H . Survival analysis of Pdx1 - Cre ; LSL - KRasG12D / + ; LSL - Trp53R172H / + ; LSL - ROCK2 : ER ( RKPC ) mice without ( n = 21 ) or with conditional ROCK activation with tamoxifen citrate ( n = 19 ) . Survival p value determined by Log - Rank test .

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

H. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; LSL-ROCK2:ER (RKPC) mice without (n=21) or with conditional ROCK activation with tamoxifen citrate (n=19). Survival p value determined by Log-Rank test.


A . H & amp ; amp ; E stained sections of cell invasion into collagen matrix after 8 days . Scale bar = 100 µm . Invasion index of KPC cells in the presence or absence of 10 µM H1152 ROCK inhibitor . Means ± SEM ( n = 9 for untreated , n = 8 for H1152 ) , p value by unpaired t test .

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

A. H&amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPC cells in the presence or absence of 10 µM H1152 ROCK inhibitor. Means ± SEM (n=9 for untreated, n=8 for H1152), p value by unpaired t test.


B . Schematic representation of ROCK domains ( RBD , Rho binding domain ; PH , pleckstrin homology domain ; CR , cysteine - rich ) . Conditionally - activated human ROCK1 , human ROCK2 and GFP control fusion proteins ( EGFP , enhanced green fluorescent protein ; hbER , estrogen receptor hormone binding domain ) were expressed in KPflC mouse PDAC cells and blotted with anti - GFP antibody .

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

B. Schematic representation of ROCK domains (RBD, Rho binding domain; PH, pleckstrin homology domain; CR, cysteine-rich). Conditionally-activated human ROCK1, human ROCK2 and GFP control fusion proteins (EGFP, enhanced green fluorescent protein; hbER, estrogen receptor hormone binding domain) were expressed in KPflC mouse PDAC cells and blotted with anti-GFP antibody.


C . KPflC cells expressing GFP : ER , ROCK1 : ER or ROCK2 : ER fusion proteins were treated for 24 h with EtOH vehicle or 1 µM 4HT in the presence or absence of 1 µM or 10 µM H1152 . Immunoblotting shows endogenous ROCK1 and ROCK2 , ER - fusions , and phosphorylation of MLC2 ( T18S19 ) . Total MLC ( MRCL3 / MRLC2 / MYL9 ) and glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) were blotted as loading controls .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 O O O O O O B-GENEPROD O O O O O O O

C. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 µM 4HT in the presence or absence of 1 µM or 10 µM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER-fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.


D . H & amp ; amp ; E stained sections of cell invasion into collagen matrix after 8 days . Scale bar = 100 µm . Invasion index of KPflC cells treated with 1 µM 4HT . Means ± SEM ( n = 6 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison test .

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

D. H&amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPflC cells treated with 1 µM 4HT. Means ± SEM (n=6), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.


E . Cell proliferation determined by Ki67 immunofluorescence . Scale bar : 20 µm . F . Quantification of cell number at the collagen matrix surface per 0 . 046 mm2 field . Means ± SEM ( n = 30 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison test . G . Ki67 positive cell percentages at the surface and within the collagen matrix . Means ± SEM ( n = 30 ; n = 12 for GFP : ER / Matrix ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison test .

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

E. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.F. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=30), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.G. Ki67 positive cell percentages at the surface and within the collagen matrix. Means ± SEM (n=30; n=12 for GFP:ER/Matrix), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.


H . Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle ( - ) or 4HT on uncoated plastic surfaces . Means ± SEM ( n = 3 ) . I . Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle ( - ) or 4HT on collagen1 - coated surfaces . Means ± SEM ( n = 3 ) .

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-SMALL_MOLECULE O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O

H. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on uncoated plastic surfaces. Means ± SEM (n=3).I. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on collagen1-coated surfaces. Means ± SEM (n=3).


A . Immunofluorescence of collagen matrix sections co - stained for cleaved collagen neo - epitope Col1 3 / 4C and DAPI ( left panels ) . Second harmonic generation ( SHG ) images of collagen fibers within 3 - dimensional collagen matrices at 15 µm and 45 µm below the cell surface ( middle panels ) . Scale bar = 20 µm . Graphs indicate the mean ± SEM fibrillary collagen areas at 2 . 5 µm increments below the cell / collagen interface ( n = 5 ) . SHG fibrillar collagen ( red ) ; cells ( green ) . B , C . Grey level correlation matrix ( GLCM ) texture analysis of SHG correlation ( n = 6 ) and contrast ( n = 5 ) . Mean ± SEM at each distance .

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

A. Immunofluorescence of collagen matrix sections co-stained for cleaved collagen neo-epitope Col1 3/4C and DAPI (left panels). Second harmonic generation (SHG) images of collagen fibers within 3-dimensional collagen matrices at 15 µm and 45 µm below the cell surface (middle panels). Scale bar = 20 µm. Graphs indicate the mean ± SEM fibrillary collagen areas at 2.5 µm increments below the cell/collagen interface (n=5). SHG fibrillar collagen (red); cells (green).B, C. Grey level correlation matrix (GLCM) texture analysis of SHG correlation (n=6) and contrast (n=5). Mean ± SEM at each distance.


D . Transmission electron microscopy of a collagen matrix section . Scale bar = 1 µm . Positions of magnified areas indicated by corresponding colored squares .

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

D. Transmission electron microscopy of a collagen matrix section. Scale bar = 1 µm. Positions of magnified areas indicated by corresponding colored squares.


E . Transmission electron microscopy of collagen matrix sections with collagen stained by Tannic acid . Yellow arrows indicate long collagen bundles , Red arrows indicate short collagen bundles . Scale bar = 0 . 5 µm .

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

E. Transmission electron microscopy of collagen matrix sections with collagen stained by Tannic acid. Yellow arrows indicate long collagen bundles, Red arrows indicate short collagen bundles. Scale bar = 0.5 µm.


A . Volcano plots of RNA sequencing data of genes that pass a threshold false discovery rate ( FDR ) < 5 % , with log10 fold - change ( FC ) in expression in 4HT treated ROCK1 : ER expressing cells relative to GFP : ER expressing cells ( left ) or 4HT treated ROCK2 : ER expressing cells relative to GFP : ER expressing cells ( right ) versus log10 adjusted p value ( n = 3 ) . ROCK1 : ER + 4HT vs GFP : ER + 4HT ; 3828 genes . ROCK2 : ER + 4HT vs GFP : ER + 4HT ; 4481 genes .

O O O O 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 O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O

A. Volcano plots of RNA sequencing data of genes that pass a threshold false discovery rate (FDR) < 5%, with log10 fold-change (FC) in expression in 4HT treated ROCK1:ER expressing cells relative to GFP:ER expressing cells (left) or 4HT treated ROCK2:ER expressing cells relative to GFP:ER expressing cells (right) versus log10 adjusted p value (n=3). ROCK1:ER+4HT vs GFP:ER+4HT; 3828 genes. ROCK2:ER+4HT vs GFP:ER+4HT; 4481 genes.


C . Heatmap of 33 differentially expressed genes in the MetaCoreTM process network Cell adhesion : Cell - matrix interaction . High relative expression in red and low relative expression in blue .

O O O O O O O O O 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. Heatmap of 33 differentially expressed genes in the MetaCoreTM process network Cell adhesion: Cell-matrix interaction. High relative expression in red and low relative expression in blue.


D . MMP average RNA sequence reads . Mean ± SEM ( n = 3 ) * adjusted p - value < 0 . 05 .

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D. MMP average RNA sequence reads. Mean ± SEM (n=3) * adjusted p-value <0.05.


E . Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) or 1 µM 4HT for 24 h ( left ) . Means ± SEM ( n = 4 ) , p value by unpaired t test . Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) , 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h ( right ) . Means ± SEM ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . F . Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) or 1 µM 4HT for 24 h ( left ) . Means ± SEM ( n = 4 ) , p value by unpaired t test . Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) , 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h ( right ) . Means ± SEM ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test .

O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE 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-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE 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-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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

E. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


G . MMP10 immunohistochemistry stained sections of human pancreas adenocarcinoma . Scale bar = 100 µm ( left ) . Histoscores of MMP10 staining ( right ) in normal pancreas ( n = 5 ) , and pancreas adenocarcinoma grade 1 ( n = 23 ) , grade 2 ( n = 27 ) and grade 3 ( n = 13 ) . One - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test .

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

G. MMP10 immunohistochemistry stained sections of human pancreas adenocarcinoma. Scale bar = 100 µm (left). Histoscores of MMP10 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma grade 1 (n=23), grade 2 (n=27) and grade 3 (n=13). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


A . Confocal microscope images of ROCK1 : ER - expressing cells co - stained for F - actin , MMP10 ( left ) or MMP13 ( right ) and DAPI following treatment with vehicle or 1 µM 4HT for 24 h . Multiple z - planes were used to generate x - z and y - z images . Scale bar = 5 µm .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O 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 microscope images of ROCK1:ER-expressing cells co-stained for F-actin, MMP10 (left) or MMP13 (right) and DAPI following treatment with vehicle or 1 µM 4HT for 24 h. Multiple z-planes were used to generate x-z and y-z images. Scale bar = 5 µm.


B . Representative immunoblot of MMP13 and MMP10 in conditioned media , as well as MMP10 and GAPDH in cell lysates . Cells were treated with vehicle ( - ) or 1 µM 4HT for 48 h .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O 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 B-SMALL_MOLECULE O O O O

B. Representative immunoblot of MMP13 and MMP10 in conditioned media, as well as MMP10 and GAPDH in cell lysates. Cells were treated with vehicle (-) or 1 µM 4HT for 48 h.


C . Representative stained gel of ultracentrifuge - enriched microvesicle protein from GFP : ER or ROCK1 : ER - expressing cell - conditioned media following treatment with vehicle ( - ) or 1 µM 4HT for 24 h ( top ) . Absolute arbitrary unit values for total microvesicle protein levels in stained gels ( bottom ) . Means ± SEM ( n = 4 ) , p value by ratio paired t - test .

O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O 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. Representative stained gel of ultracentrifuge-enriched microvesicle protein from GFP:ER or ROCK1:ER-expressing cell-conditioned media following treatment with vehicle (-) or 1 µM 4HT for 24 h (top). Absolute arbitrary unit values for total microvesicle protein levels in stained gels (bottom). Means ± SEM (n=4), p value by ratio paired t-test.


D . Transmission electron microscopy of ultracentrifuge - enriched microvesicles ( top center ) , and immunogold labeling of MMP10 in ROCK1 : ER 4HT - treated microvesicles , indicated by red arrows ( top right ) .

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

D. Transmission electron microscopy of ultracentrifuge-enriched microvesicles (top center), and immunogold labeling of MMP10 in ROCK1:ER 4HT-treated microvesicles, indicated by red arrows (top right).


D . Representative immunoblot of MMP10 and caveolin in ultracentrifuge - enriched microvesicle proteins from cell - conditioned media following treatment with vehicle ( - ) or 1 µM 4HT for 24 h as well as MMP10 , caveolin and GAPDH in cell lysates ( top left ) . Absolute arbitrary unit values for MMP10 levels in western blots of ultracentrifugation - enriched microvesicle proteins ( bottom left ) . Means ± SEM ( n = 4 ) , p value by ratio paired t test . Ratios of MMP10 to caveolin ( bottom center ) or to total microvesicle proteins ( bottom right ) . Means ± SEM ( n = 4 ) , p value by ratio paired t test .

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

D. Representative immunoblot of MMP10 and caveolin in ultracentrifuge-enriched microvesicle proteins from cell-conditioned media following treatment with vehicle (-) or 1 µM 4HT for 24 h as well as MMP10, caveolin and GAPDH in cell lysates (top left). Absolute arbitrary unit values for MMP10 levels in western blots of ultracentrifugation-enriched microvesicle proteins (bottom left). Means ± SEM (n=4), p value by ratio paired t test. Ratios of MMP10 to caveolin (bottom center) or to total microvesicle proteins (bottom right). Means ± SEM (n=4), p value by ratio paired t test.


E . Absolute arbitrary unit values for caveolin levels in western blots of ultracentrifugation - enriched microvesicle proteins . Means ± SEM ( n = 4 ) , p value by ratio paired t test . Ratio of caveolin to total microvesicle proteins . Means ± SEM ( n = 4 ) , p value by ratio paired t test . F . Ratios of MMP10 to GAPDH and caveolin to GAPDH in western blots of whole cell lysates . Means ± SEM ( n = 3 ) , p value by ratio paired t test .

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

E. Absolute arbitrary unit values for caveolin levels in western blots of ultracentrifugation-enriched microvesicle proteins. Means ± SEM (n=4), p value by ratio paired t test. Ratio of caveolin to total microvesicle proteins. Means ± SEM (n=4), p value by ratio paired t test.F. Ratios of MMP10 to GAPDH and caveolin to GAPDH in western blots of whole cell lysates. Means ± SEM (n=3), p value by ratio paired t test.


A . Representative fluorescence images of cells treated with 1 µM 4HT in the absence or presence of 10 µM H1152 for 18 h on Collagen1 - FITC ( Col1 - FITC ) . Co - staining for F - actin , MMP10 ( red ) and DAPI ( blue ) . Scale bar = 10 µm . B . Quantification of collagen degradation by cells treated with 1 µM 4HT or 4HT + 10 µM H1152 for 16 h . Means ± SEM ( n = 15 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test .

O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O 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 O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O 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. Representative fluorescence images of cells treated with 1 µM 4HT in the absence or presence of 10 µM H1152 for 18 h on Collagen1-FITC (Col1-FITC). Co-staining for F-actin, MMP10 (red) and DAPI (blue). Scale bar = 10 µm.B. Quantification of collagen degradation by cells treated with 1 µM 4HT or 4HT + 10 µM H1152 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


C . Quantification of collagen degradation by cells treated with 1 µM 4HT , 4HT + DMSO vehicle , 4HT + 50 µM Blebbistatin or 4HT + 10 µM GM6001 for 16 h . Means ± SEM ( n = 15 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test .

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C. Quantification of collagen degradation by cells treated with 1 µM 4HT, 4HT + DMSO vehicle, 4HT + 50 µM Blebbistatin or 4HT + 10 µM GM6001 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


D . Immunofluorescence of collagen matrix sections co - stained for MMP10 and DAPI . Scale bar = 20 µm .

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

D. Immunofluorescence of collagen matrix sections co-stained for MMP10 and DAPI. Scale bar = 20 µm.


E . Mmp13 immunohistochemistry stained sections of collagen matrices . Scale bar = 50 µm .

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

E. Mmp13 immunohistochemistry stained sections of collagen matrices. Scale bar = 50 µm.


A . H & amp ; amp ; E stained sections of cell invasion into collagen matrix after 8 days , in the absence ( top ) or presence of 10 µM GM6001 ( bottom ) . Scale bar = 100 µm . B . Invasion index of KPflC cells . Means ± SEM ( n = 4 ) , p value by unpaired t test . C . Quantification of cell number at the collagen matrix surface per 0 . 046 mm2 field . Means ± SEM ( n = 20 ) , p value by unpaired t test .

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

A. H&amp;E stained sections of cell invasion into collagen matrix after 8 days, in the absence (top) or presence of 10 µM GM6001 (bottom). Scale bar = 100 µm.B. Invasion index of KPflC cells. Means ± SEM (n=4), p value by unpaired t test.C. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=20), p value by unpaired t test.


D . Cell proliferation determined by Ki67 immunofluorescence . Scale bar : 20 µm . E . Ki67 positive cell percentages at the collagen matrix surface . Means ± SEM ( n = 20 ; n = 19 for GFP : ER / vehicle , n = 18 for GFP / GM6001 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . F . Ki67 positive cell percentages in collagen matrix . Means ± SEM ( n = 20 ; n = 8 for GFP : ER / vehicle , n = 10 for GFP : ER / GM6001 , n = 17 for ROCK1 : ER / GM6001 , n = 15 for ROCK2 : ER / GM6001 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-GENEPROD 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 B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE 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-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

D. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.E. Ki67 positive cell percentages at the collagen matrix surface. Means ± SEM (n=20; n=19 for GFP:ER/vehicle, n=18 for GFP/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Ki67 positive cell percentages in collagen matrix. Means ± SEM (n=20; n=8 for GFP:ER/ vehicle, n=10 for GFP:ER/GM6001, n=17 for ROCK1:ER/GM6001, n=15 for ROCK2:ER/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


G . The increase in viable cell numbers of cells plated on uncoated plastic surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µm GM6001 . Means ± SEM ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . H . The increase in viable cell numbers of cells plated on collagen1 ( Col1 ) coated surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µM GM6001 . Means ± SEM ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test .

O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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 O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. The increase in viable cell numbers of cells plated on uncoated plastic surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µm GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.H. The increase in viable cell numbers of cells plated on collagen1 (Col1) coated surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µM GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


A . Survival analysis of Pdx1 - Cre ; LSL - KRasG12D / + ; LSL - Trp53R172H / + ; ( KPC ) mice without ( n = 13 ) or with Fasudil treatment ( n = 13 ) . Survival p - value determined by Gehan - Breslow - Wilcoxon test .

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

A. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; (KPC) mice without (n=13) or with Fasudil treatment (n=13). Survival p-value determined by Gehan-Breslow-Wilcoxon test.


B . Representative images of picrosirius red stained endpoint tumors from KPC mice . Scale bar = 1 mm . C . Picrosirius red percentage positive staining area ( top ) or average staining intensity of positive area ( bottom ) in pancreatic tumors from vehicle ( n = 8 ) and Fasudil ( n = 7 ) treated mice . Box plots with exact p value by Mann - Whitney test .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE 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 B-TISSUE I-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O

B. Representative images of picrosirius red stained endpoint tumors from KPC mice. Scale bar = 1 mm.C. Picrosirius red percentage positive staining area (top) or average staining intensity of positive area (bottom) in pancreatic tumors from vehicle (n=8) and Fasudil (n=7) treated mice. Box plots with exact p value by Mann-Whitney test.


E . Ectopic expression of CG14044 in the wing pouch ( GFP area indicated by the dotted line ) induces caspase activation and DNA fragmentation ( TUNEL ) in the larval disc , melanization in the pupal wing ( arrow head ) and a structure defect in the adult wing . Data information : Scale bars , 100 μm in E ( cleaved caspase ) and 20 μm in E ( TUNEL ) and 500 μm in E ( pupae and adult wing ) .

O O O O O B-GENEPROD O O B-TISSUE I-TISSUE O B-GENEPROD O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

E. Ectopic expression of CG14044 in the wing pouch (GFP area indicated by the dotted line) induces caspase activation and DNA fragmentation (TUNEL) in the larval disc, melanization in the pupal wing (arrow head) and a structure defect in the adult wing. Data information: Scale bars, 100 μm in E (cleaved caspase) and 20 μm in E (TUNEL) and 500 μm in E (pupae and adult wing).


H . Deletion of the BH3 motif suppresses Sayonara - induced caspase activation , which was detected by the caspase sensor GC3Ai . I . Quantification of GC3Ai positive areas with Synr WT and the BH3 motif deletion mutant . Data information : Statistical significance was determined using one - way ANOVA with Dunnett ' s post hoc test . Scale bars , 100 μm

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

H. Deletion of the BH3 motif suppresses Sayonara-induced caspase activation, which was detected by the caspase sensor GC3Ai. I. Quantification of GC3Ai positive areas with Synr WT and the BH3 motif deletion mutant. Data information: Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test. Scale bars, 100 μm in


A - D . The Synr - induced wing structural defect is suppressed by knockdown of Debcl , Buffy or DCP1 . Data information : Scale bars , 500 μm in

O O O O O B-GENEPROD O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O

A-D. The Synr-induced wing structural defect is suppressed by knockdown of Debcl, Buffy or DCP1. Data information: Scale bars, 500 μm in A


F . Synr - induced caspase activation is suppressed by knockdown of Debcl . G . GC3Ai signals are suppressed by knockdown of Debcl or Buffy Data information : Scale bars , 50 μm in F and

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

F. Synr-induced caspase activation is suppressed by knockdown of Debcl. G. GC3Ai signals are suppressed by knockdown of Debcl or Buffy Data information: Scale bars, 50 μm in F and G


H - I . Lysates from HEK293T cells that express Flag - Debcl or Flag - Buffy were incubated with GST or GST - Synr bound to glutathione - Sepharose . Flag - Debcl or Flag - Buffy is pulled down with GST - Synr but not with GST .

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

H-I. Lysates from HEK293T cells that express Flag-Debcl or Flag-Buffy were incubated with GST or GST-Synr bound to glutathione-Sepharose. Flag-Debcl or Flag-Buffy is pulled down with GST-Synr but not with GST.


J . Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding Flag - Debcl to GST - Synr .

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

J. Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding Flag-Debcl to GST-Synr.


O . Debcl can be photo - crosslinked with Synr at BPA that corresponds to Y124 and F141 . BPA at Y340 was used as a negative control . Red arrowheads indicate covalently crosslinked products . P . Buffy is also photo - crosslinked with Synr at BPA that corresponds to Y124 and F141 . Red arrowheads indicate covalently crosslinked products .

O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O

O. Debcl can be photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. BPA at Y340 was used as a negative control. Red arrowheads indicate covalently crosslinked products. P. Buffy is also photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. Red arrowheads indicate covalently crosslinked products.


R . HA - Debcl is detected after two series of immunoprecipitation . The first immunoprecipitation was performed for Flag - Buffy , followed by Flag peptide - mediated elution and secondary precipitation for Myc - Synr . HA - Debcl can be detected after this sequential immunoprecipitation .

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

R. HA-Debcl is detected after two series of immunoprecipitation. The first immunoprecipitation was performed for Flag-Buffy, followed by Flag peptide-mediated elution and secondary precipitation for Myc-Synr. HA-Debcl can be detected after this sequential immunoprecipitation.


A . Ectopic expression of Synr induces autolysosome accumulation , which was detected by Lysotracker , in the wing disc . B . Ectopic expression of Synr also induces autophagosome accumulation , which was detected by mCherry - atg8a . The dotted square regions are magnified in the right pictures . Data information : Scale bars , 100 μm in A and H , 50 μm in

O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O B-SUBCELLULAR 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

A. Ectopic expression of Synr induces autolysosome accumulation, which was detected by Lysotracker, in the wing disc. B. Ectopic expression of Synr also induces autophagosome accumulation, which was detected by mCherry-atg8a. The dotted square regions are magnified in the right pictures. Data information: Scale bars, 100 μm in A and H, 50 μm in B


D - F . Knockdown of Atg2 or Atg8a suppresses the wing structural defect that is induced by Synr . Data information : Scale bars , 500 μm in D .

O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O

D-F. Knockdown of Atg2 or Atg8a suppresses the wing structural defect that is induced by Synr. Data information: Scale bars, 500 μm in D.


Synr - induced caspase activation is suppressed by inhibition of autophagy ( atg2 , atg8a RNAis ) in a similar manner to apoptosis inhibition ( Debcl , Dcp - 1 RNAis ) . Data information : Scale bars , 100 μm

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

Synr-induced caspase activation is suppressed by inhibition of autophagy (atg2, atg8a RNAis) in a similar manner to apoptosis inhibition (Debcl, Dcp-1 RNAis). Data information: Scale bars, 100 μm in


A . Inhibition of Synr suppresses the wing defect that is induced by p53 , a potent apoptosis inducer . B . Inhibition of Synr suppresses p53 - induced caspase activation . Data information : Statistical significance was determined using a two - tailed unpaired t - test ( B ) , Scale bar , 500 μm in

O O O O B-GENEPROD O O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD 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

A. Inhibition of Synr suppresses the wing defect that is induced by p53, a potent apoptosis inducer. B. Inhibition of Synr suppresses p53-induced caspase activation. Data information: Statistical significance was determined using a two-tailed unpaired t-test (B), Scale bar, 500 μm in A


D . Inhibition of Synr in the midgut enterocytes enhances resistance to starvation . E . The synr mutant also exhibits enhanced resistance to starvation . Data information : Statistical significance was determined using a log - rank ( Mantel - Cox ) test ( D - E ) .

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

D. Inhibition of Synr in the midgut enterocytes enhances resistance to starvation. E. The synr mutant also exhibits enhanced resistance to starvation. Data information: Statistical significance was determined using a log-rank (Mantel-Cox) test (D-E).


Four to six - week - old C57BL / 6 mice were injected i . p . with 104 - 107 PFU of WNV - poly ( A ) , 104 PFU of WT WNV or PBS ( negative control ) in a volume of 200 μL . Survival ( n = 5 ) in infected mice were monitored daily Data information : Log - rank test was

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

Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Survival (n=5) in infected mice were monitored daily Data information: Log-rank test was used


Four to six - week - old C57BL / 6 mice were injected i . p . with 104 - 107 PFU of WNV - poly ( A ) , 104 PFU of WT WNV or PBS ( negative control ) in a volume of 200 μL . Histological analysis of mouse brain . Scale bars represent 100 μm and 50 μm for left and right panels , respectively . Severe vacuolization ( black arrows ) and inflammation ( yellow arrow ) in brain small vessels as well as degenerative changes in neurons with cytoplasmic rarefaction ( blue arrow ) and necrosis with nuclear compaction ( red arrow ) were indicated . Scale bars represent 100 μm and 50 μm for left and right panels ,

O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM 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 B-DISEASE O O O O O B-TISSUE O B-TISSUE O O O O O O B-CELL_TYPE O B-SUBCELLULAR O O O O O O B-DISEASE 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

Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Histological analysis of mouse brain. Scale bars represent 100 μm and 50 μm for left and right panels, respectively. Severe vacuolization (black arrows) and inflammation (yellow arrow) in brain small vessels as well as degenerative changes in neurons with cytoplasmic rarefaction (blue arrow) and necrosis with nuclear compaction (red arrow) were indicated. Scale bars represent 100 μm and 50 μm for left and right panels, respectively


E Viral load and replication kinetics in different tissues of C57BL / 6 mice . Six - week - old female C57BL / 6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV - poly ( A ) in the left rear footpad , and tissues were harvested from two mice at the indicated times post - inoculation . Viral loads in brain , spleen , left lymph node , right lymph node and liver were measured by plaque assay and expressed as PFU per gram of tissue . n = 3 , values are mean ± sd , the dotted line corresponds to the limit of detection ( LOD ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O 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 O O O O O O O O O O O O O

E Viral load and replication kinetics in different tissues of C57BL/6 mice. Six-week-old female C57BL/6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV-poly(A) in the left rear footpad, and tissues were harvested from two mice at the indicated times post-inoculation. Viral loads in brain, spleen, left lymph node, right lymph node and liver were measured by plaque assay and expressed as PFU per gram of tissue. n=3, values are mean ± sd, the dotted line corresponds to the limit of detection (LOD).


B - C Total anti - WNV IgG antibody ( B ) and neutralizing antibody titers ( C ) in mouse serum at the indicated days post immunization . ( For B and C , the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection ) Data information : For statistical analysis , one - way ANOVA analysis were used * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

O O O O O O B-ORGANISM B-SUBCELLULAR O O O O O O O O O O O O B-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-C Total anti-WNV IgG antibody (B) and neutralizing antibody titers (C) in mouse serum at the indicated days post immunization. (For B and C, the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection) Data information: For statistical analysis, one-way ANOVA analysis were used *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


D - E Survival ( D ) and body - weight changes were monitored daily after challenge . ( The values represent mean ± sd in each group ) ( E ) . Data information : For statistical analysis , two - way ANOVA analysis were used Log - rank test was performed * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

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

D-E Survival (D) and body-weight changes were monitored daily after challenge. (The values represent mean ± sd in each group) (E). Data information: For statistical analysis, two-way ANOVA analysis were used Log-rank test was performed *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


G - H T - cell responses in vaccinated mice were measured through ELISpot assay . Two groups of C57BL / 6 mice ( n = 4 / group ) were i . p . inoculated with 107 of WNV - poly ( A ) or equal volume of PBS . ELISpot assay specific for IFN - γ in splenocytes was performed at 7 days post - immunization . Representative images were shown in ( G ) and the numbers of antibody secreting cells ( ASCs ) were counted ( The values represent mean ± sd in each group , each symbol represents a mouse ) ( H ) . Data information : Mann - Whitney test was used for H . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

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

G-H T-cell responses in vaccinated mice were measured through ELISpot assay. Two groups of C57BL/6 mice (n = 4 / group) were i.p. inoculated with 107 of WNV-poly(A) or equal volume of PBS. ELISpot assay specific for IFN-γ in splenocytes was performed at 7 days post-immunization. Representative images were shown in (G) and the numbers of antibody secreting cells (ASCs) were counted (The values represent mean ± sd in each group, each symbol represents a mouse) (H). Data information: Mann-Whitney test was used for H. *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


C Comparison of growth kinetics between WT WNV and WNV - poly ( A ) viruses in MEF cells . MEF cells were infected with either WT WNV or WNV - poly ( A ) virus at an MOI of 0 . 01 . Values indicate mean ± sd from two replicates of one representative experiment . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm .

O O O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O B-ORGANISM O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Comparison of growth kinetics between WT WNV and WNV-poly(A) viruses in MEF cells. MEF cells were infected with either WT WNV or WNV-poly(A) virus at an MOI of 0.01. Values indicate mean ± sd from two replicates of one representative experiment. Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


D Survival rate of IFNAR - / - mice infected with different dosages of WNV - poly ( A ) ( n = 5 ) . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm .

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

D Survival rate of IFNAR-/- mice infected with different dosages of WNV-poly(A) (n=5). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


E Cell morphology changes of MEF cells infected with WT WNV and WNV - poly ( A ) . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm .

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

E Cell morphology changes of MEF cells infected with WT WNV and WNV-poly(A). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


Virulence and efficacy of the passaged WNV - poly ( A ) ( WNV - poly ( A ) - P50 ) in mice ( n = 5 ) . ( E ) Survival of mice infected with 107 PFU of WNV - poly ( A ) - P50 . Data information : For statistical analysis , one - way ANOVA analysis and Log - rank test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

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

Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (E) Survival of mice infected with 107 PFU of WNV-poly(A)-P50. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


Virulence and efficacy of the passaged WNV - poly ( A ) ( WNV - poly ( A ) - P50 ) in mice ( n = 5 ) . ( F ) Antibody titers of mice infected with WNV - poly ( A ) - P50 at different days post immunization . ( For the values represent mean ± sd in each group ( n = 5 ) and the horizontal dotted line represents the limit of detection ) . Data information : For statistical analysis , Mann - Whitney test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

O O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (F) Antibody titers of mice infected with WNV-poly(A)-P50 at different days post immunization. (For the values represent mean ± sd in each group (n=5) and the horizontal dotted line represents the limit of detection). Data information: For statistical analysis, Mann-Whitney test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


( I ) Survival of mice after WT WNV challenge . Data information : For statistical analysis , one - way ANOVA analysis and Log - rank test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

O O O B-EXP_ASSAY O B-ORGANISM 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

(I) Survival of mice after WT WNV challenge. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


( A ) Immunoblots for LC3 using whole cell lysates of Huh7 cells before and after treatment with iron chelators ( DFP , DFX or DFO ) . β - actin was used as loading control . Bafilomycin ( - ) : without Bafilomycin , Bafilomycin ( + ) : with 100 mM of Bafilomycin .

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

(A) Immunoblots for LC3 using whole cell lysates of Huh7 cells before and after treatment with iron chelators (DFP, DFX or DFO). β-actin was used as loading control. Bafilomycin (-): without Bafilomycin, Bafilomycin (+): with 100 mM of Bafilomycin.


( B ) Dual excitation ( 550 / 488 nm ) of mt - mKeima transfected - Huh7 cells shows red fluorescence indicating mitophagy with DFP treatment at 24 h and green fluorescence without DFP treatment . Scale bar : 10 μm .

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

(B) Dual excitation (550/488 nm) of mt-mKeima transfected-Huh7 cells shows red fluorescence indicating mitophagy with DFP treatment at 24 h and green fluorescence without DFP treatment. Scale bar: 10 μm.


( C ) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt - mKeima at 633 / 488 nm for untreated ( left panel ) and DFP - treated ( 24 h , right panel ) Huh7 cells .

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

(C) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt-mKeima at 633/488 nm for untreated (left panel) and DFP-treated (24 h, right panel) Huh7 cells.


( D ) Untreated Huh7 cells or those treated with DFP ( 10 μM ) , DFX ( 10 μM ) or DFO ( 10 μM ) were quantified for mitophagy using mt - mKeima transfection and FACS analysis ( n = 4 , biological replicates ) . A high signal ( 633 / 488 ) area / cell area indicates the proportion of cells undergoing mitophagy . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * : P < 0 . 01 .

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

(D) Untreated Huh7 cells or those treated with DFP (10 μM), DFX (10 μM) or DFO (10 μM) were quantified for mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates). A high signal (633/488) area/ cell area indicates the proportion of cells undergoing mitophagy. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.


( E ) Electron microscopic images of untreated ( left panel ) and DFP - treated ( right panel ) Huh7 cells . Arrows indicate mitophagosome - like structures . Scale bar : 500 nm . The number of mitophagosome - like structures / 100 μm2 was compared for four randomly selected areas ( technical replicates ) between cells treated with DFP ( 1 . 0 mM ) and those without DFP . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O B-SUBCELLULAR 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Electron microscopic images of untreated (left panel) and DFP-treated (right panel) Huh7 cells. Arrows indicate mitophagosome-like structures. Scale bar: 500 nm. The number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) between cells treated with DFP (1.0 mM) and those without DFP. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.


( F ) Quantification of mitophagy using mt - mKeima transfection and FACS analysis for Huh7 cells treated with DFP ( 0 , 0 . 01 . 0 . 5 or 1 . 0 mM as indicated ) in the absence or presence of bafilomycin ( n = 4 , biological replicates ) . The central horizontal bar and the error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O B-CELL_LINE O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O O O O O O O O O O O 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) Quantification of mitophagy using mt-mKeima transfection and FACS analysis for Huh7 cells treated with DFP (0, 0.01. 0.5 or 1.0 mM as indicated) in the absence or presence of bafilomycin (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.


( G ) Immunoblots for Parkin , PINK1 , phosphorylated p - 62 ( p - p62 ) , HSP60 , LC3 , p62 / SQSTM , and Atg5 using the mitochondrial fraction and whole cell lysates of Huh7 cells . HSP60 and β - actin were used as loading controls for mitochondria and whole cell lysates , respectively .

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

(G) Immunoblots for Parkin, PINK1, phosphorylated p-62 (p-p62), HSP60, LC3, p62/SQSTM, and Atg5 using the mitochondrial fraction and whole cell lysates of Huh7 cells. HSP60 and β-actin were used as loading controls for mitochondria and whole cell lysates, respectively.


( A ) Cytoplasmic and mitochondrial iron content was measured by a ferrozine - based assay for Huh7 cells with DFP , DFX or DFO treatment ( 0 . 01 mM ) for 24 h ( n = 5 , biological replicates ) . The cytoplasmic iron content was measured by subtracting the iron content of the mitochondrial fraction from the iron content of the whole lysate . The central horizontal bar and the error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 .

O O O B-SUBCELLULAR O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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) Cytoplasmic and mitochondrial iron content was measured by a ferrozine-based assay for Huh7 cells with DFP, DFX or DFO treatment (0.01 mM) for 24 h (n=5, biological replicates). The cytoplasmic iron content was measured by subtracting the iron content of the mitochondrial fraction from the iron content of the whole lysate. The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.


( B ) Cytoplasmic and mitochondrial iron content was measured for Huh7 cells with DFP treatment ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM as indicated ) for 24 h . ( n = 5 , biological replicates ) The central horizontal bar and the error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 .

O O O B-SUBCELLULAR O B-SUBCELLULAR B-SMALL_MOLECULE O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cytoplasmic and mitochondrial iron content was measured for Huh7 cells with DFP treatment (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated) for 24 h. (n=5, biological replicates) The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.


( C ) Immunoblots ( left panel ) for mitochondrial ferritin ( FTMT ) , frataxin , mitoferrin , heat shock protein 60 ( HSP60 ) , ferritin heavy chain ( FTH1 ) and transferrin receptor ( TfR ) and β - actin using the mitochondrial fraction or whole cell lysate of Huh7 cells following treatment with DFP ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM as indicated ) . HSP60 and β - actin were used as loading controls for mitochondrial and whole cell lysates , respectively . The expression level of FTMT was normalized to HSP60 ( n = 4 , biological replicates , right panel ) . The central horizontal bar and the error bards indicate mean ± SD . * : P < 0 . 05 .

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

(C) Immunoblots (left panel) for mitochondrial ferritin (FTMT), frataxin, mitoferrin, heat shock protein 60 (HSP60), ferritin heavy chain (FTH1) and transferrin receptor (TfR) and β-actin using the mitochondrial fraction or whole cell lysate of Huh7 cells following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated). HSP60 and β-actin were used as loading controls for mitochondrial and whole cell lysates, respectively. The expression level of FTMT was normalized to HSP60 (n=4, biological replicates, right panel). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05.


( D ) The mRNA expression of FTMT in Huh7 cells was quantified by real - time RT - PCR before and after DFP ( 1 . 0 mM ) treatment ( n = 4 , biological replicates ) . The expression of FTMT mRNA level was normalized to GAPDH mRNA . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . * * : P < 0 . 01 .

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

(D) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before and after DFP (1.0 mM) treatment (n=4, biological replicates). The expression of FTMT mRNA level was normalized to GAPDH mRNA. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.


( E ) The mRNA expression of FTMT in Huh7 cells was quantified by real - time RT - PCR before DFP ( 1 . 0 mM ) treatment and 3 , 6 , 12 , and 24 h after DFP treatment ( n = 5 , biological replicates ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * : P < 0 . 01 , * * * : P < 0 . 001 .

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

(E) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before DFP (1.0 mM) treatment and 3, 6, 12, and 24 h after DFP treatment (n=5, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01, ***: P<0.001.


( F ) DNA - binding activity of six transcription factors ( specific protein 1 [ SP1 ] , cAMP response element binding protein [ CREB ] , Ying yang 1 [ YY1 ] , GATA2 , forkhead box protein A1 [ FoxA1 ] and CCAAT - enhancer - binding protein β [ C / EBPβ ] ) to the FTMT promoter region was compared by chromatin immunoprecipitation before and after DFP ( 1 . 0 mM ) treatment in Huh7 cells ( n = 4 , biological replicates ) . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . * : P < 0 . 05 .

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

(F) DNA-binding activity of six transcription factors (specific protein 1 [SP1], cAMP response element binding protein [CREB], Ying yang 1 [YY1], GATA2, forkhead box protein A1 [FoxA1] and CCAAT-enhancer-binding protein β [ C/EBPβ]) to the FTMT promoter region was compared by chromatin immunoprecipitation before and after DFP (1.0 mM) treatment in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. *: P<0.05.


( G ) The DNA - binding activity of SP1 to the FTMT promoter region was assayed by chromatin immunoprecipitation following treatment with DFP ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM ) as indicated in Huh7 cells ( n = 4 , biological replicates ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 .

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

(G) The DNA-binding activity of SP1 to the FTMT promoter region was assayed by chromatin immunoprecipitation following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM) as indicated in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.


( H ) FTMT mRNA expression was quantified for Huh7 cells by real - time RT - PCR before and after DFP ( 1 . 0 mM ) treatment with or without SP1 knockdown by siRNA ( n = 5 , biological replicates ) . mRNA expression of FTMT level was normalized to GAPDH . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . NC : negative control siRNA , * * : P < 0 . 01 .

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

(H) FTMT mRNA expression was quantified for Huh7 cells by real-time RT-PCR before and after DFP (1.0 mM) treatment with or without SP1 knockdown by siRNA (n=5, biological replicates). mRNA expression of FTMT level was normalized to GAPDH. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.


( I ) Immunoblots for SP1 using the cytoplasmic fraction and nuclear fraction before and after DFP ( 1 . 0 mM ) treatment . β - actin and Lam B were used as loading control for the cytoplasmic and nuclear fractions , respectively .

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

(I) Immunoblots for SP1 using the cytoplasmic fraction and nuclear fraction before and after DFP (1.0 mM) treatment. β-actin and Lam B were used as loading control for the cytoplasmic and nuclear fractions, respectively.


( J ) Immunoblots for HIF1α using Huh7 cells and HepG2 cells before and after DFP ( 0 . 1 mM ) treatment . β - actin was used as the loading control .

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

(J) Immunoblots for HIF1α using Huh7 cells and HepG2 cells before and after DFP (0.1 mM) treatment. β-actin was used as the loading control.


( K ) Immunoblots for FTMT , HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP ( 1 . 0 mM ) treatment with or without HIF1α or SP1 knockdown by siRNA . HSP60 and β - actin were used as loading controls for the mitochondrial fraction and whole cell lysates , respectively .

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

(K) Immunoblots for FTMT, HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP (1.0 mM) treatment with or without HIF1α or SP1 knockdown by siRNA. HSP60 and β-actin were used as loading controls for the mitochondrial fraction and whole cell lysates, respectively.


( A ) Immunoblots for mitochondrial ferritin ( FTMT ) with HSP60 as the loading control using the mitochondrial fraction of Huh7 cells and HepG2 cells ( upper panels ) and the quantification of mitophagy using mt - mKeima transfection and FACS analysis ( n = 4 , biological replicates ) ( lower panels ) before and after DFP ( 1 . 0 mM ) treatment with or without FTMT knockdown by siRNA . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . NC : negative control siRNA , * * : P < 0 . 01 .

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

(A) Immunoblots for mitochondrial ferritin (FTMT) with HSP60 as the loading control using the mitochondrial fraction of Huh7 cells and HepG2 cells (upper panels) and the quantification of mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates) (lower panels) before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.


( B ) Immunofluorescence staining for LC3 ( red ) , the mitochondrial marker Tom20 ( green ) and FTMT ( blue ) in Huh7 cells before and after DFP ( 1 . 0 mM ) treatment with or without FTMT knockdown by siRNA . FTMT colocalize with LC3 at the mitochondria ( white puncta indicated by arrows ) . Boxed areas are enlarged below . Scale bar : 5

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

(B) Immunofluorescence staining for LC3 (red), the mitochondrial marker Tom20 (green) and FTMT (blue) in Huh7 cells before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. FTMT colocalize with LC3 at the mitochondria (white puncta indicated by arrows). Boxed areas are enlarged below. Scale bar: 5 μm


( C ) Immunoblots for mitochondrial ferritin ( FTMT ) following FTMT knockdown in the absence or presence of DFP using the mitochondrial fraction of Huh7 cells and those transfected with FTMT siRNA - resistant FTMT ( left panel ) . HSP60 as the loading control . Those cells were subjected to the quantification of mitophagy using mt - mKeima transfection and FACS analysis before and after DFP ( 1 . 0 mM ) treatment ( n = 5 , biological replicates ) ( right panel ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * * : P < 0 . 001 .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-CELL_LINE O O O O O B-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots for mitochondrial ferritin (FTMT) following FTMT knockdown in the absence or presence of DFP using the mitochondrial fraction of Huh7 cells and those transfected with FTMT siRNA-resistant FTMT (left panel). HSP60 as the loading control. Those cells were subjected to the quantification of mitophagy using mt-mKeima transfection and FACS analysis before and after DFP (1.0 mM) treatment (n=5, biological replicates) (right panel). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. ***: P<0.001.


( D ) Super resolution images of immunofluorescence staining for LC3 ( red ) , FTMT ( green ) and Tom20 ( blue ) in FTMT knockdown - Huh7 cells transfected with FTMT - GFP or FTMT siRNA - resistant FTMT - GFP before and after DFP ( 1 . 0 mM ) treatment . White arrows indicate colocalization of LC3 , FTMT , and Tom20 .

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

(D) Super resolution images of immunofluorescence staining for LC3 (red), FTMT (green) and Tom20 (blue) in FTMT knockdown-Huh7 cells transfected with FTMT-GFP or FTMT siRNA-resistant FTMT-GFP before and after DFP (1.0 mM) treatment. White arrows indicate colocalization of LC3, FTMT, and Tom20.


( A ) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti - FTMT - coupled magnetic beads and immunoblotted with the indicated antibodies ( anti - NCOA4 or anti - LC3 ) .

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

(A) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti-FTMT-coupled magnetic beads and immunoblotted with the indicated antibodies (anti-NCOA4 or anti-LC3).


( B ) GST pulldown assay using recombinant GST - NCOA4 mixed with recombinant His - FTMT . Bound FTMT was analyzed by immunoblotting using either anti - FTMT or anti - His antibodies .

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

(B) GST pulldown assay using recombinant GST-NCOA4 mixed with recombinant His-FTMT. Bound FTMT was analyzed by immunoblotting using either anti-FTMT or anti-His antibodies.


( C ) Immunofluorescence staining for Tom20 ( red ) , NCOA4 ( green ) and FTMT ( blue ) in Huh7 cells before and after DFP ( 1 mM ) treatment . Boxed areas are enlarged on the right . The white puncta indicated by white arrows show the colocalization of FTMT , NCOA4 and Tom20 . Scale bar : 10 μm and 5 μm in enlarged images .

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

(C) Immunofluorescence staining for Tom20 (red), NCOA4 (green) and FTMT (blue) in Huh7 cells before and after DFP (1 mM) treatment. Boxed areas are enlarged on the right. The white puncta indicated by white arrows show the colocalization of FTMT, NCOA4 and Tom20. Scale bar: 10 μm and 5 μm in enlarged images.


( D ) Super - resolution images of immunofluorescence staining for Tom20 ( blue ) , NCOA4 ( red ) and FTMT ( green ) in Huh7 cells after DFP ( 1 mM ) treatment , using the single - molecule fluorescence microscope . The white arrows indicate colocalization of these proteins .

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

(D) Super-resolution images of immunofluorescence staining for Tom20 (blue), NCOA4 (red) and FTMT (green) in Huh7 cells after DFP (1 mM) treatment, using the single-molecule fluorescence microscope. The white arrows indicate colocalization of these proteins.


( E ) Immunofluorescence staining for Tom20 ( blue ) , FTMT - GFP ( green ) , recombinant FTMTR82A - GFP ( green ) , NCOA4 - mCherry ( red ) and NCOA4I489A / W497A - mCherry ( red ) in Huh7 cells . Boxed areas are enlarged on the right . The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20 , FTMT - GFP and NCOA4 - mCherry . The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT - GFP and NCOA4 - mCherry . Scale bar : 10 μm and 5 μm in enlarged images .

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

(E) Immunofluorescence staining for Tom20 (blue), FTMT-GFP (green), recombinant FTMTR82A-GFP (green), NCOA4-mCherry (red) and NCOA4I489A/W497A-mCherry (red) in Huh7 cells. Boxed areas are enlarged on the right. The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20, FTMT-GFP and NCOA4-mCherry. The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT-GFP and NCOA4-mCherry. Scale bar: 10 μm and 5 μm in enlarged images.


( F ) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti - GFP - coupled magnetic beads and immunoblotted with anti - mCherry and anti - GFP antibodies . Co - immunoprecipitation revealed a specific interaction of NCOA4 - mCherry with FTMT - GFP or FTMTD57A - GFP , which has a substitution of alanine ( A ) for aspartic acid ( D ) at position 57 in the FTMT mitochondrial leader sequence ( lanes 1 and 3 ) .

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

(F) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies. Co-immunoprecipitation revealed a specific interaction of NCOA4-mCherry with FTMT-GFP or FTMTD57A-GFP, which has a substitution of alanine (A) for aspartic acid (D) at position 57 in the FTMT mitochondrial leader sequence (lanes 1 and 3).


( G ) Following DFP ( 1 . 0 mM ) or ammonium iron sulfate ( FAS ) treatment as indicated , extracted proteins from Huh7 cells expressing FTMT - GFP and NCOA4 - mCherry were mixed with anti - GFP - coupled magnetic beads and immunoblotted with anti - mCherry and anti - GFP antibodies .

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

(G) Following DFP (1.0 mM) or ammonium iron sulfate (FAS) treatment as indicated, extracted proteins from Huh7 cells expressing FTMT-GFP and NCOA4-mCherry were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies.


( A ) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria . The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC - 1 for 1 h at 30 ° C and subjected to flow cytometric analysis ( n = 4 , biological replicates ) by excitation with a blue laser ( 488 nm ) with simultaneous emission assessment using a 585 / 42 detector for JC - 1 red and a 530 / 30 for JC - 1 green . The trapezoid areas show the population of depolarized mitochondria . Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . * * : P < 0 . 01 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O 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 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 O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria. The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC-1 for 1 h at 30°C and subjected to flow cytometric analysis (n=4, biological replicates) by excitation with a blue laser (488 nm) with simultaneous emission assessment using a 585/42 detector for JC-1 red and a 530/30 for JC-1 green. The trapezoid areas show the population of depolarized mitochondria. Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.


( B ) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer ( n = 4 , technical replicates ) . Huh7 cells were incubated with 1 mM of DFP for different lengths of time ( 0 , 3 , 6 , 18 , and 24 h ) . Oxygen consumption was measured , and the respiration rate was analyzed by injecting of 1μM oligomycin , 1 μM carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP ) and 10 μM rotenone / antimycin A . OCR : oxygen consumption rate . The central point and the error bards indicate mean ± SD .

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

(B) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer (n=4, technical replicates). Huh7 cells were incubated with 1 mM of DFP for different lengths of time (0, 3, 6, 18, and 24 h). Oxygen consumption was measured, and the respiration rate was analyzed by injecting of 1μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 10 μM rotenone/antimycin A. OCR: oxygen consumption rate. The central point and the error bards indicate mean ± SD.


( C ) Immunofluorescence staining for FTMT - GFP ( green ) and LC3 ( red ) in Huh7 cells transfected with FTMT - GFP . The proportion of colocalization was quantified for four randomly selected areas ( technical replicates ) using ImageJ software version 1 . 46 ( NIH , Bethesda , MD ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * : P < 0 . 01 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O 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 O O O O O O O O O O O O O O O O O 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 for FTMT-GFP (green) and LC3 (red) in Huh7 cells transfected with FTMT-GFP. The proportion of colocalization was quantified for four randomly selected areas (technical replicates) using ImageJ software version 1.46 (NIH, Bethesda, MD). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.


( D ) Immunofluorescence staining for Ferritin - GFP heavy chain ( FTH1 ) ( green ) and LC3 ( red ) in Huh7 cells transfected with FTH1 - GFP . The proportion of colocalization was quantified as described in ( C ) . * : P < 0 . 05 , * * : P < 0 . 01 .

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

(D) Immunofluorescence staining for Ferritin-GFP heavy chain (FTH1) (green) and LC3 (red) in Huh7 cells transfected with FTH1-GFP. The proportion of colocalization was quantified as described in (C). *: P<0.05, **: P<0.01.


( E ) Immunoblots for the precursor form and processed form of FTMT - GFP , endogenous FTMT , NCOA4 , LC3 , and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT - GFP with or without digestion with proteinase K in the presence or absence of DFP . mtHSP70 was used as the loading control .

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

(E) Immunoblots for the precursor form and processed form of FTMT-GFP, endogenous FTMT, NCOA4, LC3, and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT-GFP with or without digestion with proteinase K in the presence or absence of DFP. mtHSP70 was used as the loading control.


( F ) Immunoblots for FTMT , VDAC , and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment . The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment . mtHSP70 was used as the non - membrane - integral protein control and VDAC as the membrane - integral protein control . mtHSP70 at the right - hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment .

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

(F) Immunoblots for FTMT, VDAC, and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment. The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment. mtHSP70 was used as the non-membrane-integral protein control and VDAC as the membrane-integral protein control. mtHSP70 at the right-hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment.


( A ) Immunofluorescence staining for the polarized mitochondria ( red ) , Tom20 ( blue ) , and LC3 ( green ) in Huh7 cells untreated or treated with DFP ( 1 mM ) for 24 h , or with CCCP ( 30 μM ) for 6 h . Boxed areas are enlarged on the right . Scale bar : 10 μm , and 5 μm in enlarged images .

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

(A) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), and LC3 (green) in Huh7 cells untreated or treated with DFP (1 mM) for 24 h, or with CCCP (30 μM) for 6 h. Boxed areas are enlarged on the right. Scale bar: 10 μm, and 5 μm in enlarged images.


( B ) Immunofluorescence staining for the polarized mitochondria ( red ) , Tom20 ( blue ) , LC3 ( blue ) , and FTMTD57A ( green ) using Huh7 cells expressing FTMTD57A . Boxed areas are enlarged on the right . The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria , Tom20 and FTMTD57A or the colocalization of the polarized mitochondria , FTMTD57A , and LC3 . Scale bar : 10 μm , and 5 μm in enlarged images .

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

(B) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), LC3 (blue), and FTMTD57A (green) using Huh7 cells expressing FTMTD57A. Boxed areas are enlarged on the right. The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria, Tom20 and FTMTD57A or the colocalization of the polarized mitochondria, FTMTD57A, and LC3. Scale bar: 10 μm, and 5 μm in enlarged images.


( C ) STAM mice were fed a high - fat diet and orally administered 0 . 0375 mg / g body weight of DFP , 0 . 075 mg / g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age ( n = 6 ) . Arrows indicate liver tumors . Scale bar : 1 . 0 cm . The number and maximum size of liver tumors were compared among the three groups . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 .

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

(C) STAM mice were fed a high-fat diet and orally administered 0.0375 mg/g body weight of DFP, 0.075 mg/g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age (n=6). Arrows indicate liver tumors. Scale bar: 1.0 cm. The number and maximum size of liver tumors were compared among the three groups. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.


( D ) Histology of the nontumorous liver ( H & E and Sirius red stain , original magnification x40 ) and superoxide anion production in the livers of STAM mice fed a high - fat diet with or without DFP ( 0 . 075 mg / g ) administration . Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion . Scale bar : 100 μm .

O O O B-EXP_ASSAY O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O B-ORGANISM O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O

(D) Histology of the nontumorous liver (H&E and Sirius red stain, original magnification x40) and superoxide anion production in the livers of STAM mice fed a high-fat diet with or without DFP (0.075 mg/g) administration. Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion. Scale bar: 100 μm.


( E ) Histology of the hepatocellular carcinoma using H & E staining in STAM mice fed a high - fat diet . The boxed area is enlarged on the right ( x40 in left panel and x400 in right panel ) . The tumor has increased cell density , an increased ratio of nucleus to cytoplasm , and thickened trabeculae Scale bar : 100 μm .

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

(E) Histology of the hepatocellular carcinoma using H&E staining in STAM mice fed a high-fat diet. The boxed area is enlarged on the right (x40 in left panel and x400 in right panel). The tumor has increased cell density, an increased ratio of nucleus to cytoplasm, and thickened trabeculae Scale bar: 100 μm.


( F ) Electron microscopic images of livers in control ( C57BL / 6 ) and DMBA + HFD mice that were orally administered 0 . 075 mg / g of DFP or distilled water . Scale bars : 2 . 0 μm . The boxed areas ( x 1 , 500 ) are enlarged below ( x6 , 000 on left and x10 , 000 on right ) . The white arrow indicates mitophagosome - like structures . The mean number of mitophagosome - like structures / 100 μm2 was compared for four randomly selected areas ( technical replicates ) among the four groups ( control , DMBA + HFD mice treated with 0 , 0 . 0375 or 0 . 075 mg / g of DFP ) ( n = 6 ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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) Electron microscopic images of livers in control (C57BL/6) and DMBA + HFD mice that were orally administered 0.075 mg/g of DFP or distilled water. Scale bars: 2.0 μm. The boxed areas (x 1,500) are enlarged below (x6,000 on left and x10,000 on right). The white arrow indicates mitophagosome-like structures. The mean number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) among the four groups (control, DMBA + HFD mice treated with 0, 0.0375 or 0.075 mg/g of DFP) (n=6). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.


( A ) Immunoblots for FTMT , frataxin , mitoferrin , HSP60 , FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control ( C57BL / 6J ) mice and STAM mice treated with DFP ( 0 , 0 . 0375 , or 0 . 075 mg / g body weight ) ( n = 6 ) . The expression of FTMT , frataxin , mitoferrin , FTH1 , and TfR were normalized to HSP60 or β - actin , respectively . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . ( B ) The mean number of mitophagosome - like structures / 100 μm2 from four randomly selected areas in the livers of DFP ( 0 . 075 mg / g body weight ) - administered mice were compared between control and FTMT siRNA - treated groups for STAM mice ( n = 6 ) and DMBA + HFD mice ( n = 6 ) . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . NC : negative control siRNA , * : P < 0 . 05 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-TISSUE B-SUBCELLULAR O O O O B-TISSUE O O O O O O O O B-ORGANISM O O B-ORGANISM O O B-SMALL_MOLECULE 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-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD 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 B-SUBCELLULAR O O O O O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE 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

(A) Immunoblots for FTMT, frataxin, mitoferrin, HSP60, FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control (C57BL/6J) mice and STAM mice treated with DFP (0, 0.0375, or 0.075 mg/g body weight) (n=6). The expression of FTMT, frataxin, mitoferrin, FTH1, and TfR were normalized to HSP60 or β-actin, respectively. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. (B) The mean number of mitophagosome-like structures/100 μm2 from four randomly selected areas in the livers of DFP (0.075 mg/g body weight)-administered mice were compared between control and FTMT siRNA-treated groups for STAM mice (n=6) and DMBA + HFD mice (n=6). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. NC: negative control siRNA, *: P<0.05.