( 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 .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

(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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

(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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) 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 .

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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 .

O O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O O O O O

(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 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(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 .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O

(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 .

O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) 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 .

O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A ) Immunofluorescence staining with anti - α - Spectrin ( red ) , anti - Vasa ( blue ) and anti - GFP ( green ) on adult ovary . The scale bar is 10 μm . ( B ) Immunofluorescence staining on PGCs of larvae ovary . The scale bar is 20 μm . ( C ) Immunofluorescence staining on GSCs and early spermatogonia in adult testis . The scale bar is 20 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O O O

(A) Immunofluorescence staining with anti-α-Spectrin (red), anti-Vasa (blue) and anti-GFP (green) on adult ovary. The scale bar is 10 μm. (B) Immunofluorescence staining on PGCs of larvae ovary. The scale bar is 20 μm. (C) Immunofluorescence staining on GSCs and early spermatogonia in adult testis. The scale bar is 20 μm.


( D ) Immunofluorescence staining on PGCs of larvae testis tissue . The scale bar is 20 μm . ( E ) Immunofluorescence staining with anti - α - Spectrin ( red ) and anti - Vasa ( green ) on testis from wild type or eggpl [ 1 ] flies . The scale bar is 10 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O B-TISSUE O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O

(D) Immunofluorescence staining on PGCs of larvae testis tissue. The scale bar is 20 μm. (E) Immunofluorescence staining with anti-α-Spectrin (red) and anti-Vasa (green) on testis from wild type or eggpl[1] flies. The scale bar is 10 μm.


( A ) Immunofluorescence staining with anti - BrdU ( red ) . The scale bar is 10 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O

(A) Immunofluorescence staining with anti-BrdU (red). The scale bar is 10 μm.


( B ) The oviposition of wild type , Nos - Gal4 > UAS - eggpl - RNAi and eggpl [ 1 ] line over 21 days . Error bars show mean ± SEM , 3 biological replicates ( n = 3 ) were performed , and there were 10 female and 5 male adult flies in each biological replicate .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O 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-ORGANISM O O O O O

(B) The oviposition of wild type, Nos-Gal4>UAS-eggpl-RNAi and eggpl[1] line over 21 days. Error bars show mean ± SEM, 3 biological replicates (n=3) were performed, and there were 10 female and 5 male adult flies in each biological replicate.


( F ) Immunofluorescence staining with anti - GFP on germaria from eggpl : : GFP knock - in flies with or without feeding fresh yeast paste on day 2 - , 7 - , 14 - and 21 . The dotted line indicates the examined area . The scale bar is 10 μm . ( G ) Quantification of the GFP intensity mean in the eggpl : : GFP knock - in line . The central band indicates the median , the box length indicates the interquartile range , and the upper and whisker indicate the maximum and minimum value respectively . The 10 biological replicates ( n = 10 ) were performed , one - way ANOVA , * * * P < 0 . 001 . The dashed line indicates the examined area .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Immunofluorescence staining with anti-GFP on germaria from eggpl::GFP knock-in flies with or without feeding fresh yeast paste on day 2-, 7-, 14- and 21. The dotted line indicates the examined area. The scale bar is 10 μm. (G) Quantification of the GFP intensity mean in the eggpl::GFP knock-in line. The central band indicates the median, the box length indicates the interquartile range, and the upper and whisker indicate the maximum and minimum value respectively. The 10 biological replicates (n=10) were performed, one-way ANOVA, ***P<0.001. The dashed line indicates the examined area.


( H - I ) Images of ovaries or mature eggs from flies of the indicated genotypes , feeding conditions , and days post - eclosion showing comparisons of the overall ovary sizes ( F ) and the numbers of retained matured eggs ( G ) . The scar bar is 500 μm . ( J - K ) Quantitative analysis of the ovary size ( H ) and the number of retained mature eggs . ( I ) under different food conditions . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . The 3 biological replicates ( n = 3 ) were performed .

O O O O O O O B-TISSUE O O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-I) Images of ovaries or mature eggs from flies of the indicated genotypes, feeding conditions, and days post-eclosion showing comparisons of the overall ovary sizes (F) and the numbers of retained matured eggs (G). The scar bar is 500 μm. (J-K) Quantitative analysis of the ovary size (H) and the number of retained mature eggs. (I) under different food conditions. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, *P<0.05, **P<0.01, ***P<0.001. The 3 biological replicates (n=3) were performed.


( B ) Quantification of the GFP signal in germaria from eggpl : : GFP knock - in flies of the indicated genotypes under different conditions . The central band indicates the median , the box length indicates the interquartile range , and the upper and whisker indicate the maximum and minimum value respectively , one - way ANOVA , * * P < 0 . 01 , * * * P < 0 . 001 , n = 10 , biological replicates .

O O O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the GFP signal in germaria from eggpl::GFP knock-in flies of the indicated genotypes under different conditions. The central band indicates the median, the box length indicates the interquartile range, and the upper and whisker indicate the maximum and minimum value respectively, one-way ANOVA, **P<0.01, ***P<0.001, n = 10, biological replicates.


( C ) Immunofluorescence staining with anti - GFP on germaria with or without feeding fresh yeast paste on 7th day . The dashed line indicates the examined area . The scale bar is 10 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Immunofluorescence staining with anti-GFP on germaria with or without feeding fresh yeast paste on 7th day. The dashed line indicates the examined area. The scale bar is 10 μm.


A . Immunoblotting of c - MYC and ATF4 in melanoma cells treated with NT - siRNA ( - ) or siASNS ( # 1 or # 2 ) for 72 hr .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O

A. Immunoblotting of c-MYC and ATF4 in melanoma cells treated with NT-siRNA (-) or siASNS (#1 or #2) for 72 hr.


Reverse transcription with quantitative PCR ( RT - qPCR ) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS ( # 1 or # 2 ) ( B )

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O

Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2) (B)


C . Reverse transcription with quantitative PCR ( RT - qPCR ) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS , siATF4 , or both ( C ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-DISEASE O O O O O O O O O O O O O O O O

C. Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS, siATF4, or both (C).


D . Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS , siATF4 , or both .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O

D. Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS, siATF4, or both.


E . Proliferation of melanoma cells over indicated times after treatment with siASNS , siATF4 , or both .

O O B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O

E. Proliferation of melanoma cells over indicated times after treatment with siASNS, siATF4, or both.


F . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS ( # 1 or # 2 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-DISEASE O O O O O O O O O O O O O O O

F. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2).


Immunoblotting of c - MYC in melanoma cells treated for 72 hr with siASNS , L - Asn , or both ( G )

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

Immunoblotting of c-MYC in melanoma cells treated for 72 hr with siASNS, L-Asn, or both (G)


H . Immunoblotting of c - MYC in A375 cells treated as indicated for 72 hr ( H ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O O O

H. Immunoblotting of c-MYC in A375 cells treated as indicated for 72 hr (H).


A . Immunoblotting of c - MYC in A375 cells treated with NT - siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints ( left ) . Plot showing relative protein levels of c - MYC ( y - axis ) as a function of time lapsed ( x - axis ) in the left panel ( right ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD 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. Immunoblotting of c-MYC in A375 cells treated with NT-siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints (left). Plot showing relative protein levels of c-MYC (y-axis) as a function of time lapsed (x-axis) in the left panel (right).


Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with NT - siRNA ( - ) or siASNS ( # 1 or # 2 ) ( B and C )

B-EXP_ASSAY O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with NT-siRNA (-) or siASNS (#1 or #2) (B and C)


Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS , PLX - 4032 , or both ( D ) .

B-EXP_ASSAY O O O O B-DISEASE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, PLX-4032, or both (D).


E . Immunoblotting of indicated proteins in A375 cells cultured in L - Asn and treated with L - A ' ase , PLX - 4032 , or both for 72 hr .

O O B-EXP_ASSAY O O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

E. Immunoblotting of indicated proteins in A375 cells cultured in L-Asn and treated with L-A'ase, PLX-4032, or both for 72 hr.


F . A375 cells were treated with siASNS , SCH772984 , or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3 - β .

O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O

F. A375 cells were treated with siASNS, SCH772984, or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3-β.


G . A375 cells were treated with siASNS , HA - GSK3 - β ( S9A ) , or both for 72 hr followed by immunoblotting of indicated proteins .

O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O

G. A375 cells were treated with siASNS, HA-GSK3-β (S9A), or both for 72 hr followed by immunoblotting of indicated proteins.


H . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-DISEASE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

H. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.


I . A375 cells were treated as indicated for 72 hr followed by immunoblotting of c - MYC .

O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O

I. A375 cells were treated as indicated for 72 hr followed by immunoblotting of c-MYC.


J . Immunoblotting of c - MYC and HA proteins in A375 cells treated as indicated for 72 hr .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O

J. Immunoblotting of c-MYC and HA proteins in A375 cells treated as indicated for 72 hr.


A . Immunoblotting of indicated proteins in melanoma cells treated with siASNS , sicMYC ( # 1 or # 2 ) , or a combination of siASNS and sicMYC ( # 1 or # 2 ) for 72 hr .

O O B-EXP_ASSAY O O O O B-DISEASE O O O O O O O O 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. Immunoblotting of indicated proteins in melanoma cells treated with siASNS, sicMYC (#1 or #2), or a combination of siASNS and sicMYC (#1 or #2) for 72 hr.


B . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS , sicMYC , or both .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-DISEASE O O O O O O O O O O O O O

B. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, sicMYC, or both.


C . Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS , sicMYC , PLX - 4032 , or a combination of siASNS and sicMYC , or siASNS and PLX - 4032 .

O O B-EXP_ASSAY O O O O B-DISEASE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

C. Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, sicMYC, PLX-4032, or a combination of siASNS and sicMYC, or siASNS and PLX-4032.


D . Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

D. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.


E . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

E. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, HA-GSK3-β (S9A), or both.


F . A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins .

O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O

F. A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins.


Proliferation of melanoma cells over indicated times after treatment with siASNS , sicMYC , or both ( G ) ,

B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O O O O

Proliferation of melanoma cells over indicated times after treatment with siASNS, sicMYC, or both (G),


Proliferation of melanoma cells over indicated times after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both ( H ) .

B-EXP_ASSAY O B-DISEASE O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

Proliferation of melanoma cells over indicated times after treatment with siASNS, HA-GSK3-β (S9A), or both (H).


A . Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS , siSLC1A5 , siSLC7A5 , or a combination of siASNS and siSLC1A5 , or siASNS and siSLC7A5 .

O O B-EXP_ASSAY O O O O B-CELL_LINE O 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. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, siSLC1A5, siSLC7A5, or a combination of siASNS and siSLC1A5, or siASNS and siSLC7A5.


B . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , siSLC7A5 , or both .

B. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC7A5, or both.


C . Proliferation of melanoma cells over indicated times after treatment with siASNS , siSLC7A5 , or both .

O O B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O

C. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC7A5, or both.


D . GC - MS - based estimation of intracellular amino acid levels in A375 cells treated with siASNS , siATF4 , or both ( top left ) ; siASNS , sicMYC , or both ( top right ) ; siASNS , siSLC1A5 , or both ( bottom left ) ; or siASNS , siSLC7A5 , or both ( bottom right ) . The fold change is relative to NT - siRNA - treated A375 cells .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE O O

D. GC-MS-based estimation of intracellular amino acid levels in A375 cells treated with siASNS, siATF4, or both (top left); siASNS, sicMYC, or both (top right); siASNS, siSLC1A5, or both (bottom left); or siASNS, siSLC7A5, or both (bottom right). The fold change is relative to NT-siRNA-treated A375 cells.


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment ( E ) ,

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Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment (E),


Immunoblotting of indicated proteins in A375 cells 72 hr after , treatment with siASNS , sicMYC , or

B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O

Immunoblotting of indicated proteins in A375 cells 72 hr after , treatment with siASNS, sicMYC, or both


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS , siSLC7A5 , or both ( G )

B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS, siSLC7A5, or both (G)


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , BCH , or both ( H )

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Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, BCH, or both (H)


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , HA - GSK3 - β ( S9A ) , or both ( I )

B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, HA-GSK3-β (S9A), or both (I)


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS or indicated combinations ( J )

B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS or indicated combinations (J)


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , siSLC3A2 , or both ( K ) .

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, siSLC3A2, or both (K).


L . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , siSLC3A2 , or both .

L. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC3A2, or both.


M . Proliferation of melanoma cells over indicated times after treatment with siASNS , siSLC3A2 , or both .

M. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC3A2, or both.


A . Confocal microscopy image of PD myotubes ( cl . 3LE8 ) infected for 72 h with adenovirus containing TFEB ( PD + Ad - TFEB 72 h ) shows a dramatic reduction in the number of large LAMP1 - positive lysosomes ( red ) compared to that in untreated ( PD ) or adenovirus ( PD + Ad - null ) - treated PD myotubes . WT myotubes are shown on the left panel . Nuclei are stained with Hoechst ( blue ) . TFEB was detected with anti - Flag antibody ( green ) . B . Distribution of lysosomal size differs significantly in Ad - null and Ad - TFEB PD myotubes ( p = 6 . 32 × 10 − 8 ; Kolmogorov - Smirnov test ) . Lysosomal size is expressed as number of pixels representing lysosomal area ( LAMP1 - positive structures ) . The median lysosomal size of Ad - TFEB infected myotubes ( m = 367 . 13 pixels , n = 703 , range 208 - 2659 ) was significantly lower than that of Ad - null infected myotubes ( m = 491 . 16 pixels , n = 1395 , range 200 - 2857 ; p = 6 . 5 × 10 − 12 ; Wilcoxon rank sum test ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-ORGANISM O O O B-ORGANISM O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O B-ORGANISM O B-GENEPROD O B-CELL_TYPE O O O 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-ORGANISM O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O 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. Confocal microscopy image of PD myotubes (cl. 3LE8) infected for 72 h with adenovirus containing TFEB (PD + Ad-TFEB 72 h) shows a dramatic reduction in the number of large LAMP1-positive lysosomes (red) compared to that in untreated (PD) or adenovirus (PD + Ad-null)-treated PD myotubes. WT myotubes are shown on the left panel. Nuclei are stained with Hoechst (blue). TFEB was detected with anti-Flag antibody (green).B. Distribution of lysosomal size differs significantly in Ad-null and Ad-TFEB PD myotubes (p = 6.32 × 10−8; Kolmogorov-Smirnov test). Lysosomal size is expressed as number of pixels representing lysosomal area (LAMP1-positive structures). The median lysosomal size of Ad-TFEB infected myotubes (m = 367.13 pixels, n = 703, range 208-2659) was significantly lower than that of Ad-null infected myotubes (m = 491.16 pixels, n = 1395, range 200-2857; p = 6.5 × 10−12; Wilcoxon rank sum test).


C . Confocal microscopy image of PD myotubes infected for 24 h with Ad - TFEB shows relocation of lysosomes to the plasma membrane ( top ) . Images showing LAMP1 staining ( red ) on plasma membrane in a PD myotube infected with Ad - TFEB ( bottom ; arrows ) but not in a non - infected cell ( middle ) . Non - permeabilized cells were incubated with anti - LAMP1 antibody at 4 ° C for 40 min , followed by fixation and staining with secondary antibody .

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C. Confocal microscopy image of PD myotubes infected for 24 h with Ad-TFEB shows relocation of lysosomes to the plasma membrane (top). Images showing LAMP1 staining (red) on plasma membrane in a PD myotube infected with Ad-TFEB (bottom; arrows) but not in a non-infected cell (middle). Non-permeabilized cells were incubated with anti-LAMP1 antibody at 4°C for 40 min, followed by fixation and staining with secondary antibody.


D . Confocal microscopy images of live non - infected PD myotubes ( left ) or PD myotubes infected for 72 h with Ad - TFEB ( right ) show a dramatic reduction in the amount of accumulated glycogen in TFEB - treated cells . The cells were incubated with the fluorescent glucose ( 2 - NBDG ; green ) , extensively washed , and analysed by confocal microscopy . Bar : 10 µm for all panels .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

D. Confocal microscopy images of live non-infected PD myotubes (left) or PD myotubes infected for 72 h with Ad-TFEB (right) show a dramatic reduction in the amount of accumulated glycogen in TFEB-treated cells. The cells were incubated with the fluorescent glucose (2-NBDG; green), extensively washed, and analysed by confocal microscopy. Bar: 10 µm for all panels.


Confocal microscopy of live unstained fibres from a GFP - LC3 : WT mouse ( WT ) and from untreated ( GAA − / − ) or ERT - treated ( GAA − / − ; + ERT ) GFP - LC3 : GAA − / − mice . Images show typical centrally located autophagic build - up with multiple clusters of LC3 - positive autophagosomes . Structures like these are never seen in WT muscle . Autophagic build - up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles ( not shown ) , but only in 60 - 70 % of FDBfibres . Labelled recombinant human GAA ( red ) was administered i . v . into 3 - 4 month - old GFP - LC3 : GAA − / − mice ( n = 5 ) at a dose of 100 mg / kg twice with a 24 h interval . Mice were sacrificed the next day , and live fibres ( shown for EDLmuscle ) were analysed by confocal microscopy . The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles . At least 500 fibres were analysed for each condition . Bar : 10 µm .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM 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-ORGANISM O O O O O O O O B-CELL_TYPE O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-CELL_TYPE O O O O O O O O O O O O

Confocal microscopy of live unstained fibres from a GFP-LC3:WT mouse (WT) and from untreated (GAA−/−) or ERT-treated (GAA−/−; +ERT) GFP-LC3:GAA−/− mice. Images show typical centrally located autophagic build-up with multiple clusters of LC3-positive autophagosomes. Structures like these are never seen in WT muscle. Autophagic build-up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles (not shown), but only in 60-70% of FDBfibres. Labelled recombinant human GAA (red) was administered i.v. into 3-4 month-old GFP-LC3:GAA−/− mice (n = 5) at a dose of 100 mg/kg twice with a 24 h interval. Mice were sacrificed the next day, and live fibres (shown for EDLmuscle) were analysed by confocal microscopy. The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles. At least 500 fibres were analysed for each condition. Bar: 10 µm.


A . Immunostaining of non - infected cells ( day 13 in differentiation medium ) and cells infected with a mutant form of TFEB ( TFEBmt ) with LAMP1 ( red ) and Flag ( green ) . Ad - TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium . Ad - TFEBmt - infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size , similar to that seen with TFEB .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O

A. Immunostaining of non-infected cells (day 13 in differentiation medium) and cells infected with a mutant form of TFEB (TFEBmt) with LAMP1 (red) and Flag (green). Ad-TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium. Ad-TFEBmt-infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size, similar to that seen with TFEB.


B . Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction ; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt - treated samples may reflect the differences in the glycosylation pattern .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O

B. Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt-treated samples may reflect the differences in the glycosylation pattern.


C . Western blot of protein lysates from untreated , Ad - null , TFEB , and TFEBmt - treated myotubes with anti - caspase - 3 antibody . No activated ( cleaved ) products are detected in any condition . α - Tubulin was used as a loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

C. Western blot of protein lysates from untreated, Ad-null, TFEB, and TFEBmt-treated myotubes with anti-caspase-3 antibody. No activated (cleaved) products are detected in any condition. α-Tubulin was used as a loading control.


D . TUNEL assay shows the presence of some apoptotic cells in Ad - TFEB - infected cultures , but not in control cultures infected with adenovirus alone ( n = 3 ) . Bar : 10 µm for all panels .

O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

D. TUNEL assay shows the presence of some apoptotic cells in Ad-TFEB-infected cultures, but not in control cultures infected with adenovirus alone (n = 3). Bar: 10 µm for all panels.


A . Confocal microscopy images of live fibres derived from 3 to 4 month - old GFP - LC3 : WT ( top left ) , untreated GFP - LC3 : GAA − / − ( bottom left ) or TFEB - treated GFP - LC3 : GAA − / − ( right ) mice . All fibres were transfected with mCherry - LAMP1 to visualize lysosomes ( red ) . The effects of TFEB are clearly visible - overall reduction in lysosomal size , appearance of normal size lysosomes ( similar to those in the WT ) , and lysosomal docking to the plasma membrane ( inset ) . Bar : 10 µm .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

A. Confocal microscopy images of live fibres derived from 3 to 4 month-old GFP-LC3:WT (top left), untreated GFP-LC3:GAA−/− (bottom left) or TFEB-treated GFP-LC3:GAA−/− (right) mice. All fibres were transfected with mCherry-LAMP1 to visualize lysosomes (red). The effects of TFEB are clearly visible - overall reduction in lysosomal size, appearance of normal size lysosomes (similar to those in the WT), and lysosomal docking to the plasma membrane (inset). Bar: 10 µm.


B . FDBmuscle of a GAA − / − mouse was transfected with both GFP - TFEB and mCherry - LAMP1 ( GAA − / − + GFP - TFEB ) . Images were taken before ( left ) and after 4 h ( right ) of time - lapse microscopy . Lysosomal clearance is visible in the TFEB - transfected fibre at both time points . Lysosomes appear to " exit " the fibre ( inset and arrow ) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation ( green nuclei ) . Bar : 10 µm . C . The mean maximum velocity of lysosomes ( top ) and the number of large ( > 3 . 5 µm ) lysosomes ( bottom ) in untreated and TFEB - treated PD fibres ( note : all data for Flag - and GFP - TFEB - treated fibres are pooled ) . Lysosomal velocities were calculated from time - lapse images using ImageJ software with the manual tracking plug - in . For each condition the trajectories of multiple lysosomes were followed ( n = 26 untreated ; n = 43 TFEB - treated ) and the three highest velocity measurements per lysosome were recorded . In TFEB - treated fibres , the maximum velocity of lysosomes was significantly increased ( p = 2 . 07 × 10 − 17 ) and the number of large lysosomes was significantly decreased ( p = 1 . 0 × 10 − 3 ) . Ten untreated and 24 TFEB - treated fibres were analysed for the size calculations . * indicates statistically significant differences ( p ≤ 0 . 001 ; Student ' s t - test ) . Error bars represent 95 % confidence intervals .

O O B-TISSUE O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O 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. FDBmuscle of a GAA−/− mouse was transfected with both GFP-TFEB and mCherry-LAMP1 (GAA−/− + GFP-TFEB). Images were taken before (left) and after 4 h (right) of time-lapse microscopy. Lysosomal clearance is visible in the TFEB-transfected fibre at both time points. Lysosomes appear to "exit" the fibre (inset and arrow) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation (green nuclei). Bar: 10 µm.C. The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated PD fibres (note: all data for Flag- and GFP-TFEB-treated fibres are pooled). Lysosomal velocities were calculated from time-lapse images using ImageJ software with the manual tracking plug-in. For each condition the trajectories of multiple lysosomes were followed (n = 26 untreated; n = 43 TFEB-treated) and the three highest velocity measurements per lysosome were recorded. In TFEB-treated fibres, the maximum velocity of lysosomes was significantly increased (p = 2.07 × 10−17) and the number of large lysosomes was significantly decreased (p = 1.0 × 10−3). Ten untreated and 24 TFEB-treated fibres were analysed for the size calculations. * indicates statistically significant differences (p ≤ 0.001; Student's t-test). Error bars represent 95% confidence intervals.


Confocal microscopy images of live fibres from GFP - LC3 : GAA − / − mice . Muscle was transfected with mCherry - LAMP1 only . The image ( a single frame from the time - lapse series presented in Movie 5 ) shows lysosomes ( red ) , LC3 - positive autophagosomes ( green ) and a number of autolysosomes ( yellow ) . Muscle was transfected with Flag - TFEB and mCherry - LAMP1 . Massive formation of autolysosomes is indicated by yellow structures ; the three lower panels provide a snapshot of the process of exocytosis . The structures at the plasma membrane are labelled with both LC3 and LAMP1 , indicating that they represent amphisomes ( a product of fusion between autophagic vesicles and late endosomes ) or autolysosomes ( a product of fusion between autophagic vesicles and lysosomes ) . Bar : 10 µm for all panels .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O

Confocal microscopy images of live fibres from GFP-LC3:GAA−/− mice.Muscle was transfected with mCherry-LAMP1 only. The image (a single frame from the time-lapse series presented in Movie 5) shows lysosomes (red), LC3-positive autophagosomes (green) and a number of autolysosomes (yellow).Muscle was transfected with Flag-TFEB and mCherry-LAMP1. Massive formation of autolysosomes is indicated by yellow structures; the three lower panels provide a snapshot of the process of exocytosis. The structures at the plasma membrane are labelled with both LC3 and LAMP1, indicating that they represent amphisomes (a product of fusion between autophagic vesicles and late endosomes) or autolysosomes (a product of fusion between autophagic vesicles and lysosomes). Bar: 10 µm for all panels.


Confocal microscopy images of live fibres from muscle - specific autophagy - deficient GAA − / − mice ( Atg7 : GAA DKO ) . Muscle was transfected with mCherry - LAMP1 only . Muscle was transfected with GFP - TFEB and mCherry - LAMP1 ( Atg7 : GAA DKO + GFP - TFEB ) . The TFEB - transfected fibres show realignment of the lysosomes and membrane detachment ( most striking in top and bottom panels ) similar to those in TFEB - transfected fibres from PD mice ( see Fig 4B inset ) . Lysosomes can be seen in the space between the fibre and plasma membrane ( arrows ) . Bar : 10 µm for all panels . The mean maximum velocity of lysosomes ( top ) and the number of large ( > 3 . 5 µm ) lysosomes ( bottom ) in untreated and TFEB - treated autophagy - deficient Atg7 : GAA DKO fibres . The increase in maximum velocity ( 41 % ) is significant ( p = 2 . 729 × 10 − 18 ; n = 57 lysosomes for untreated ; n = 52 lysosomes for TFEB - treated ) , but there is only a slight trend toward smaller lysosomal size ( p = 7 . 0 × 10 − 2 ; n = 10 fibres for untreated ; n = 21 fibres for TFEB - treated ) . The corresponding values from GAA − / − mice are presented for comparison ( for TFEB - treated condition : n = 19 lysosomes for velocity measurements , and n = 10 fibres for the lysosomal size measurements ) . * indicates statistically significant differences ( p ≤ 1 . 0 × 10 − 5 for the top panel and p ≤ 0 . 01 for the lower panel ; Student ' s t - test ) .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O B-TISSUE O O O O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O 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-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal microscopy images of live fibres from muscle-specific autophagy-deficient GAA−/− mice (Atg7:GAA DKO).Muscle was transfected with mCherry-LAMP1 only.Muscle was transfected with GFP-TFEB and mCherry-LAMP1 (Atg7:GAA DKO +GFP-TFEB). The TFEB-transfected fibres show realignment of the lysosomes and membrane detachment (most striking in top and bottom panels) similar to those in TFEB-transfected fibres from PD mice (see Fig 4B inset). Lysosomes can be seen in the space between the fibre and plasma membrane (arrows). Bar: 10 µm for all panels.The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated autophagy-deficient Atg7:GAA DKO fibres. The increase in maximum velocity (41%) is significant (p = 2.729 × 10−18; n = 57 lysosomes for untreated; n = 52 lysosomes for TFEB-treated), but there is only a slight trend toward smaller lysosomal size (p = 7.0 × 10−2; n = 10 fibres for untreated; n = 21 fibres for TFEB-treated). The corresponding values from GAA−/− mice are presented for comparison (for TFEB-treated condition: n = 19 lysosomes for velocity measurements, and n = 10 fibres for the lysosomal size measurements). * indicates statistically significant differences (p ≤ 1.0 × 10−5 for the top panel and p ≤ 0.01 for the lower panel; Student's t-test).


A . Glycogen assay in TFEB - injected gastrocnemii and in the contralateral muscles . In TFEB - injected musclesglycogen levels were significantly decreased compared to those in untreated muscles . * indicates statistically significant differences ( p = 1 . 0 × 10 − 4 ; n = 6 ; Student ' s t - test ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O B-TISSUE O O B-GENEPROD O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O 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. Glycogen assay in TFEB-injected gastrocnemii and in the contralateral muscles. In TFEB-injected musclesglycogen levels were significantly decreased compared to those in untreated muscles. * indicates statistically significant differences (p = 1.0 × 10−4; n = 6; Student's t-test).


B . PAS staining of TFEB - treated muscle shows a reduction of lysosomalglycogen stores ( puncta ) compared to those in untreated muscle . Original magnification : 20 × .

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B. PAS staining of TFEB-treated muscle shows a reduction of lysosomalglycogen stores (puncta) compared to those in untreated muscle. Original magnification: 20×.


C . LAMP1 staining of TFEB - injected gastrocnemii and of the contralateral untreated muscles . In TFEB - treated muscles , the size of LAMP1 - positive vesicles was reduced . Bar : 2 µm .

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C. LAMP1 staining of TFEB-injected gastrocnemii and of the contralateral untreated muscles. In TFEB-treated muscles, the size of LAMP1-positive vesicles was reduced. Bar: 2 µm.