the same gene promoters and plotted as in ( E ) .

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(F) FAIRE assay was performed in cells treated as in (D) and qRT-PCRs were performed for the same gene promoters and plotted as in (E).


JNK - induced transcription factors are critically required for EMT . ( A ) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors . Right panel shows RNA levels ( normalized tag counts ) for these factors .

JNK-induced transcription factors are critically required for EMT. (A) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors. Right panel shows RNA levels (normalized tag counts) for these factors.


( B ) Same overlap as in ( A ) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT . Right panel displays RNA levels as in ( A ) for 10 transcription factors identified in this overlap .

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(B) Same overlap as in (A) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT. Right panel displays RNA levels as in (A) for 10 transcription factors identified in this overlap.


( C ) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days ( d4 ) of siRNA - mediated depletion of factors compared to non - targeting control ( NTC ) . Staining was performed with antibodies against epithelial markers E - cadherin and ZO1 , mesenchymal markers Fibronectin 1 , with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques . Scale bar , 100 μm , 40X magnification . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(C) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days (d4) of siRNA-mediated depletion of factors compared to non-targeting control (NTC). Staining was performed with antibodies against epithelial markers E-cadherin and ZO1, mesenchymal markers Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. Scale bar, 100 μm, 40X magnification. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Depletion of Prkrir , Pcbp4 and Tsc22d1 rescues TGF - β - induced reduction in the rate of cell division . ( A ) Lineage trees tracked by live imaging under control , Non treated control ( NTC ) and siRNA - mediated depletion of factors ( X = cell death / cells could not be tracked till the end ) .

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Depletion of Prkrir, Pcbp4 and Tsc22d1 rescues TGF-β-induced reduction in the rate of cell division. (A) Lineage trees tracked by live imaging under control, Non treated control (NTC) and siRNA-mediated depletion of factors (X= cell death/cells could not be tracked till the end).


( B ) Average number of rounds of division per clone in NTC compared with control and siRNA - mediated depletion of factors ( ANOVA , Tukeys post - test , * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(B) Average number of rounds of division per clone in NTC compared with control and siRNA-mediated depletion of factors (ANOVA, Tukeys post-test, *=p<0.05,**=p<0.01).


PRKRIR , PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate . ( A ) Scratch assay in MDA - MB - 231 cells treated for four days with DMSO or JNKi . Scale bar , 200 μm ; 20 X magnification . Mean and SEM is plotted from three independent biological replicates .

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PRKRIR, PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate. (A) Scratch assay in MDA-MB-231 cells treated for four days with DMSO or JNKi. Scale bar, 200 μm; 20 X magnification. Mean and SEM is plotted from three independent biological replicates.


( B ) mRNA levels for key EMT marker genes in MDA - MB - 231 treated with DMSO or JNKi for 4 days were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological

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(B) mRNA levels for key EMT marker genes in MDA-MB-231 treated with DMSO or JNKi for 4 days were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates


( C ) mRNA levels for transcription factors in isogenic epithelial ( MDA - MB - 361 ) and mesenchymal ( MDA - MB - 231 ) breast cancer cells were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates .

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(C) mRNA levels for transcription factors in isogenic epithelial (MDA-MB-361) and mesenchymal (MDA-MB-231) breast cancer cells were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.


( D ) Scratch assay in MDA - MB - 231 cells transfected with NTC or siRNA against PRKRIR , NFIL3 , PCBP4 and TSC22D1 for 4 days as in A . Mean and SEM is plotted from three independent biological replicates as bar plot on right .

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(D) Scratch assay in MDA-MB-231 cells transfected with NTC or siRNA against PRKRIR, NFIL3, PCBP4 and TSC22D1 for 4 days as in A. Mean and SEM is plotted from three independent biological replicates as bar plot on right .


( E ) Invasion assay in MDA - MB - 231 cells treated as in ( D ) . Mean and SEM is plotted from three independent biological replicates .

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(E) Invasion assay in MDA-MB-231 cells treated as in (D). Mean and SEM is plotted from three independent biological replicates.


( F ) mRNA levels of key EMT marker genes in MDA - MB - 231 and MDA - MB - 231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor ( SP600125 ) for 24 hours were measured by qRT - PCRs relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(F) mRNA levels of key EMT marker genes in MDA-MB-231 and MDA-MB-231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor (SP600125) for 24 hours were measured by qRT-PCRs relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


( C ) mRNA levels for representative genes highlighted in ( A ) for cells treated like in ( Fig 5D ) were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates .

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(C) mRNA levels for representative genes highlighted in (A) for cells treated like in (Fig 5D) were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.


F ) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag - HA tagged Tsc22d1 and detection using an anti - Flag antibody , Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus . Y - axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions . SEM is derived from independent biological replicates .

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F) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag-HA tagged Tsc22d1 and detection using an anti-Flag antibody, Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus. Y-axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions. SEM is derived from independent biological replicates.


( G ) ChIP assay using anti - Flag antibody following expression of Flag - HA tagged TSC22D1 in MDA - MB - 231 cells . Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y - axis as ratio of precipitated DNA ( bound ) to total input DNA and then further divided by the same obtained in the empty vector transfected cells . SEM is derived from independent biological replicates .

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(G) ChIP assay using anti-Flag antibody following expression of Flag-HA tagged TSC22D1 in MDA-MB-231 cells. Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y-axis as ratio of precipitated DNA (bound) to total input DNA and then further divided by the same obtained in the empty vector transfected cells. SEM is derived from independent biological replicates.


( H ) mRNA levels for PRKRIR , PCBP4 , TSC22D1 in non - invasive ( DCIS ) ( n = 20 ) and invasive tumor ( n = 20 ) samples were measured by qRT PCRs relative to TBP and plotted on the y - axis . ZEB1 was used as an established positive control for invasive tumors . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(H) mRNA levels for PRKRIR, PCBP4, TSC22D1 in non-invasive (DCIS) (n=20) and invasive tumor (n=20) samples were measured by qRT PCRs relative to TBP and plotted on the y-axis. ZEB1 was used as an established positive control for invasive tumors. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration . ( A ) RT - qPCR analysis for key EMT markers in embryonic stem cells ( ES ) , neuronal progenitors ( NP ) and terminally differentiated neurons ( TN ) . mRNA levels were measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

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Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration. (A) RT-qPCR analysis for key EMT markers in embryonic stem cells (ES), neuronal progenitors (NP) and terminally differentiated neurons (TN). mRNA levels were measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( B ) Western blot analyzing total JNK and phospho - JNK ( p - JNK ) during neuronal differentiation of stem cells . β - actin serve as a loading control .

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(B) Western blot analyzing total JNK and phospho-JNK (p-JNK) during neuronal differentiation of stem cells. β-actin serve as a loading control.


( C ) RNA levels of Pcbp4 , Prkrir and Tsc22d1 during neuronal differentiation as shown in A , measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

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(C) RNA levels of Pcbp4, Prkrir and Tsc22d1 during neuronal differentiation as shown in A, measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( D ) RNA levels of Pcbp4 , Prkrir , Tsc22d1 , represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 ( JNKi ) for 6 hours .

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(D) RNA levels of Pcbp4, Prkrir, Tsc22d1, represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 (JNKi) for 6 hours.


( E ) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in ( A ) , measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

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(E) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in (A), measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( F ) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA - seq data from Ventricular Zone ( VZ ) , Sub - Ventricular Zone ( SVZ ) and Cortical plate ( CP ) of E14 . 5 mouse Cortex .

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(F) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA-seq data from Ventricular Zone (VZ), Sub-Ventricular Zone (SVZ) and Cortical plate (CP) of E14.5 mouse Cortex.


( G ) RNA levels of genes shown in Figure ( E ) , represented by average normalized tag counts derived from RNA - seq data of TGF - β - induced EMT in NMuMG cells and plotted on the y - axis . Error bars represent SEM from three biological replicates .

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(G) RNA levels of genes shown in Figure (E), represented by average normalized tag counts derived from RNA-seq data of TGF-β-induced EMT in NMuMG cells and plotted on the y-axis. Error bars represent SEM from three biological replicates.


( I ) A representative image from immunofluorescence analysis performed with anti - GFP and anti - Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain . The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining . y axis represent percentage of cells above or below the Tbr1 stain region . Error bars represent SEM from 3 independent biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(I) A representative image from immunofluorescence analysis performed with anti-GFP and anti-Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain. The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining. y axis represent percentage of cells above or below the Tbr1 stain region. Error bars represent SEM from 3 independent biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


( C ) To assign identities to these germ cell subclusters , the violin plots were used to visualize the distribution of normalized typical marker genes expression levels .

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(C) To assign identities to these germ cell subclusters, the violin plots were used to visualize the distribution of normalized typical marker genes expression levels.


( E ) The expression of GSC maintenance genes ( aret , Mei - P26 , egg , Myc and Hrb98DE ) and differentiation genes ( bam , bgcn , out , twin and Set1 ) along the primary branch ( dotted circle line ) in pseudotime .

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(E) The expression of GSC maintenance genes (aret, Mei-P26, egg, Myc and Hrb98DE) and differentiation genes (bam, bgcn, out, twin and Set1) along the primary branch (dotted circle line) in pseudotime.


( C ) The typical phenotypes of germaria in selected RNAi lines stained with anti - α - Spectrin ( green ) and anti - pMad ( red ) . The scale bar is 10 μm .

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(C) The typical phenotypes of germaria in selected RNAi lines stained with anti-α-Spectrin (green) and anti-pMad (red). The scale bar is 10 μm.


( E ) The heat map of SCENIC analysis of the GSC , undifferentiated germ cell - 1 , undifferentiated germ cell - 2 , early nurse cell , oocyte and later nurse cell subclusters .

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(E) The heat map of SCENIC analysis of the GSC, undifferentiated germ cell-1, undifferentiated germ cell-2, early nurse cell, oocyte and later nurse cell subclusters.


( B ) The expression patterns of candidate marker genes were validated by using in situ hybridization . The scale bar is 10 μm .

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(B) The expression patterns of candidate marker genes were validated by using in situ hybridization. The scale bar is 10 μm.


( C - C ' ) Immunofluorescence staining with anti - TfIIA - S on wild type and nos - Gal4 > UAS - TfIIA - S - RNAi ( negative control ) . The scale bar is 10 μm .

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(C-C') Immunofluorescence staining with anti-TfIIA-S on wild type and nos-Gal4>UAS-TfIIA-S-RNAi (negative control). The scale bar is 10 μm.


( E - E ' ) Immunofluorescence staining with anti - eggpl on wild type and nos - Gal4 > UAS - eggpl - RNAi ( negative control ) ovary . The scale bar is 10 μm .

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(E-E') Immunofluorescence staining with anti-eggpl on wild type and nos-Gal4>UAS-eggpl-RNAi (negative control) ovary. The scale bar is 10 μm.


( F ) The overexpression of GFP ( green ) and eggpl in Sf9 cell line in vitro . The anti - eggpl ( red ) was used to detect the eggpl protein , and Hoechst ( blue ) was used to label the nucleus . The scale bar is 10 μm .

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(F) The overexpression of GFP (green) and eggpl in Sf9 cell line in vitro. The anti-eggpl (red) was used to detect the eggpl protein, and Hoechst (blue) was used to label the nucleus. The scale bar is 10 μm.


( A - A ' ) The mRNA in situ hybridization of eggpl ( green ) on wild type and nos - Gal4 > UAS - eggpl - RNAi ( negative control ) line . The scale bar is 10 μm . ( B - C ) Immunofluorescence staining with anti - α - Spectrin ( red ) and anti - Vasa ( red ) on eggpl - in situ ( green ) labeled tissues respectively . The scale bar is 10 μm . The dashed line indicates the eggpl - in situ ( green ) signals .

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(A-A') The mRNA in situ hybridization of eggpl (green) on wild type and nos-Gal4>UAS-eggpl-RNAi (negative control) line. The scale bar is 10 μm. (B-C) Immunofluorescence staining with anti-α-Spectrin (red) and anti-Vasa (red) on eggpl-in situ (green) labeled tissues respectively. The scale bar is 10 μm. The dashed line indicates the eggpl-in situ (green) signals.


( F and H ) The mRNA in situ hybridization of eggpl ( green ) on nos - Gal4 > UAS - eggpl - RNAi ( negative control ) ( F ) and nos - Gal4 > UAS - eggpl - GFP lines ( H ) . The scale bar is 10 μm . ( G ) The anti - GFP , anti - α - Spectrin ( red ) and anti - Bam ( blue ) were used to stain on eggpl : : GFP knock - in line . The scale bar is 10 μm . ( I ) Double staining with eggpl in situ and anti - GFP on eggpl knock - in lines . The scale bar is 10 μm . The dashed line indicates the eggpl - in situ ( green ) signals .

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(F and H) The mRNA in situ hybridization of eggpl (green) on nos-Gal4>UAS-eggpl-RNAi (negative control) (F) and nos-Gal4>UAS-eggpl-GFP lines (H). The scale bar is 10 μm. (G) The anti-GFP, anti-α-Spectrin (red) and anti-Bam (blue) were used to stain on eggpl::GFP knock-in line. The scale bar is 10 μm. (I) Double staining with eggpl in situ and anti-GFP on eggpl knock-in lines. The scale bar is 10 μm. The dashed line indicates the eggpl-in situ (green) signals.


( K ) The phenotypes of wild type , nos - Gal4 > UAS - eggpl - RNAi line and nos - Gal4 > UAS - eggpl : : GFP line staining with anti - α - Spectrin ( green ) , anti - pMad ( red ) . The scale bar is 10 μm .

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(K) The phenotypes of wild type, nos-Gal4>UAS-eggpl-RNAi line and nos-Gal4>UAS-eggpl::GFP line staining with anti-α-Spectrin (green), anti-pMad (red). The scale bar is 10 μm.


( L ) The average number of GSC and CB in nos - Gal4 line , nos - Gal4 > UAS - eggpl - RNAi line and nos - Gal4 > UAS - eggpl : : GFP line on 2 day , 7 day , 14 day and 21 day . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference , * P < 0 . 05 , * * * P < 0 . 001 , n ≥ 72 , biological replicates .

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(L) The average number of GSC and CB in nos-Gal4 line, nos-Gal4>UAS-eggpl-RNAi line and nos-Gal4>UAS-eggpl::GFP line on 2 day, 7 day, 14 day and 21 day. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, *P<0.05, ***P<0.001, n ≥ 72, biological replicates.


( Q ) The statistical analysis of the average number of GSCs and CBs and Cysts in eggpl [ 1 ] line . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference , * * * P < 0 . 001 , n ≥ 104 , biological replicates .

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(Q) The statistical analysis of the average number of GSCs and CBs and Cysts in eggpl[1] line. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, ***P<0.001, n ≥ 104, biological replicates.


( R ) The average number of apoptotic germ cells in wild type , nos - Gal4 > UAS - eggpl - RNAi line and eggpl [ 1 ] line . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference ( biological replicates , n = 3 ) , and N represents the total number of counted ovarioles .

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(R) The average number of apoptotic germ cells in wild type, nos-Gal4>UAS-eggpl-RNAi line and eggpl[1] line. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference (biological replicates, n=3), and N represents the total number of counted ovarioles.