( D ) Using the dual color labeling system ( esg - Gal4 > UASp - FRT - H3 - eGFP - FRT - H3 - mCherry ) , old and new H3 distribution in prophase and prometaphase ISCs shows large , separable domains of H3 - eGFP ( old ) and H3 - mCherry ( new ) . Note : all figure panels in this work are maximum intensity projection images to show signals from all Z - stacks . ( E ) Using the UASp - FRT - histone - eGFP - T2A - histone - mCherry - PolyA transgene that co - expresses H3 - eGFP and H3 - mCherry , significant overlap between the two signals were detected in ISCs during prophase and prometaphase . ( F ) Quantification of the colocalization between old and new H3 , as well as between the co - expressed eGFP - and mCherry - tagged H3 in prophase and prometaphase ISCs . Pearson ' s correlation coefficients were measured , where 1 represents complete colocalization and 0 stands for no colocalization . Old and new H3 showed significantly less colocalization ( Pearson ' s correlation coefficient for H3 = 0 . 44 + 0 . 02 , n = 81 ISCs ) when compared to the co - expressed eGFP - and mCherry - tagged H3 ( Pearson ' s correlation coefficient for H3 co - expressed = 0 . 86 + 0 . 01 , n = 19 ISCs ) . Individual data points and mean values are shown . Error bars represent SEM . * * * * p < 0 . 0001 ; unpaired t test to compare two individual datasets to each other . Data information : n for individual ISCs . Scale bar in ( D ) , ( E ) : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Using the dual color labeling system (esg-Gal4>UASp-FRT-H3-eGFP-FRT-H3-mCherry), old and new H3 distribution in prophase and prometaphase ISCs shows large, separable domains of H3-eGFP (old) and H3-mCherry (new). Note: all figure panels in this work are maximum intensity projection images to show signals from all Z-stacks. (E) Using the UASp-FRT-histone-eGFP-T2A-histone-mCherry-PolyA transgene that co-expresses H3-eGFP and H3-mCherry, significant overlap between the two signals were detected in ISCs during prophase and prometaphase. (F) Quantification of the colocalization between old and new H3, as well as between the co-expressed eGFP- and mCherry-tagged H3 in prophase and prometaphase ISCs. Pearson's correlation coefficients were measured, where 1 represents complete colocalization and 0 stands for no colocalization. Old and new H3 showed significantly less colocalization (Pearson's correlation coefficient for H3 = 0.44 + 0.02, n = 81 ISCs) when compared to the co-expressed eGFP- and mCherry-tagged H3 (Pearson's correlation coefficient for H3 co-expressed = 0.86 + 0.01, n = 19 ISCs). Individual data points and mean values are shown. Error bars represent SEM. **** p < 0.0001; unpaired t test to compare two individual datasets to each other. Data information: n for individual ISCs. Scale bar in (D), (E): 5 µm.


( A ) Old and new H4 distribution in prophase and prometaphase ISCs ( esg - Gal4 > UASp - FRT - H4 - eGFP - FRT - H4 - mCherry ) . Separable domains of H4 - eGFP ( old ) and H4 - mCherry ( new ) were observed ( B ) Old and new H2A distribution in prophase and prometaphase ISCs ( esg - Gal4 > UASp - FRT - H2A - eGFP - FRT - H2A - mCherry ) , showing largely overlapping signals between H2A - eGFP ( old ) and H2A - mCherry ( new ) . Data information : n for individual ISCs . Scale bar in ( A ) , ( B ) 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Old and new H4 distribution in prophase and prometaphase ISCs (esg-Gal4>UASp-FRT-H4-eGFP-FRT-H4-mCherry). Separable domains of H4-eGFP (old) and H4-mCherry (new) were observed (B) Old and new H2A distribution in prophase and prometaphase ISCs (esg-Gal4>UASp-FRT-H2A-eGFP-FRT-H2A-mCherry), showing largely overlapping signals between H2A-eGFP (old) and H2A-mCherry (new). Data information: n for individual ISCs. Scale bar in (A), (B) 5 µm.


( D ) Late - stage mitotic ISC showing asymmetric segregation of H3 between the two sets of sister chromatids at the opposite poles of the ISC . ( E ) Late - stage mitotic ISC showing less asymmetric segregation of H4 , compared to H3 , between the two sets of sister chromatids at the opposite poles of the ISC . ( F ) Late - stage mitotic ISC showing symmetric segregation of H2A between the two sets of sister chromatids at the opposite poles of the ISC . Data information : n for individual ISCs . Scale bar in ( D ) , ( E ) , and ( F ) : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Late-stage mitotic ISC showing asymmetric segregation of H3 between the two sets of sister chromatids at the opposite poles of the ISC. (E) Late-stage mitotic ISC showing less asymmetric segregation of H4, compared to H3, between the two sets of sister chromatids at the opposite poles of the ISC. (F) Late-stage mitotic ISC showing symmetric segregation of H2A between the two sets of sister chromatids at the opposite poles of the ISC. Data information: n for individual ISCs. Scale bar in (D), (E), and (F): 5 µm.


( A ) Old and new H3 distribution in a post - mitotic pair of cells with asymmetric Dl - nLacZ labeling , showing that H3 - eGFP ( old ) is asymmetrically inherited by the Delta - high cell , while H3 - mCherry ( new ) is enriched in the Delta - low cell ( esg - Gal4 > UASp - FRT - H3 - eGFP - FRT - H3 - mCherry ) . ( B ) Old and new H3 distribution in a post - mitotic pair of cells with symmetric Dl - nLacZ labeling , showing that both H3 - eGFP ( old ) and H3 - mCherry ( new ) are more symmetrically distributed between the two cells . Scale bar in ( A ) , ( B ) , 5 µm ; asterisk , ISC side .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Old and new H3 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H3-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H3-mCherry (new) is enriched in the Delta-low cell (esg-Gal4>UASp-FRT-H3-eGFP-FRT-H3-mCherry). (B) Old and new H3 distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that both H3-eGFP (old) and H3-mCherry (new) are more symmetrically distributed between the two cells. Scale bar in (A), (B), 5 µm; asterisk, ISC side.


( E ) Old and new H4 distribution in a post - mitotic pair of cells with asymmetric Dl - nLacZ labeling , showing that H4 - eGFP ( old ) is asymmetrically inherited by the Delta - high cell , while H4 - mCherry ( new ) is enriched toward the Delta - low cell ( esg - Gal4 > UASp - FRT - H4 - eGFP - FRT - H4 - mCherry ) . ( F ) Old and new H4 distribution in a post - mitotic pair with symmetric Dl - nLacZ labeling , showing that H4 - eGFP ( old ) and H4 - mCherry ( new ) are symmetrically distributed between the two cellss . Scale bar in ( E ) , ( F ) , 5 µm ; asterisk , ISC side .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Old and new H4 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H4-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H4-mCherry (new) is enriched toward the Delta-low cell (esg-Gal4>UASp-FRT-H4-eGFP-FRT-H4-mCherry). (F) Old and new H4 distribution in a post-mitotic pair with symmetric Dl-nLacZ labeling, showing that H4-eGFP (old) and H4-mCherry (new) are symmetrically distributed between the two cellss. Scale bar in (E), (F), 5 µm; asterisk, ISC side.


( I ) Old and new H2A distribution in a post - mitotic pair of cells with asymmetric Dl - nLacZ labeling , showing that H2A - eGFP ( old ) and H2A - mCherry ( new ) are symmetrically inherited by the two cells ( esg - Gal4 > UASp - FRT - H2A - eGFP - FRT - H2A - mCherry ) . ( J ) Old and new H2A distribution in a post - mitotic pair of cells with symmetric Dl - nLacZ labeling , showing that H2A - eGFP ( old ) and H2A - mCherry ( new ) are symmetrically distributed between the two cells . Scale bar in ( I ) , ( J ) , 5 µm ; asterisk , ISC side .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Old and new H2A distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H2A-eGFP (old) and H2A-mCherry (new) are symmetrically inherited by the two cells (esg-Gal4>UASp-FRT-H2A-eGFP-FRT-H2A-mCherry). (J) Old and new H2A distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that H2A-eGFP (old) and H2A-mCherry (new) are symmetrically distributed between the two cells. Scale bar in (I), (J), 5 µm; asterisk, ISC side.


( M ) The ISC - specific combination of drivers esg - Gal4 , Su ( H ) - Gal80 drive H3 transgene [ esg - Gal4 , Su ( H ) - Gal80 > UASp - FRT - H3 - eGFP - FRT - H3 - mCherry ] with heat shock treatment and recovery ( see Materials and Methods ) in a Delta - asymmetric pair of cells . Old and new H3 distribution in a post - mitotic pair of cells with asymmetric Dl - nLacZ labeling , showing that H3 - eGFP ( old ) is asymmetrically inherited by the Delta - high cell , while H3 - mCherry ( new ) is enriched in the Delta - low cell . ( N ) ISC specific drivers express the H3 transgene in a Delta - symmetric pair of cells . Old and new H3 distribution in a post - mitotic pair of cells with symmetric Dl - nLacZ labeling , showing that both H3 - eGFP ( old ) and H3 - mCherry ( new ) are more symmetrically distributed between the two cells . Scale bar in ( M ) , and ( N ) : 5 µm ; asterisk , ISC side .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) The ISC-specific combination of drivers esg-Gal4, Su(H)-Gal80 drive H3 transgene [esg-Gal4, Su(H)-Gal80>UASp-FRT-H3-eGFP-FRT-H3-mCherry] with heat shock treatment and recovery (see Materials and Methods) in a Delta-asymmetric pair of cells. Old and new H3 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H3-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H3-mCherry (new) is enriched in the Delta-low cell. (N) ISC specific drivers express the H3 transgene in a Delta-symmetric pair of cells. Old and new H3 distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that both H3-eGFP (old) and H3-mCherry (new) are more symmetrically distributed between the two cells. Scale bar in (M), and (N): 5 µm; asterisk, ISC side.


( A ) Old and new H3T3A distribution in prophase ISC , showing separable domains between H3T3A - GFP ( old ) and H3T3A - mKO ( new ) ( esg - Gal4 > UASp - FRT - H3T3A - eGFP - FRT - H3T3A - mCherry ) . Data information : Scale bar in ( A ) , ( C ) , ( E ) , ( F ) : 5 µm ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Old and new H3T3A distribution in prophase ISC, showing separable domains between H3T3A-GFP (old) and H3T3A-mKO (new) (esg-Gal4>UASp-FRT-H3T3A-eGFP-FRT-H3T3A-mCherry). Data information: Scale bar in (A), (C), (E), (F): 5 µm,


( B ) Quantification of colocalization between H3T3A - GFP ( old ) and H3T3A - mKO ( new ) in prophase and prometaphase ISCs using Pearson ' s correlation coefficients . The H3 and H2A data are from Figure 2C for direct comparison . Old and new H3T3A distribution is more similar to that of H3 ( Pearson ' s correlation coefficient for H3T3A = 0 . 49 + 0 . 02 , n = 55 ISCs ) . Individual data points and mean values are shown . Error bars represent SEM . * * * * p < 0 . 0001 , * p < 0 . 05 ; unpaired t test to compare two individual datasets to each other .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of colocalization between H3T3A-GFP (old) and H3T3A-mKO (new) in prophase and prometaphase ISCs using Pearson's correlation coefficients. The H3 and H2A data are from Figure 2C for direct comparison. Old and new H3T3A distribution is more similar to that of H3 (Pearson's correlation coefficient for H3T3A= 0.49 + 0.02, n = 55 ISCs). Individual data points and mean values are shown. Error bars represent SEM. **** p < 0.0001, * p < 0.05; unpaired t test to compare two individual datasets to each other.


( E ) In a post - mitotic pair of cells with comparable Dl - nLacZ , both H3T3A - GFP ( old ) and H3T3A - mKO ( new ) are symmetrically distributed between the two cells . ( F ) In a post - mitotic pair of cells with asymmetric Dl - nLacZ expression , H3T3A - GFP ( old ) is enriched in the Delta - high cell , while H3T3A - mKO ( new ) is more toward the Delta - low cell . Data information : Scale bar in ( E ) , ( F ) : 5 µm , asterisk , ISC side .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In a post-mitotic pair of cells with comparable Dl-nLacZ, both H3T3A-GFP (old) and H3T3A-mKO (new) are symmetrically distributed between the two cells. (F) In a post-mitotic pair of cells with asymmetric Dl-nLacZ expression, H3T3A-GFP (old) is enriched in the Delta-high cell, while H3T3A-mKO (new) is more toward the Delta-low cell. Data information: Scale bar in (E), (F): 5 µm, asterisk, ISC side.


( H ) Distribution of the Dl - nLacZ - positive ( magenta ) cells in an H3 - expressing midgut , which are well spaced and interspersed within the intestinal epithelium . ( I ) Distribution of the Dl - nLacZ - positive ( magenta ) cells in an H3T3A - expressing midgut , which are unevenly distributed in clusters with two or more Delta - positive cells . DAPI : white in ( H - I ) . Data information : Scale bar in ( H ) and ( I ) : 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of the Dl-nLacZ-positive (magenta) cells in an H3-expressing midgut, which are well spaced and interspersed within the intestinal epithelium. (I) Distribution of the Dl-nLacZ-positive (magenta) cells in an H3T3A-expressing midgut, which are unevenly distributed in clusters with two or more Delta-positive cells. DAPI: white in (H-I). Data information: Scale bar in (H) and (I): 50 µm


( K ) Percentages of clusters with 1 - , 2 - , or > 3 - Delta - positive cell clusters for H3 - expressing midguts ( avg . percentage of 1 - Dl + cells = 87 . 7 % + 1 . 1 % , avg . percentage of 2 - Dl + cells = 11 . 9 % + 0 . 9 % , avg . percentage of > 3 - Dl + cells = 0 . 4 % + 0 . 2 % , N = 4 midguts , n = 1 , 482 clusters ) and H3T3A - expressing midguts ( avg . percentage of 1 - Dl + cells = 68 . 2 % + 1 . 2 % , avg . percentage of 2 - Dl + cells = 28 . 5 % + 0 . 9 % , avg . percentage of > 3 - Dl + cells = 3 . 3 % + 0 . 4 % , N = 5 midguts , n = 1 , 862 clusters ) . Bars represent mean proportions of cell clusters with 1 , 2 , or 3 + Dl + cells out of all cell clusters counted . Error bars represent SEM . Comparing H3 - expressing midguts with H3T3A - expressing midguts , there was a statistically significant difference in the distributions of Delta - positive cells per cluster , determined by a Chi - Square test ( X2 ( 2 , n = 1862 ) = 908 . 75 , * * * * p < 0 . 0001 ) . As shown , there are significant increases in 2 and > 3 - Delta - positive cells per cluster in the H3T3A - expressing intestines when compared to the H3 - expressing intestines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Percentages of clusters with 1-, 2-, or >3-Delta-positive cell clusters for H3-expressing midguts (avg. percentage of 1-Dl+ cells = 87.7% + 1.1%, avg. percentage of 2-Dl+ cells = 11.9% + 0.9%, avg. percentage of >3-Dl+ cells = 0.4% + 0.2%, N = 4 midguts, n = 1,482 clusters) and H3T3A-expressing midguts (avg. percentage of 1-Dl+ cells = 68.2% + 1.2%, avg. percentage of 2-Dl+ cells = 28.5% + 0.9%, avg. percentage of >3-Dl+ cells = 3.3% + 0.4%, N = 5 midguts, n = 1,862 clusters). Bars represent mean proportions of cell clusters with 1, 2, or 3+ Dl+ cells out of all cell clusters counted. Error bars represent SEM. Comparing H3-expressing midguts with H3T3A-expressing midguts, there was a statistically significant difference in the distributions of Delta-positive cells per cluster, determined by a Chi-Square test (X2(2, n = 1862) = 908.75, **** p < 0.0001). As shown, there are significant increases in 2 and >3-Delta-positive cells per cluster in the H3T3A-expressing intestines when compared to the H3-expressing intestines.


( L ) Percentages of single Delta - positive cells out of total Delta - positive cells for H3 - expressing midguts ( avg . single Dl + cells = 77 . 9 % + 1 . 9 % , N = 4 midguts , n = 1 , 674 Dl + cells ) and H3T3A - expressing midguts ( avg . single Dl + cells = 50 . 4 % + 1 . 5 % , N = 5 midguts , n = 2 , 522 Dl + cells ) . Bars represent mean proportion of single Dl + ISCs out of all ISCs counted . Error bars represent SEM . There are fewer single Delta - positive cells in H3T3A - expressing midguts compared to H3 - expressing midguts ( two sample t test , * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Percentages of single Delta-positive cells out of total Delta-positive cells for H3-expressing midguts (avg. single Dl+ cells = 77.9% + 1.9%, N = 4 midguts, n = 1,674 Dl+ cells) and H3T3A-expressing midguts (avg. single Dl+ cells = 50.4% + 1.5%, N = 5 midguts, n = 2,522 Dl+ cells). Bars represent mean proportion of single Dl+ ISCs out of all ISCs counted. Error bars represent SEM. There are fewer single Delta-positive cells in H3T3A-expressing midguts compared to H3-expressing midguts (two sample t test, *** p < 0.001).


( M ) Comparison of the mitotic index in H3 - expressing ( N = 8 midguts ) and H3T3A - expressing ( N = 8 midguts ) midguts . Total number of mitotic ISCs were quantified per midgut by the presence of H3S10 phosphorylation mark . H3T3A - expressing midguts had a significantly higher mitotic index when compared to H3 - expressing midguts ( two sample t test , * p < 0 . 05 ) . Bars represent mean number of mitotic cells per midgut . Error bars represent SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Comparison of the mitotic index in H3-expressing (N = 8 midguts) and H3T3A-expressing (N = 8 midguts) midguts. Total number of mitotic ISCs were quantified per midgut by the presence of H3S10 phosphorylation mark. H3T3A-expressing midguts had a significantly higher mitotic index when compared to H3-expressing midguts (two sample t test, * p < 0.05). Bars represent mean number of mitotic cells per midgut. Error bars represent SEM.


( A ) A view of the midgut of an UAS - H3 - mCherry transgene driven by esg - Gal4 , Su ( H ) - Gal80 , tub - Gal80ts at the Gal80ts permissive temperature ( 18 ° C ) , showing no expression of the histone transgene or the UAS - eYFP reporter that marks ISCs . ( B ) A view of the midgut of an UAS - H3 - mCherry transgene driven by esg - Gal4 , SuH - Gal80 , tub - Gal80ts at the Gal80ts restrictive temperature ( 29 ° C ) , showing expression of the H3 - mCherry transgene ( red ) and the UAS - eYFP reporter ( green ) . ( C ) A view of the midgut of an UAS - H3T3A - mCherry transgene driven by esg - Gal4 , SuH - Gal80 , tub - Gal80ts at the Gal80ts permissive temperature ( 18 ° C ) , showing no expression of the histone transgene or the UAS - eYFP reporter . ( D ) A view of the midgut of an UAS - H3T3A - mCherry transgene driven by esg - Gal4 , SuH - Gal80 , tub - Gal80ts at the Gal80ts restrictive temperature ( 29 ° C ) , showing expression of the H3T3A - mCherry transgene ( red ) and the UAS - eYFP reporter ( green ) . Data information : Scale bars in ( A ) , ( B ) , ( C ) , and ( D ) : 50

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(A) A view of the midgut of an UAS-H3-mCherry transgene driven by esg-Gal4, Su(H)-Gal80, tub-Gal80ts at the Gal80ts permissive temperature (18°C), showing no expression of the histone transgene or the UAS-eYFP reporter that marks ISCs. (B) A view of the midgut of an UAS-H3-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts restrictive temperature (29°C), showing expression of the H3-mCherry transgene (red) and the UAS-eYFP reporter (green). (C) A view of the midgut of an UAS-H3T3A-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts permissive temperature (18°C), showing no expression of the histone transgene or the UAS-eYFP reporter. (D) A view of the midgut of an UAS-H3T3A-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts restrictive temperature (29°C), showing expression of the H3T3A-mCherry transgene (red) and the UAS-eYFP reporter (green). Data information: Scale bars in (A), (B), (C), and (D): 50 µm


( I ) Comparison of the mitotic index in H3 - expressing ( N = 11 midguts ) and H3T3A - expressing ( N = 11 midguts ) midguts . Total number of mitotic ISCs were quantified per midgut by the presence of H3S10 phosphorylation mark . Bars represent mean number of mitotic cells per midgut . Error bars represent SEM . H3T3A - expressing midguts had a significantly higher mitotic index when compared to H3 - expressing midguts ( two sample t test , * p < 0 . 05 ) .

(I) Comparison of the mitotic index in H3-expressing (N = 11 midguts) and H3T3A-expressing (N = 11 midguts) midguts. Total number of mitotic ISCs were quantified per midgut by the presence of H3S10 phosphorylation mark. Bars represent mean number of mitotic cells per midgut. Error bars represent SEM. H3T3A-expressing midguts had a significantly higher mitotic index when compared to H3-expressing midguts (two sample t test, * p < 0.05).


( J ) Qualitative analysis of midgut phenotypes . No phenotype is called when the ISCs appear normally distributed based on eYFP reporter signal . A mild phenotype is called when there is a small region of eYFP - positive cells clustered within a region , showing mild disorganization . A strong phenotype is called when there is a large region of eYFP - positive cells clustered , showing significant disorganization . ( K ) Percentage of H3 - expressing midguts ( N = 11 midguts ) and H3T3A - expressing midguts ( N = 11 midguts ) with no phenotype , a mild phenotype , or a strong phenotype . Data information : Scale bars in ( J ) : 500 µm .

(J) Qualitative analysis of midgut phenotypes. No phenotype is called when the ISCs appear normally distributed based on eYFP reporter signal. A mild phenotype is called when there is a small region of eYFP-positive cells clustered within a region, showing mild disorganization. A strong phenotype is called when there is a large region of eYFP-positive cells clustered, showing significant disorganization. (K) Percentage of H3- expressing midguts (N = 11 midguts) and H3T3A- expressing midguts (N = 11 midguts) with no phenotype, a mild phenotype, or a strong phenotype. Data information: Scale bars in (J): 500 µm.


( A ) A view of the midgut of an UAS - H3 - mCherry transgene driven by esg - Gal4 , SuH - Gal80 , tub - Gal80ts at the Gal80ts restrictive temperature ( 29 ° C ) , showing expression of the H3 - mCherry transgene ( grey ) and the UAS - eYFP reporter ( green ) from the driver as well as expression of the Su ( H ) - LacZ reporter line ( magenta ) . eYFP and Su ( H ) - LacZ signals are organized and separable . ( B ) A view of the midgut of an UAS - H3T3A - mCherry transgene driven by esg - Gal4 , SuH - Gal80 , tub - Gal80ts at the Gal80ts restrictive temperature ( 29 ° C ) , showing expression of the H3T3A - mCherry transgene ( grey ) and the UAS - eYFP reporter ( green ) from the driver as well as expression of the Su ( H ) - LacZ reporter line ( magenta ) . eYFP and Su ( H ) - LacZ signals are disorganized and overlapping . Data information : Scale bars in ( A ) and ( B ) : 50 µm .

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(A) A view of the midgut of an UAS-H3-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts restrictive temperature (29°C), showing expression of the H3-mCherry transgene (grey) and the UAS-eYFP reporter (green) from the driver as well as expression of the Su(H)-LacZ reporter line (magenta). eYFP and Su(H)-LacZ signals are organized and separable. (B) A view of the midgut of an UAS-H3T3A-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts restrictive temperature (29°C), showing expression of the H3T3A-mCherry transgene (grey) and the UAS-eYFP reporter (green) from the driver as well as expression of the Su(H)-LacZ reporter line (magenta). eYFP and Su(H)-LacZ signals are disorganized and overlapping. Data information: Scale bars in (A) and (B): 50 µm.


Figure 1 . Ret - eGFP is Expressed in Multiple Sensory Neuron Subsets ( A − L ) Ret - positive neurons largely overlap with markers for myelinated neurons ( NF200 ) , non - peptidergic nociceptors ( IB4 ) and C - fiber low - threshold mechanoreceptors ( TH ) . However some Ret positive neurons are negative for these markers ( indicated by the arrows in D , H and L ) suggesting the existence of a further subset of Ret + neurons . ( M ) Quantification of the proportion of Ret - eGFP positive neurons in DRG . ( N ) Quantification of Ret - eGFP co - expression with other markers . ( n = 2278 cells from 3 mice ) Scale bars , 50 μm .

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Figure 1. Ret-eGFP is Expressed in Multiple Sensory Neuron Subsets(A−L) Ret-positive neurons largely overlap with markers for myelinated neurons (NF200), non-peptidergic nociceptors (IB4) and C-fiber low-threshold mechanoreceptors (TH). However some Ret positive neurons are negative for these markers (indicated by the arrows in D,H and L) suggesting the existence of a further subset of Ret+ neurons. (M) Quantification of the proportion of Ret-eGFP positive neurons in DRG . (N) Quantification of Ret-eGFP co-expression with other markers. (n=2278 cells from 3 mice) Scale bars, 50 μm.


( A ) Flow cytometric analysis of dissociated sensory neurons plotted according to their level of endogenous Ret - eGFP expression and IB4 binding ( also indicated in Figure EV1 ) . There are 5 well - defined subsets , 2 of which do not bind to IB4 but display a differential level of eGFP intensity ( termed Ret - eGFPLo : IB4Neg , A , and Ret - eGFPHi : IB4Neg , B , respectively ) . The other 3 subsets bind IB4 and display a range of eGFP intensities , termed Ret - eGFPLo : IB4Lo , C , Ret - eGFPHi : IB4Lo , D , and Ret - eGFPHi : IB4Hi , E . Percent of total events are indicated in each box .

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(A) Flow cytometric analysis of dissociated sensory neurons plotted according to their level of endogenous Ret-eGFP expression and IB4 binding (also indicated in Figure EV1). There are 5 well-defined subsets, 2 of which do not bind to IB4 but display a differential level of eGFP intensity (termed Ret-eGFPLo:IB4Neg, A, and Ret-eGFPHi:IB4Neg ,B, respectively). The other 3 subsets bind IB4 and display a range of eGFP intensities, termed Ret-eGFPLo:IB4Lo, C, Ret-eGFPHi:IB4Lo, D, and Ret-eGFPHi:IB4Hi, E. Percent of total events are indicated in each box.


( B ) No correlation between the median cell size of different Ret + subsets and eGFP intensity or IB4 binding . The graph shows the forward scatter values plotted against the normalized number of cells , displayed as the percent of Max . ( C − D ) Variations in endogenous eGFP intensity are clearly visible with fluorescent microscopy .

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(B) No correlation between the median cell size of different Ret+ subsets and eGFP intensity or IB4 binding. The graph shows the forward scatter values plotted against the normalized number of cells, displayed as the percent of Max. (C−D) Variations in endogenous eGFP intensity are clearly visible with fluorescent microscopy.


( C ) Cultured neurons and ( D ) DRG section from Avil - Cre : : Ret + / eGFPmice displaying native eGFPfluorescence and stained with IB4 . Different levels of eGFP intensity ( high - indicated by arrows and low - indicated by arrowheads ) are detected across IB4 + and IB4 - cells . Scale bar , 50 μm .

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(C) Cultured neurons and (D) DRG section from Avil-Cre::Ret+/eGFPmice displaying native eGFPfluorescence and stained with IB4. Different levels of eGFP intensity (high -indicated by arrows and low -indicated by arrowheads) are detected across IB4+ and IB4- cells. Scale bar, 50 μm.


( A ) Ret - eGFPHi : IB4Neg and Ret - eGFPLo : IB4Negneurons display distinct expression profiles . A volcano plot of fold change expression in Ret - eGFPLo : IB4Neg versus Ret - eGFPHi : IB4Neg against probability . Ret has higher expression in the Ret - eGFPHi : IB4Neg subset , which also shows an up regulation of the Ret co - receptor Gfra2 and Fam38b , encoding for the mechanosensitive ion channel Piezo2 . The Ret - eGFPLo : IB4Neg subset , displays an array of molecules previously associated with itch ( marked in red ) .

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(A) Ret-eGFPHi:IB4Neg and Ret-eGFPLo:IB4Negneurons display distinct expression profiles. A volcano plot of fold change expression in Ret-eGFPLo:IB4Neg versus Ret-eGFPHi:IB4Neg against probability. Ret has higher expression in the Ret-eGFPHi:IB4Neg subset, which also shows an up regulation of the Ret co-receptor Gfra2 and Fam38b, encoding for the mechanosensitive ion channel Piezo2. The Ret-eGFPLo:IB4Neg subset, displays an array of molecules previously associated with itch (marked in red).


( B ) Itch - associated transcripts are enriched in the Ret - eGFPLo : IB4Neg population compared to all DRGneurons . Volcano plot of fold change expression in Ret - eGFPLo : IB4Negneurons versus sorted neurons from Avil - Cre : : R26tdRFPmice against probability . Molecules linked to itch perception are significantly upregulated in the Ret - eGFPLo : IB4Neg subset .

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(B) Itch-associated transcripts are enriched in the Ret-eGFPLo:IB4Neg population compared to all DRGneurons. Volcano plot of fold change expression in Ret-eGFPLo:IB4Negneurons versus sorted neurons from Avil-Cre::R26tdRFPmice against probability. Molecules linked to itch perception are significantly upregulated in the Ret-eGFPLo:IB4Neg subset.


( C ) Gene expression within the Ret - eGFPLo : IB4Neg population is confirmed by triple comparison between Ret - eGFPLo : IB4Neg , Ret - eGFPHi : IB4Neg and Avil - Cre : : R26tdRFPdatasets .

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(C) Gene expression within the Ret-eGFPLo:IB4Neg population is confirmed by triple comparison between Ret-eGFPLo:IB4Neg, Ret-eGFPHi:IB4Neg and Avil-Cre::R26tdRFPdatasets.


( D ) Validation of differential microarray screening by quantitative RT - PCR . Transcripts encoding ion channels , receptors , neuropeptides , signaling molecules and transcription factors were selected for quantitative RT - PCR . Differential expression between Ret - eGFPLo : IB4Neg and Ret - eGFPHi : IB4Neg populations correlates with microarray analysis ( n = 3 ) .

(D) Validation of differential microarray screening by quantitative RT-PCR. Transcripts encoding ion channels, receptors, neuropeptides, signaling molecules and transcription factors were selected for quantitative RT-PCR. Differential expression between Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg populations correlates with microarray analysis (n=3).


( A − H ) SstCre mediated recombination of the Ret locus drives eGFP expression in sensory neurons that do not bind to IB4 or co - express NF200 or TH . Triple immunostaining of DRG from Sst - Cre : : ReteGFP / + mice with RetGFP ( A ) , IB4 ( B ) and NF200 ( C ) , and RetGFP ( E ) , IB4 ( F ) and TH ( G ) .

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(A−H) SstCre mediated recombination of the Ret locus drives eGFP expression in sensory neurons that do not bind to IB4 or co-express NF200 or TH. Triple immunostaining of DRG from Sst-Cre::ReteGFP/+mice with RetGFP (A), IB4 (B) and NF200 (C), and RetGFP (E), IB4 (F) and TH (G).


( I ) Quantification of SstCre : : Ret - eGFP expression in DRG . ( J ) Quantification of co - expression of SstCre : : Ret - eGFP with neuronal markers ( n = 8827 cells from 3 mice ) .

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(I) Quantification of SstCre::Ret-eGFP expression in DRG. (J) Quantification of co-expression of SstCre::Ret-eGFP with neuronal markers (n=8827 cells from 3 mice).


( K − M ) Peripheral projections of sensory neurons from Sst - Cre : : Rosa26SnapCaaXmice terminate in the hairy skin as free nerve endings . Non - innervated hair follicles are indicated by arrows . Double labelling using TMRStar to label SNAP - tag ( K ) and DAPI ( L ) . Scale bars , 50 μm .

(K−M) Peripheral projections of sensory neurons from Sst-Cre::Rosa26SnapCaaXmice terminate in the hairy skin as free nerve endings. Non-innervated hair follicles are indicated by arrows. Double labelling using TMRStar to label SNAP-tag (K) and DAPI (L). Scale bars, 50 μm.


( A - B ) Contour diagram of dissociated sensory neurons from Sst - Cre : : ReteGFP / + ( A ) and Avil - Cre : : ReteGFP / + neurons ( B ) , plotted according to their native eGFP expression and IB4 binding . Only one subset of Ret - eGFP cells can be defined within the Sst - Cre : : ReteGFP / + dissociated DRGneurons . This displays the same range of eGFPfluorescence as the Ret - eGFPLo : IB4Neg population and doesn ' t bind to IB4 .

(A-B) Contour diagram of dissociated sensory neurons from Sst-Cre::ReteGFP/+ (A) and Avil-Cre::ReteGFP/+neurons (B), plotted according to their native eGFP expression and IB4 binding. Only one subset of Ret-eGFP cells can be defined within the Sst-Cre::ReteGFP/+ dissociated DRGneurons. This displays the same range of eGFPfluorescence as the Ret-eGFPLo:IB4Neg population and doesn't bind to IB4.


( C ) Quantitative RT - PCR analysis of selected transcripts in the Sst - Cre : : ReteGFP ( darker colors ) and Ret - eGFPHi : IB4Neg ( lighter colors ) subsets displayed as differential expression compared to the Ret - eGFPHi : IB4Neg subset . Almost all the genes that are up - regulated in the Ret - eGFPLo : IB4Neg compared to the Ret - eGFPHi : IB4Neg subset are also enriched in the Sst - Cre : : ReteGFP population ( n = 3 )

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(C) Quantitative RT-PCR analysis of selected transcripts in the Sst-Cre::ReteGFP (darker colors) and Ret-eGFPHi:IB4Neg (lighter colors) subsets displayed as differential expression compared to the Ret-eGFPHi:IB4Neg subset. Almost all the genes that are up-regulated in the Ret-eGFPLo:IB4Neg compared to the Ret-eGFPHi:IB4Neg subset are also enriched in the Sst-Cre::ReteGFP population (n=3)


( A - G ) Ablation of Sst - Cre : : AviliDTR positive sensory neurons by injection of diphtheria toxin ( DTX ) . Immunostaining of DRG from Sst - Cre : : AviliDTRmice with an antibody against the diphtheria toxin receptor and IB4 in the absence of diphtheria toxin ( A - C ) and after systemic injection of diphtheria toxin ( D - F ) .

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(A-G) Ablation of Sst-Cre::AviliDTR positive sensory neurons by injection of diphtheria toxin (DTX). Immunostaining of DRG from Sst-Cre::AviliDTRmice with an antibody against the diphtheria toxin receptor and IB4 in the absence of diphtheria toxin (A-C) and after systemic injection of diphtheria toxin (D-F).


( G ) Quantification of diphtheria toxin receptor positive neurons before and after injection of diphtheria toxin .

(G) Quantification of diphtheria toxin receptor positive neurons before and after injection of diphtheria toxin.


( H - K ) Examples of calcium flux in dissociated DRG upon application of histamine ( H ) , chloroquine ( I ) , IL -

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(H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31


( H - K ) Examples of calcium flux in dissociated DRG upon application of histamine ( H ) , chloroquine ( I ) , IL - 31 ( J ) and 5HT1F agonist Ly344864 ( K ) .

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(H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31 (J) and 5HT1F agonist Ly344864 (K).


( L ) Quantification of percent responding neurons to the indicated pruritogens before or after systemic injection of diphtheria toxin in Sst - Cre : : AviliDTRmice . Scale bars , 50 μm , asterisk denotes p < 0 . 05 , n = 4 - 6 mice , Student ' s t - test , error bars indicate SEM .

(L) Quantification of percent responding neurons to the indicated pruritogens before or after systemic injection of diphtheria toxin in Sst-Cre::AviliDTRmice. Scale bars, 50 μm, asterisk denotes p<0.05, n=4-6 mice, Student's t-test, error bars indicate SEM.


( A - B ) Nociceptive withdrawal thresholds in control ( AviliDTR without the Cre ) and Sst - Cre : : AviliDTRmice after systemic diphtheria toxin injection . No significant differences in thermal ( A ) or mechanical ( B ) withdrawal reflexes after ablation of SstCre positive neurons .

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(A-B) Nociceptive withdrawal thresholds in control (AviliDTR without the Cre) and Sst-Cre::AviliDTRmice after systemic diphtheria toxin injection. No significant differences in thermal (A) or mechanical (B) withdrawal reflexes after ablation of SstCre positive neurons.


( C - F ) Scratching behavior after a single intradermal injection of the indicated pruritogen observed for 1 hour . All compounds evoked a significant increase in the number of scratching bouts compared to vehicle alone in control ( AviliDTR ) or Sst - Cre : : AviliDTRmice . Diphtheria toxin injection in control mice ( AviliDTR ) had no effect on scratching behavior evoked by histamine ( C ) , chloroquine ( D ) , IL - 31 ( E ) or 5HT1F agonist Ly344864 ( F ) but significantly reduced responses to IL31 , or Ly344864 in Sst - Cre : : AviliDTRmice . n - numbers indicated in brackets , asterisk denotes p < 0 . 05 , two way RM ANOVA , Holm - Sidak multiple comparison . Error bars indicate SEM .

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(C-F) Scratching behavior after a single intradermal injection of the indicated pruritogen observed for 1 hour. All compounds evoked a significant increase in the number of scratching bouts compared to vehicle alone in control (AviliDTR) or Sst-Cre::AviliDTRmice. Diphtheria toxin injection in control mice (AviliDTR) had no effect on scratching behavior evoked by histamine (C), chloroquine (D), IL-31 (E) or 5HT1F agonist Ly344864 (F) but significantly reduced responses to IL31, or Ly344864 in Sst-Cre::AviliDTRmice. n-numbers indicated in brackets, asterisk denotes p<0.05, two way RM ANOVA, Holm-Sidak multiple comparison. Error bars indicate SEM.


W0 , W6 and W12 refer to the week of study . Serum FGF - 21 and GDF - 15 levels before , during and after treatment ( two - sided Wilcoxon signed - rank test , P - value = 0 . 031 in all significant cases ) ( see Fig . EV2 for details where P1 - P6 refer to the individual patients ) .

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W0, W6 and W12 refer to the week of study. Serum FGF-21 and GDF-15 levels before, during and after treatment (two-sided Wilcoxon signed-rank test, P-value = 0.031 in all significant cases) (see Fig. EV2 for details where P1 - P6 refer to the individual patients).


Volcano plot showing differences in metabolite levels between 0 and 6 & 12 weeks of treatment . Metabolites in histidine , alanine , aspartate and glutamine metabolism , and the TCA cycle pathways are annotated at 10 % False Discovery Rate ( see Dataset EV1 for the whole list of differentially regulated metabolites . Although some other metabolites showed a more pronounced difference , these did not fit into a recognised pathway ) . Metabolite labels are colour - coded according to the identified KEGG pathways ( dark red = " Alanine , aspartate and glutamate metabolism " , dark green = " Histidine metabolism " and dark blue = " Citrate cycle ( TCA cycle ) " , with transition colour - code if the metabolite belongs to more than one KEGG pathways ) .

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Volcano plot showing differences in metabolite levels between 0 and 6&12 weeks of treatment. Metabolites in histidine, alanine, aspartate and glutamine metabolism, and the TCA cycle pathways are annotated at 10% False Discovery Rate (see Dataset EV1 for the whole list of differentially regulated metabolites. Although some other metabolites showed a more pronounced difference, these did not fit into a recognised pathway). Metabolite labels are colour-coded according to the identified KEGG pathways (dark red = "Alanine, aspartate and glutamate metabolism", dark green = "Histidine metabolism" and dark blue = "Citrate cycle (TCA cycle)", with transition colour-code if the metabolite belongs to more than one KEGG pathways).


Scatterplot depicting P - values from the metabolites Pathway Enrichment Analysis ( y - axis ) and impact values from Pathway Topology Analysis ( x - axis ) ( see Dataset EV2 for details ) . KEGG pathways at 10 % adjusted Holm - Bonferroni P - value are highlighted .

Scatterplot depicting P-values from the metabolites Pathway Enrichment Analysis (y-axis) and impact values from Pathway Topology Analysis (x-axis) (see Dataset EV2 for details). KEGG pathways at 10% adjusted Holm-Bonferroni P-value are highlighted.


Volcano plot of acyl - carnitine metabolites between 0 and 6 & 12 weeks of treatment with highlighted differential metabolites at 10 % FDR .

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Volcano plot of acyl-carnitine metabolites between 0 and 6&12 weeks of treatment with highlighted differential metabolites at 10% FDR.


Respiratory chain complex and citrate synthase activity in skeletal muscle before and after 12 weeks of treatment . No significant differences were detected before and after 12 weeks of treatment by using the two - sided Wilcoxon signed - rank test with an empirical level of significance .

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Respiratory chain complex and citrate synthase activity in skeletal muscle before and after 12 weeks of treatment. No significant differences were detected before and after 12 weeks of treatment by using the two-sided Wilcoxon signed-rank test with an empirical level of significance.


P1 - P6 refer to individual patients . Representative examples of the quadruple immunofluorescence quantification of skeletal muscle fibres before and after treatment . The box in the top right - hand corner indicates the normal range , using two antibodies to NDUFB8 and COX I .

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P1 - P6 refer to individual patients. Representative examples of the quadruple immunofluorescence quantification of skeletal muscle fibres before and after treatment. The box in the top right-hand corner indicates the normal range, using two antibodies to NDUFB8 and COX I.


Quantitative quadruple immunofluorescence of skeletal muscle fibres in the six patients showing a decrease ( two - sided Wilcoxon signed - rank test with an empirical level of significance ) in the proportion of complex IV deficient skeletal muscle fibres before and after 12 weeks of treatment ( empirical P - value = 0 . 048 ) .

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Quantitative quadruple immunofluorescence of skeletal muscle fibres in the six patients showing a decrease (two-sided Wilcoxon signed-rank test with an empirical level of significance) in the proportion of complex IV deficient skeletal muscle fibres before and after 12 weeks of treatment (empirical P-value = 0.048).


Hierarchical cluster analysis of skeletal muscle RNA - seq transcripts before and after treatment showing no clear separation between untreated and treated patients .

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Hierarchical cluster analysis of skeletal muscle RNA-seq transcripts before and after treatment showing no clear separation between untreated and treated patients.


Volcano plot depicting differentially expressed RNA - seq genes at 10 % FDR after 12 weeks of treatment . Significantly different mammalian mitochondrial genes ( MitoCarta 2 . 0 ) are annotated .

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Volcano plot depicting differentially expressed RNA-seq genes at 10% FDR after 12 weeks of treatment. Significantly different mammalian mitochondrial genes (MitoCarta 2.0) are annotated.


Percentage level m . 3243A > G in blood , urinary sediment and skeletal muscle . No differences were detected by using the two - sided Wilcoxon signed - rank test with an empirical level of significance . W = week of the study .

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Percentage level m.3243A>G in blood, urinary sediment and skeletal muscle. No differences were detected by using the two-sided Wilcoxon signed-rank test with an empirical level of significance. W = week of the study.


Cardiac parameters before and after treatment ( two - sided Wilcoxon signed - rank test with an empirical level of significance ) . ES = end - systolic volume ( ml ) ( empirical P - value = 0 . 047 ) and index ( ml / m2 ) ( empirical P - value = 0 . 046 ) , measured by MRI . Horizontal lines on ( B ) , ( F ) and ( G ) refer to statistical comparisons of the mean values , where ns = non - significant , * P - value ≤ 0 . 05 . For normal reference ranges see ( Hollingsworth et al . , 2012 )

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Cardiac parameters before and after treatment (two-sided Wilcoxon signed-rank test with an empirical level of significance). ES = end-systolic volume (ml) (empirical P-value = 0.047) and index (ml/m2) (empirical P-value = 0.046), measured by MRI. Horizontal lines on (B), (F) and (G) refer to statistical comparisons of the mean values, where ns = non-significant, *P-value ≤ 0.05. For normal reference ranges see (Hollingsworth et al., 2012)


A ) Representative confocal images showing perinuclear clustering of mitochondria and Parkin aggregates on the mitochondria in Huh7 cells infected with HCVcc . At 3 days post - infection , cells prestained with MitoTracker ( Mito , red ) were immunostained with anti - Parkin ( green ) and HCV core ( orange ) antibodies . Nuclei were stained with DAPI ( blue ) . Infected ( + ) and uninfected cells ( − ) are marked . The illustrated images display typical distribution of mitochondria in uninfected cells and altered distribution of mitochondria in the perinuclear region of infected cells ( gray ) . In the zoomed images , the white arrows indicate accumulation of endogenous Parkin recruited to the mitochondria in HCV - infected cells ( yellow ) .

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A) Representative confocal images showing perinuclear clustering of mitochondria and Parkin aggregates on the mitochondria in Huh7 cells infected with HCVcc. At 3 days post-infection, cells prestained with MitoTracker (Mito, red) were immunostained with anti-Parkin (green) and HCV core (orange) antibodies. Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked. The illustrated images display typical distribution of mitochondria in uninfected cells and altered distribution of mitochondria in the perinuclear region of infected cells (gray). In the zoomed images, the white arrows indicate accumulation of endogenous Parkin recruited to the mitochondria in HCV-infected cells (yellow).


( B ) Ultrastructure of HCV - infected cells showing perinuclear clustering of damaged mitochondria . Control naïve Huh7 cells ( left ) and stable cells harboring HCV full - length replicon FLR - JFH1 ( right ) were examined by electron microscopy . In the zoomed images , typical ultrastructure of mitochondria in naïve cells and ultrastructural abnormalities of mitochondria in HCV replicon cells are shown . Organelle mark : N , nucleus ; M and white arrow , mitochondria . Scale bar = 1 µM . Fluorescent images ( below ) indicate the expression of HCV core protein in HCV replicon cells . Cells were immunostained with anti - HCV core antibody ( red ) . Nuclei were stained with DAPI ( blue ) .

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(B) Ultrastructure of HCV-infected cells showing perinuclear clustering of damaged mitochondria. Control naïve Huh7 cells (left) and stable cells harboring HCV full-length replicon FLR-JFH1 (right) were examined by electron microscopy. In the zoomed images, typical ultrastructure of mitochondria in naïve cells and ultrastructural abnormalities of mitochondria in HCV replicon cells are shown. Organelle mark: N, nucleus; M and white arrow, mitochondria. Scale bar = 1 µM. Fluorescent images (below) indicate the expression of HCV core protein in HCV replicon cells. Cells were immunostained with anti-HCV core antibody (red). Nuclei were stained with DAPI (blue).


( C ) Quantification of the number of mitochondria in the perinuclear region ( mean ± SEM ; n ≥ 5 cells ; * p < 0 . 05 ) .

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(C) Quantification of the number of mitochondria in the perinuclear region (mean ± SEM; n≥5 cells; *p<0.05).


( D ) Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in CCCP - treated ( see Figure S1 ) or HCV - infected cells ( A ) and those treated with 3 - MA or BafA1 , respectively ( see Figure 4A ) ( mean ± SEM ; n = 10 cells ; * p < 0 . 01 ) . P values were calculated by using an unpaired Student ' s t - test .

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(D) Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in CCCP-treated (see Figure S1) or HCV-infected cells (A) and those treated with 3-MA or BafA1, respectively (see Figure 4A) (mean ± SEM; n = 10 cells; *p<0.01). P values were calculated by using an unpaired Student's t-test.


( E ) Western blot analysis showing endogenous Parkin recruitment to mitochondria in HCV - infected cells . Huh7 cells were infected with HCVcc . At 5 days post - infection , pure cytoplasm and mitochondria fractions were isolated by ultracentrifugation as described in Materials and Methods . Cellular fractions of HCV - infected cells were analyzed by immunoblotting using antibodies specific for the indicated proteins . Fractions : whole cell lysates , WCL ; purified cytoplasm , pCyto ; purified mitochondria , pMito . Organelle markers : VDAC1 , mitochondria ; Calreticulin , ER ; GAPDH , cytoplasm .

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(E) Western blot analysis showing endogenous Parkin recruitment to mitochondria in HCV-infected cells. Huh7 cells were infected with HCVcc. At 5 days post-infection, pure cytoplasm and mitochondria fractions were isolated by ultracentrifugation as described in Materials and Methods. Cellular fractions of HCV-infected cells were analyzed by immunoblotting using antibodies specific for the indicated proteins. Fractions: whole cell lysates, WCL; purified cytoplasm, pCyto; purified mitochondria, pMito. Organelle markers: VDAC1, mitochondria; Calreticulin, ER; GAPDH, cytoplasm.


( A ) Parkin ubiquitination in the purified mitochondria isolated from HCV - infected cells . Parkin protein in pCyto and pMito fractions , respectively , was immunoprecipitated by anti - Parkin antibody , followed by immunoblotting with anti - ubiquitin ( Ub ) antibody . Normal rabbit IgG was used as a negative control for immunoprecipitation ( IP ) .

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(A) Parkin ubiquitination in the purified mitochondria isolated from HCV-infected cells. Parkin protein in pCyto and pMito fractions, respectively, was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a negative control for immunoprecipitation (IP).


( B , E , and F ) At the indicated time points post - infection , WCL were extracted from HCV - infected Huh7 cells . ( B ) Parkin ubiquitination in WCL extracted from HCV - infected cells . All ubiquitinated proteins were immunoprecipitated with anti - Ub antibody and analyzed by immunoblotting with anti - Parkin antibody . HCV infection was verified by immunoblotting with anti - HCV core antibody . β - actin was used as an internal loading control .

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(B, E, and F) At the indicated time points post-infection, WCL were extracted from HCV-infected Huh7 cells. (B) Parkin ubiquitination in WCL extracted from HCV-infected cells. All ubiquitinated proteins were immunoprecipitated with anti-Ub antibody and analyzed by immunoblotting with anti-Parkin antibody. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( C ) Confocal microscopy showing Parkin ubiquitination in HCV - infected cells . HCV - infected cells prestained with MitoTracker ( Mito ) were immunostained with anti - Parkin and ubiquitin ( Ub ) antibodies . In the zoomed images , the arrows ( white spots ) indicate the merge of Mito ( green ) , Parkin ( red ) , and Ub ( blue ) . Nuclei are demarcated with white dot circles . Fluorescence image of HCV E2 verifies HCV - infected cells ( light gray ) .

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(C) Confocal microscopy showing Parkin ubiquitination in HCV-infected cells. HCV-infected cells prestained with MitoTracker (Mito) were immunostained with anti-Parkin and ubiquitin (Ub) antibodies. In the zoomed images, the arrows (white spots) indicate the merge of Mito (green), Parkin (red), and Ub (blue). Nuclei are demarcated with white dot circles. Fluorescence image of HCV E2 verifies HCV-infected cells (light gray).


( D ) Quantitative analysis of the ubiquitination of endogenous Parkin colocalized with mitochondria in the panels ( C ) ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 05 ) .

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(D) Quantitative analysis of the ubiquitination of endogenous Parkin colocalized with mitochondria in the panels (C) (mean ± SEM; n≥10 cells; *p<0.05).


( E ) Western blot analyses of Mfn2 and VDAC1 expression , mitochondrial substrate of Parkin , in HCV - infected cells . Intracellular HCV RNA levels were analyzed by real - time qRT - PCR , as described in Materials and Methods ( mean ± SD ; n = 3 ; * p < 0 . 01 ) . WCL extracted from HCV - infected cells were analyzed by immunoblotting with anti - Parkin , Mfn2 , and VDAC1 antibodies , respectively . HCV infection was verified by immunoblotting with anti - HCV core antibody . β - actin was used as an internal loading control .

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(E) Western blot analyses of Mfn2 and VDAC1 expression, mitochondrial substrate of Parkin, in HCV-infected cells. Intracellular HCV RNA levels were analyzed by real-time qRT-PCR, as described in Materials and Methods (mean ± SD; n = 3; *p<0.01). WCL extracted from HCV-infected cells were analyzed by immunoblotting with anti-Parkin, Mfn2, and VDAC1 antibodies, respectively. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( F ) Western blot analysis of p62 expression , the autophagy - associated factor , in HCV - infected cells . WCL extracted from HCV - infected cells were analyzed by immunoblotting with anti - p62 antibody . β - actin was used as an internal loading control . ( E and F ) The relative intensity of Mfn2 , VDAC1 , and p62 expression normalized to β - actin was analyzed by ImageJ .

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(F) Western blot analysis of p62 expression, the autophagy-associated factor, in HCV-infected cells. WCL extracted from HCV-infected cells were analyzed by immunoblotting with anti-p62 antibody. β-actin was used as an internal loading control. (E and F) The relative intensity of Mfn2, VDAC1, and p62 expression normalized to β-actin was analyzed by ImageJ.


( G ) Parkin - mediated ubiquitination of Mfn2 and VDAC1 in HCV - infected cells . The ubiquitinated Mfn2 and VDAC1 proteins were analyzed by immunoprecipitation with anti - Mfn2 and VDAC1 antibodies , respectively , followed by immunoblotting with anti - Ub antibody . The protein expression levels of Parkin were analyzed by immunoblotting with anti - Parkin antibody . HCV infection was verified by immunoblotting with anti - HCV core antibody . β - actin was used as an internal loading control . Normal mouse IgG was used as a negative control for immunoprecipitation ( IP ) . ( D and E ) P values were calculated by using an unpaired Student ' s t - test .

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(G) Parkin-mediated ubiquitination of Mfn2 and VDAC1 in HCV-infected cells. The ubiquitinated Mfn2 and VDAC1 proteins were analyzed by immunoprecipitation with anti-Mfn2 and VDAC1 antibodies, respectively, followed by immunoblotting with anti-Ub antibody. The protein expression levels of Parkin were analyzed by immunoblotting with anti-Parkin antibody. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control. Normal mouse IgG was used as a negative control for immunoprecipitation (IP). (D and E) P values were calculated by using an unpaired Student's t-test.


( A and B ) Quantitative analyses of ATF4 , Parkin , and PINK1 gene expression in HCV - infected cells . Huh7 cells infected with HCVcc for 5 days or treated with CCCP ( 10 or 20 µM ) for 24 h were used for analysis of ATF4 , Parkin , and PINK1 gene expression . ( A ) Intracellular mRNA levels of ATF4 , Parkin , and PINK1 were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in Parkin and PINK1 gene expression ( mean ± SD ; n = 3 ; * p < 0 . 01 , * * p < 0 . 05 ) . P values were calculated by using an unpaired Student ' s t - test .

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(A and B) Quantitative analyses of ATF4, Parkin, and PINK1 gene expression in HCV-infected cells. Huh7 cells infected with HCVcc for 5 days or treated with CCCP (10 or 20 µM) for 24 h were used for analysis of ATF4, Parkin, and PINK1 gene expression. (A) Intracellular mRNA levels of ATF4, Parkin, and PINK1 were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin and PINK1 gene expression (mean ± SD; n = 3; *p<0.01, **p<0.05). P values were calculated by using an unpaired Student's t-test.


B ) The protein expression levels of Parkin and PINK1 were analyzed by immunoblotting with anti - Parkin and PINK1 antibodies . The expression of HCV core protein was analyzed by immunoblotting with anti - HCV core antibody . β - actin was used as an internal loading control .

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B) The protein expression levels of Parkin and PINK1 were analyzed by immunoblotting with anti-Parkin and PINK1 antibodies. The expression of HCV core protein was analyzed by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( C ) Western blot analysis of Parkin expression in liver biopsies of chronic hepatitis C patients . Samples 1 and 2 represent liver biopsies of HCV - negative patients and samples 3 - 7 represent HCV - positive chronic hepatitis patients . ( B and C ) The relative intensity of Parkin and PINK1 expression normalized to β - actin analyzed by ImageJ .

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(C) Western blot analysis of Parkin expression in liver biopsies of chronic hepatitis C patients. Samples 1 and 2 represent liver biopsies of HCV-negative patients and samples 3-7 represent HCV-positive chronic hepatitis patients. (B and C) The relative intensity of Parkin and PINK1 expression normalized to β-actin analyzed by ImageJ.


( D ) Confocal microscopy showing Parkin overexpression in HCV - infected cells . Huh7 cells infected with HCVcc were immunostained with anti - Parkin antibody ( red ) . Fluorescence image of HCV E2 verifies HCV - infected cells ( light gray ) . Nuclei were stained with DAPI ( blue ) . Infected ( + ) and uninfected cells ( − ) are marked .

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(D) Confocal microscopy showing Parkin overexpression in HCV-infected cells. Huh7 cells infected with HCVcc were immunostained with anti-Parkin antibody (red). Fluorescence image of HCV E2 verifies HCV-infected cells (light gray). Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked.


Confocal microscopy showing mitophagosome formation associated with Parkin in HCV - infected cells . ( A ) Huh7 cells transiently expressing GFP - LC3 protein were infected with HCVcc in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 12 h before fixation . At 3 day post - infection , cells were immunostained with antibodies against HCV E2 ( orange ) , TOM20 ( blue ) , and Parkin ( red ) . In the zoom images , the arrows ( white puncta ) indicate GFP - LC3 puncta ( green ) colocalized with TOM20 and Parkin . Nuclei are demarcated with white dot circles . (

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Confocal microscopy showing mitophagosome formation associated with Parkin in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 day post-infection, cells were immunostained with antibodies against HCV E2 (orange), TOM20 (blue), and Parkin (red). In the zoom images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin. Nuclei are demarcated with white dot circles. (


( B ) Quantification of the number of GFP - LC3 puncta colocalized with TOM20 in the panel ( A ) ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 01 ) .

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(B) Quantification of the number of GFP-LC3 puncta colocalized with TOM20 in the panel (A) (mean ± SEM; n≥10 cells; *p<0.01).


( C ) Quantitative analysis of the area of GFP - LC3 puncta ( white ) representing merge of GFP - LC3 puncta , TOM20 , and Parkin in the panel ( A ) ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 001 ) . ( B and C ) P values were calculated by using an unpaired Student ' s t - test .

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(C) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A) (mean ± SEM; n≥10 cells; *p<0.001). (B and C) P values were calculated by using an unpaired Student's t-test.


. ( D ) Ultrastructure of HCV - infected cells showing the formation of mitophagosome . Control naïve Huh7 cells ( uninfected ) , HCV FLR - JFH1 replicon cells , and Huh7 cells infected with HCVcc ( Jc1 ) were examined by electron microscopy . In the zoomed image , the formation of mitophagosome in HCV - infected cells is shown . Organelle mark : N , nucleus ; MF , mitophagosome formation . Scale bar = 0 . 2 µM ( uninfected ) ; 0 . 1 µM ( infected ) .

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. (D) Ultrastructure of HCV-infected cells showing the formation of mitophagosome. Control naïve Huh7 cells (uninfected), HCV FLR-JFH1 replicon cells, and Huh7 cells infected with HCVcc (Jc1) were examined by electron microscopy. In the zoomed image, the formation of mitophagosome in HCV-infected cells is shown. Organelle mark: N, nucleus; MF, mitophagosome formation. Scale bar = 0.2 µM (uninfected); 0.1 µM (infected).


( E ) Immunoelectron microscopic analysis showing the formation of mitophagosome in HCV - infected cells . Huh7 cells infected with HCVcc were stained by triple immunogold labeling method , as described in Materials and Methods . The immunogold particles of GFP - LC3 ( white arrow , 18 nm ) , HCV E2 ( blue arrow , 12 nm ) , and TOM20 ( red arrow , 6 nm ) are shown . Fluorescence image of HCV core identifies HCV - infected cells ( red ) . Nuclei were stained with DAPI ( blue ) . Scale bar = 0 . 2 µM .

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(E) Immunoelectron microscopic analysis showing the formation of mitophagosome in HCV-infected cells. Huh7 cells infected with HCVcc were stained by triple immunogold labeling method, as described in Materials and Methods. The immunogold particles of GFP-LC3 (white arrow, 18 nm), HCV E2 (blue arrow, 12 nm), and TOM20 (red arrow, 6 nm) are shown. Fluorescence image of HCV core identifies HCV-infected cells (red). Nuclei were stained with DAPI (blue). Scale bar = 0.2 µM.


Confocal microscopy showing the formation of mitophagolysosome in HCV - infected cells . ( A ) Huh7 cells transiently expressing GFP - LC3 protein were infected with HCVcc in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 12 h before fixation . At 3 days post - infection , cells prestained with LysoTracker ( red ) were immunostained with antibodies against TOM20 ( blue ) and HCV core ( cyan ) . Nuclei are demarcated with white dot circles . In the zoomed images , the arrows indicate the colocalization of GFP - LC3 puncta ( green ) , lysosome , and mitochondria in HCV - infected cells ( white puncta ) .

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Confocal microscopy showing the formation of mitophagolysosome in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 days post-infection, cells prestained with LysoTracker (red) were immunostained with antibodies against TOM20 (blue) and HCV core (cyan). Nuclei are demarcated with white dot circles. In the zoomed images, the arrows indicate the colocalization of GFP-LC3 puncta (green), lysosome, and mitochondria in HCV-infected cells (white puncta).


( B ) Ultrastructure of HCV - infected cells showing the formation of mitophagolysosome . HCV FLR - JFH1 replicon cells were examined by electron microscopy . In the zoomed image , the formation of mitophagolysosome in HCV - infected cells is shown . Organelle marker : ML , mitophagolysosome . Scale bar = 0 . 1 µM .

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(B) Ultrastructure of HCV-infected cells showing the formation of mitophagolysosome. HCV FLR-JFH1 replicon cells were examined by electron microscopy. In the zoomed image, the formation of mitophagolysosome in HCV-infected cells is shown. Organelle marker: ML, mitophagolysosome. Scale bar = 0.1 µM.


( C and D ) Quantitative analyses of the colocalization of GFP - LC3 containing lysosomes with mitochondria ( C ) or Parkin ( D ) in the panels ( A ) and Figure S5 ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 001 , * * p < 0 . 05 ) . ( E ) ImageJ quantitative analysis of the number of mitochondria ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 001 , * * p < 0 . 05 ) . P values were calculated by using an unpaired Student ' s t - test .

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(C and D) Quantitative analyses of the colocalization of GFP-LC3 containing lysosomes with mitochondria (C) or Parkin (D) in the panels (A) and Figure S5 (mean ± SEM; n≥10 cells; *p<0.001, **p<0.05). (E) ImageJ quantitative analysis of the number of mitochondria (mean ± SEM; n≥10 cells; *p<0.001, **p<0.05). P values were calculated by using an unpaired Student's t-test.


A and B ) Inhibitory effect of Parkin , PINKI , and ATG5 silencing on HCV replication . Huh7 cells transfected with Non - Targeting ( NT ) or gene - specific siRNA pools targeting Parkin , PINK1 , and ATG5 genes , respectively , were infected with HCVcc . At day 3 post - infection , the levels of HCV RNA ( A ) and targeted gene

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Inhibitory effect of Parkin, PINKI, and ATG5 silencing on HCV replication. Huh7 cells transfected with Non-Targeting (NT) or gene-specific siRNA pools targeting Parkin, PINK1, and ATG5 genes, respectively, were infected with HCVcc. At day 3 post-infection, the levels of HCV RNA (A) and targeted gene mRNA


A ( B ) were analyzed by real - time qRT - PCR , as described in Materials and Methods ( mean ± SD ; n = 3 ; * p < 0 . 01 ) . GAPDH was used to normalize changes in Parkin , PINK1 , and ATG5 gene expression .

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A (B) were analyzed by real-time qRT-PCR, as described in Materials and Methods (mean ± SD; n = 3; *p<0.01). GAPDH was used to normalize changes in Parkin, PINK1, and ATG5 gene expression.


( C and D ) Rescue of HCV RNA replication by Parkin overexpression . Huh7 cells stably expressing mock vector ( M - KD ) , Non - target - shRNA ( NT - KD ) , and Parkin - shRNA ( P - KD ) , respectively , were infected with HCVcc . P - KD cells were further transfected with two different concentration of the plasmid DNA encoding wild - type Parkin for 2 days before harvest . At 3 days post - infection , intracellular HCV RNA levels were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in HCV RNA expression ( C ) ( mean ± SD ; n = 3 ; * p < 0 . 01 , * * p < 0 . 05 ) .

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(C and D) Rescue of HCV RNA replication by Parkin overexpression. Huh7 cells stably expressing mock vector (M-KD), Non-target-shRNA (NT-KD), and Parkin-shRNA (P-KD), respectively, were infected with HCVcc. P-KD cells were further transfected with two different concentration of the plasmid DNA encoding wild-type Parkin for 2 days before harvest. At 3 days post-infection, intracellular HCV RNA levels were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in HCV RNA expression (C) (mean ± SD; n = 3; *p<0.01, **p<0.05).


( D ) The expression levels of Parkin and HCV core protein were analyzed by immunoblotting with anti - Parkin and HCV core antibodies . Ectopic expression level of HA - tagged wild - type Parkin was detected by immunoblotting with anti - HA antibody . β - actin was used as an internal loading control . P values were calculated by using an unpaired Student ' s t - test . The relative intensity of HCV core expression normalized to β - actin was analyzed by ImageJ .

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(D) The expression levels of Parkin and HCV core protein were analyzed by immunoblotting with anti-Parkin and HCV core antibodies. Ectopic expression level of HA-tagged wild-type Parkin was detected by immunoblotting with anti-HA antibody. β-actin was used as an internal loading control. P values were calculated by using an unpaired Student's t-test. The relative intensity of HCV core expression normalized to β-actin was analyzed by ImageJ.


( A ) Rescue effect of 3 - MA and BafA1 on reduction of mitochondrial complex I enzyme activity caused by HCV infection . Huh7 cells infected with HCVcc were treated with 3 - MA ( 10 mM ) or BafA1 ( 100 nM ) for 12 h before harvest . At 3 days post - infection , the activity of mitochondrial complex I enzyme was measured according to manufacturer ' s instructions ( mean ± SD ; n = 3 ; * p < 0 . 01 ) .

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

(A) Rescue effect of 3-MA and BafA1 on reduction of mitochondrial complex I enzyme activity caused by HCV infection. Huh7 cells infected with HCVcc were treated with 3-MA (10 mM) or BafA1 (100 nM) for 12 h before harvest. At 3 days post-infection, the activity of mitochondrial complex I enzyme was measured according to manufacturer's instructions (mean ± SD; n = 3; *p<0.01).


. ( B ) Rescue effect of Parkin knockdown on reduction of mitochondrial complex I enzyme activity caused by HCV infection . NT - KD and P - KD cells infected with HCVcc were harvested on day 3 post - infection and used for analysis of the activity of mitochondrial complex I enzyme ( mean ± SD ; n = 3 ; * p < 0 . 05 ) .

. (B) Rescue effect of Parkin knockdown on reduction of mitochondrial complex I enzyme activity caused by HCV infection. NT-KD and P-KD cells infected with HCVcc were harvested on day 3 post-infection and used for analysis of the activity of mitochondrial complex I enzyme (mean ± SD; n = 3; *p<0.05).


( C ) Western blot analysis of mitochondrial respiratory chain complex enzyme expression . NT - KD and P - KD cells were infected with HCVcc and at 3 days post - infection , the expression levels of complex I and IV enzyme were analyzed by immunoblotting with anti - Hu total OxPhos complex antibody . Huh7 cells treated with CCCP ( 10 µM ) for 12 h were also analyzed as a control . β - actin was used as an internal loading control . P values were calculated by using an unpaired Student ' s t - test .

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(C) Western blot analysis of mitochondrial respiratory chain complex enzyme expression. NT-KD and P-KD cells were infected with HCVcc and at 3 days post-infection, the expression levels of complex I and IV enzyme were analyzed by immunoblotting with anti-Hu total OxPhos complex antibody. Huh7 cells treated with CCCP (10 µM) for 12 h were also analyzed as a control. β-actin was used as an internal loading control. P values were calculated by using an unpaired Student's t-test.


( D ) Effect of Parkin silencing on depletion of mitochondrial DNA caused by HCV infection . Huh7 cells transfected with NT or Parkin - specific siRNA pools were infected with HCVcc . At day 3 post - infection , mitochondrial DNA levels of ND - 2 and COX - 2 were analyzed by real - time qPCR . GAPDH was used to normalize changes in ND - 2 and COX - 2 gene expression ( mean ± SD ; n = 3 ; * p < 0 . 01 ) . ( A , B , and D ) P values were calculated by using an unpaired Student ' s t - test .

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(D) Effect of Parkin silencing on depletion of mitochondrial DNA caused by HCV infection. Huh7 cells transfected with NT or Parkin-specific siRNA pools were infected with HCVcc. At day 3 post-infection, mitochondrial DNA levels of ND-2 and COX-2 were analyzed by real-time qPCR. GAPDH was used to normalize changes in ND-2 and COX-2 gene expression (mean ± SD; n = 3; *p<0.01). (A, B, and D) P values were calculated by using an unpaired Student's t-test.


( B ) Immunogold electron micrograph of GFP or ORF6 - GFP expressed HeLa cells . Blue arrows mark ORF6 - GFP dots are enriched on the surface of LDs . Green arrows mark GFP dots are distributed in the cytoplasm . Scale bar represents 500 nm .

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(B) Immunogold electron micrograph of GFP or ORF6-GFP expressed HeLa cells. Blue arrows mark ORF6-GFP dots are enriched on the surface of LDs. Green arrows mark GFP dots are distributed in the cytoplasm. Scale bar represents 500 nm.


( C , D and E ) HeLa cells stable expressing ORF6 - Flag were treated with or without 200 μM OA for 12 h , then fixed and stained with anti - Flag ( red ) . Initial LDs were labeled with GFP - GPAT4152 - 208 ( green ) , mature LDs were labeled with BODIPY - 493 / 503 ( green ) or GFP - Plin2 ( green ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm .

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(C, D and E) HeLa cells stable expressing ORF6-Flag were treated with or without 200 μM OA for 12 h, then fixed and stained with anti-Flag (red). Initial LDs were labeled with GFP-GPAT4152-208 (green), mature LDs were labeled with BODIPY-493/503 (green) or GFP-Plin2 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.


( B ) Analysis of the homodimerization of ORF6 by cross - linking with DSS . HEK293T cells expressing ORF6 - Flag were treated with 0 , 0 . 1 , 0 . 2 and 0 . 3 mM DSS for 30 min . Cell lysates were analyzed via WB .

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(B) Analysis of the homodimerization of ORF6 by cross-linking with DSS. HEK293T cells expressing ORF6-Flag were treated with 0, 0.1, 0.2 and 0.3 mM DSS for 30 min. Cell lysates were analyzed via WB.


( D ) The indicated Flag tagged ORF6 or its mutants were co - expressed in HEK293T cells with HA tagged ORF6 . Protein interactions were analyzed by immunoprecipitation with anti - Flag beads and immunoblotting analysis .

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(D) The indicated Flag tagged ORF6 or its mutants were co-expressed in HEK293T cells with HA tagged ORF6. Protein interactions were analyzed by immunoprecipitation with anti-Flag beads and immunoblotting analysis.


( E ) Purified GST - ORF6 or indicated mutants were incubated with purified His tagged ORF6 , and analysis of the self - interaction by GST pull - down .

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(E) Purified GST-ORF6 or indicated mutants were incubated with purified His tagged ORF6, and analysis of the self-interaction by GST pull-down.


( F ) HEK293T cells were transfected with ORF6 - Flag or ΔNTD - Flag or ΔAH1 - Flag or ΔAH2 - Flag and were treated with 0 . 1 mM DSS for 30 min . Cell lysates were analyzed via WB .

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(F) HEK293T cells were transfected with ORF6-Flag or ΔNTD-Flag or ΔAH1-Flag or ΔAH2-Flag and were treated with 0.1 mM DSS for 30 min. Cell lysates were analyzed via WB.


( G ) HeLa cells were transfected with ORF6 - Flag or indicated mutants and were treated with 200 μM OA for 12 h , and then fixed and stained with anti - Flag ( red ) . LDs were labeled with BODIPY - 493 / 503 ( green ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm .

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(G) HeLa cells were transfected with ORF6-Flag or indicated mutants and were treated with 200 μM OA for 12 h, and then fixed and stained with anti-Flag (red). LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.


( A ) HeLa cells were transfected with vector or ORF6 - Flag for 24 h , and then treated with 200 μM OA for indicated times . Cells were fixed and stained with anti - Flag ( green ) . LDs were labeled with LipidTox Deep Red ( red ) . The nuclei were stained with DAPI . Cells were imaged by confocal microscopy . Scale bar represents 10 μm . White ROIs indicate cells expressing ORF6 and yellow ROIs indicate the cells without ORF6 expression .

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(A) HeLa cells were transfected with vector or ORF6-Flag for 24 h, and then treated with 200 μM OA for indicated times. Cells were fixed and stained with anti-Flag (green). LDs were labeled with LipidTox Deep Red (red). The nuclei were stained with DAPI. Cells were imaged by confocal microscopy. Scale bar represents 10 μm. White ROIs indicate cells expressing ORF6 and yellow ROIs indicate the cells without ORF6 expression.


( D ) HeLa cells stable expressing vector or ORF6 - Flag were treated or untreated with 50 μM ATGL inhibitor ( ATGLi ) for 24 h , and then were fixed and stained with anti - Flag ( red ) . LDs were labeled with BODIPY - 493 / 503 ( green ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm . ( E ) Mean number of LDs in each cell in ( D ) was counted from 25 cells of three independent experiments . Two - tailed Unpaired Student ' s t - test , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Error bars represent the mean ± SD .

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(D) HeLa cells stable expressing vector or ORF6-Flag were treated or untreated with 50 μM ATGL inhibitor (ATGLi) for 24 h, and then were fixed and stained with anti-Flag (red). LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (E) Mean number of LDs in each cell in (D) was counted from 25 cells of three independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ****p < 0.0001. Error bars represent the mean ± SD.


( H ) HeLa cells stable expressing vector or ORF6 - Flag were treated or untreated with 50 μM ATGL inhibitor , along with DMSO or 1 μM DGAT1 inhibitor ( DGAT1i ) or / and 2 μM DGAT2 inhibitor ( DGAT2i ) for 24 h . LDs were labeled with BODIPY - 493 / 503 ( green ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm . ( I ) Mean number of LDs in each cell in ( H ) was counted from 35 cells of three independent experiments . Two - tailed Unpaired Student ' s t - test , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns means no significance . Error bars represent the mean ± SD .

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(H) HeLa cells stable expressing vector or ORF6-Flag were treated or untreated with 50 μM ATGL inhibitor, along with DMSO or 1 μM DGAT1 inhibitor (DGAT1i) or/and 2 μM DGAT2 inhibitor (DGAT2i) for 24 h. LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (I) Mean number of LDs in each cell in (H) was counted from 35 cells of three independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ****p < 0.0001, ns means no significance. Error bars represent the mean ± SD.


( J ) SARS - CoV - 2 - infected Vero - E6 cells were treated with 1 μM DGAT1 inhibitor or / and 2 μM DGAT2 inhibitor for 24 h . Cell viability was analyzed . Cell lysates were analyzed via WB . Supernatants were determined by plaque assays . Virus titer values represent mean ± SD for three independent experiments . Two - tailed Unpaired Student ' s t - test , * * * p < 0 . 001 , ns means no significance . Error bars represent the mean ± SD .

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(J) SARS-CoV-2-infected Vero-E6 cells were treated with 1 μM DGAT1 inhibitor or/and 2 μM DGAT2 inhibitor for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ns means no significance. Error bars represent the mean ± SD.


( A ) Subcellular fractions were isolated from GFP or ORF6 - GFP over - expressed cells . Calnexin represent the ER , Plin2 represent LDs , Rab11 represent endosomes , VDAC represent mitochondria , and Tubulin represents cytoplasm , respectively .

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(A) Subcellular fractions were isolated from GFP or ORF6-GFP over-expressed cells. Calnexin represent the ER, Plin2 represent LDs, Rab11 represent endosomes, VDAC represent mitochondria, and Tubulin represents cytoplasm, respectively.


( C ) Representative transmission electron micrograph of non - infected or SARS - CoV - 2 infected Vero - E6 cells . Red arrows mark the ER . LD , lipid droplets . Scale bar represents 1 μm . Quantification of number of ER engaged in ER - LD contact per LD . 20 LDs of two independent experiments were counted . Two - tailed Unpaired Student ' s t - test , * * * * p < 0 . 0001 . Error bars represent as the mean ± SD .

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(C) Representative transmission electron micrograph of non-infected or SARS-CoV-2 infected Vero-E6 cells. Red arrows mark the ER. LD, lipid droplets. Scale bar represents 1 μm. Quantification of number of ER engaged in ER-LD contact per LD. 20 LDs of two independent experiments were counted. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent as the mean ± SD.


( E ) ORF6 - Flag or the mutants were co - expressed with mCheey - RAMP4 . Cells were treated with 200 μM OA for 12 h , and then fixed and stained with anti - Flag ( red ) . The ER was visualized with mCheey - RAMP4 ( red ) . LDs were labeled with LipidTOX Deep Red ( white ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm .

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(E) ORF6-Flag or the mutants were co-expressed with mCheey-RAMP4. Cells were treated with 200 μM OA for 12 h, and then fixed and stained with anti-Flag (red). The ER was visualized with mCheey-RAMP4 (red). LDs were labeled with LipidTOX Deep Red (white). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.


( J ) HeLa cells stable expressing ORF6 - Flag were transfected with si - BAP31 and / or si - USE1 for 24 h , and were further treated with 50 μM ATGL inhibitor for 24h . Cells were fixed and stained with anti - Flag ( red ) . LDs were labeled with BODIPY - 493 / 503 ( green ) . The nuclei were stained with DAPI . Cells were imaged by confocal microscopy . Scale bar represents 10 μm . ( K ) Mean number of LDs in each cell in ( J ) was counted from 25 cells of three independent experiments . Two - tailed Unpaired Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 . Error bars represent the mean ± SD .

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(J) HeLa cells stable expressing ORF6-Flag were transfected with si-BAP31 and/or si-USE1 for 24 h, and were further treated with 50 μM ATGL inhibitor for 24h. Cells were fixed and stained with anti-Flag (red). LDs were labeled with BODIPY-493/503 (green). The nuclei were stained with DAPI. Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (K) Mean number of LDs in each cell in (J) was counted from 25 cells of three independent experiments. Two-tailed Unpaired Student's t-test, *p < 0.05, **p < 0.01. Error bars represent the mean ± SD.


( L ) SARS - CoV - 2 - infected Vero - E6 cells were transfected with control , or si - BAP31 and / or si - USE1 for 48 h . Cell lysates were analyzed via WB . Supernatants were determined by plaque assays . Virus titer values represent mean ± SD for three independent replicates . Two - tailed Unpaired Student ' s t - test , * * * * p < 0 . 0001 , ns means no significance . Error bars represent the mean ± SD .

(L) SARS-CoV-2-infected Vero-E6 cells were transfected with control, or si-BAP31 and/or si-USE1 for 48 h. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two-tailed Unpaired Student's t-test, ****p < 0.0001, ns means no significance. Error bars represent the mean ± SD.