A ) HT29 cells were treated with or without 300 nM 5 - AZA - CdR for 2 days and subsequently washed and transfected with the indicated siRNAs . Cells were harvested 72h post - transfection and the type I IFN response was analyzed by RT - qPCR analysis of IFN - β , IFIT1 , and ISG15 transcript expression , normalized to ACTB . B ) LIM1215 cells were treated with or without 300 nM 5 - AZA - CdR for 2 days and subsequently washed and transfected with the indicated siRNAs . Cells were harvested 72h post - transfection and the type I IFN response was analyzed as in ( A ) . C ) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days . Three days after treatment initiation , cells were transfected with the indicated siRNAs and cultured for an additional 72h . The type I IFN response was monitored as in ( A ) . Data information : Data from three biological independent experiments are shown with mean ± s . d . . Statistical analysis was performed using ordinary two - way ANOVA with Tukey ' s post hoc test . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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A) HT29 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed by RT-qPCR analysis of IFN-β, IFIT1, and ISG15 transcript expression, normalized to ACTB. B) LIM1215 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed as in (A). C) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days. Three days after treatment initiation, cells were transfected with the indicated siRNAs and cultured for an additional 72h. The type I IFN response was monitored as in (A). Data information: Data from three biological independent experiments are shown with mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


ALP assay . The cells expressing Pho8Δ60p were cultured in SD + CA medium ( open bars ) and shifted to SD ( − N ) medium for three hours ( filled bars ) . The ALP activity of the lysates was measured to estimate autophagic activity . Wild - type cells , TN124 ; Δapg8 mutant harboring vector , TK303 ; Δapg8 mutant harboring APG8 on a centromeric vector , TK301 ; Δapg8 mutant harboring 3 × HA - tagged APG8 plasmid , TK307 .

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ALP assay. The cells expressing Pho8Δ60p were cultured in SD+CA medium (open bars) and shifted to SD(−N) medium for three hours (filled bars). The ALP activity of the lysates was measured to estimate autophagic activity. Wild-type cells, TN124; Δapg8 mutant harboring vector, TK303; Δapg8 mutant harboring APG8 on a centromeric vector, TK301; Δapg8 mutant harboring 3 × HA-tagged APG8 plasmid, TK307.


Expression of APG8 . A , The lysates prepared from the growing cells were subjected to immunoblotting with the anti - Apg8p antibody . Lane 1 , wild - type cells ( YW5 - 1B ) ; lane 2 , Δapg8 cells harboring APG8 on a multicopy vector ( TK201 ) ; lane 3 , Δapg8 cells ( TK404 ) .

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Expression of APG8. A, The lysates prepared from the growing cells were subjected to immunoblotting with the anti-Apg8p antibody. Lane 1, wild-type cells (YW5-1B); lane 2, Δapg8 cells harboring APG8 on a multicopy vector (TK201); lane 3, Δapg8 cells (TK404).


B , The wild - type ( YW5 - 1B ) cells were cultured in YEPD medium until 1 - 2 × 107 cells / ml ( 0 h ) and shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , 3 , 4 . 5 , and 6 h at 30 ° C . The amount of Apg8p was estimated by immunoblotting with anti - Apg8p antibody . Each lane has 10 μg of total protein .

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B, The wild-type (YW5-1B) cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, 3, 4.5, and 6 h at 30°C. The amount of Apg8p was estimated by immunoblotting with anti-Apg8p antibody. Each lane has 10 μg of total protein.


C , YW5 - 1B cells were cultured in YEPD medium until 1 - 2 × 107 cells / ml ( 0 h ) and shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , and 6 h at 30 ° C , and mRNA was prepared from each culture as described in Materials and Methods . APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe . Each lane has 4 μg of total mRNA .

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C, YW5-1B cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, and 6 h at 30°C, and mRNA was prepared from each culture as described in Materials and Methods. APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe. Each lane has 4 μg of total mRNA.


D , YW5 - 1B cells were cultured in YEPD medium until 1 × 107 cells / ml at 30 ° C . Rapamycin was added to the culture at a final concentration of 0 . 2 μg / ml and the cells were further incubated in YEPD medium for two hours at 30 ° C . Western blotting was performed with the anti - Apg8p antibody . Each lane has 20 μg of total protein .

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D, YW5-1B cells were cultured in YEPD medium until 1 × 107 cells/ml at 30°C. Rapamycin was added to the culture at a final concentration of 0.2 μg/ml and the cells were further incubated in YEPD medium for two hours at 30°C. Western blotting was performed with the anti-Apg8p antibody. Each lane has 20 μg of total protein.


Subcellular fractionation and solubilization of Apg8p . The wild - type cells ( YW5 - 1B ) growing logarithmically in YEPD medium or starved in SD ( − N ) medium for three hours were lysed . A , The lysates ( T ) were centrifuged at 13 , 000 g for 15 min to generate pellet ( LSP ) and supernatant ( LSS ) . LSS fraction was further centrifuged at 100 , 000 g for one hour and separated to pellet ( HSP ) and supernatant ( HSS ) . LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates . Equal volume of each sample was applied to each well , and immunoblotting was performed with anti - Apg8p antibody .

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Subcellular fractionation and solubilization of Apg8p. The wild-type cells (YW5-1B) growing logarithmically in YEPD medium or starved in SD(−N) medium for three hours were lysed. A, The lysates (T) were centrifuged at 13,000 g for 15 min to generate pellet (LSP) and supernatant (LSS). LSS fraction was further centrifuged at 100,000 g for one hour and separated to pellet (HSP) and supernatant (HSS). LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates. Equal volume of each sample was applied to each well, and immunoblotting was performed with anti-Apg8p antibody.


B , The lysates ( T ) were sonicated and centrifuged at 100 , 000 g for one hour to separate pellet ( P ) and supernatant ( S ) . The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody , respectively .

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B, The lysates (T) were sonicated and centrifuged at 100,000 g for one hour to separate pellet (P) and supernatant (S). The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody, respectively.


C , The sonicated lysates were centrifuged at 100 , 000 g for one hour to generate pellet fraction . The pellet fraction was treated with 2 % Triton X - 100 ( TX - 100 ) for 30 min on ice . The sample was centrifuged at 100 , 000 g for one hour and separated to supernatant ( S ) and pellet ( P ) fractions . The distribution of Apg8p was examined by immunoblotting with the anti - Apg8p antibody .

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C, The sonicated lysates were centrifuged at 100,000 g for one hour to generate pellet fraction. The pellet fraction was treated with 2% Triton X-100 (TX-100) for 30 min on ice. The sample was centrifuged at 100,000 g for one hour and separated to supernatant (S) and pellet (P) fractions. The distribution of Apg8p was examined by immunoblotting with the anti-Apg8p antibody.


Immunofluorescent staining of Δapg8 cells expressing 3 × HA - Apg8p . A , The Δapg8 cells harboring 3 × HA - tagged APG8 plasmid , TK114 cells , were grown until logarithmic phase . The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts . The spheroplasts were permeabilized by 0 . 5 % Triton X - 100 , and incubated with the anti - HA antibody , 16B12 , followed with the FITC - conjugated anti - mouse IgG . Left , Fluorescence image of 3 × HA - Apg8p ; right , Nomarski image of the cells . Arrows show punctate signals that are a little larger than tiny dot signals . Bars , 10 μm . B , Immunofluorescent staining of YW5 - 1B cells before ( 0 h ) and after ( 3 h ) shift to starvation ( negative control ) . Left , Fluorescence image ; right , Nomarski image . Bars , 10 μm . C , The cells were shifted to starvation for 0 , 0 . 5 , and 3 h . Upper panels , Fluorescence images of 3 × HA - Apg8p ; lower panels , Nomarski images of the cell . Bars , 5 μm .

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Immunofluorescent staining of Δapg8 cells expressing 3 × HA-Apg8p. A, The Δapg8 cells harboring 3 × HA-tagged APG8 plasmid, TK114 cells, were grown until logarithmic phase. The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts. The spheroplasts were permeabilized by 0.5% Triton X-100, and incubated with the anti-HA antibody, 16B12, followed with the FITC-conjugated anti-mouse IgG. Left, Fluorescence image of 3 × HA-Apg8p; right, Nomarski image of the cells. Arrows show punctate signals that are a little larger than tiny dot signals. Bars, 10 μm. B, Immunofluorescent staining of YW5-1B cells before (0 h) and after (3 h) shift to starvation (negative control). Left, Fluorescence image; right, Nomarski image. Bars, 10 μm. C, The cells were shifted to starvation for 0, 0.5, and 3 h. Upper panels, Fluorescence images of 3 × HA-Apg8p; lower panels, Nomarski images of the cell. Bars, 5 μm.


Change of Apg8p localization in Δpep4 cells after shift to starvation . The Δapg8Δpep4 cells harboring 3 × HA - tagged APG8 plasmid , TK116 cells , were shifted to starvation for 0 , 0 . 5 , 1 , 3 , and 6 h . Immunofluorescence microscopy was performed as described in Fig . 5 . A - E , FITC staining of 3 × HA - Apg8p . F - J , Nomarski images of the cells . Arrows show an autophagic body . Bars , 5 μm .

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Change of Apg8p localization in Δpep4 cells after shift to starvation. The Δapg8Δpep4 cells harboring 3 × HA-tagged APG8 plasmid, TK116 cells, were shifted to starvation for 0, 0.5, 1, 3, and 6 h. Immunofluorescence microscopy was performed as described in Fig. 5. A-E, FITC staining of 3 × HA-Apg8p. F-J, Nomarski images of the cells. Arrows show an autophagic body. Bars, 5 μm.


Immunostaining image of a cell containing autophagic bodies in the vacuole . The Δapg8Δpep4 cells expressing 3 × HA - Apg8p , TK116 cells , were shifted to SD ( − N ) medium for two hours . Cells were immunolabeled with anti - N mAb , 16B12 , followed by 10 - nm gold - conjugated goat anti - mouse IgG . AB , vacuole ; N , nucleus ; V , vacuole .

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Immunostaining image of a cell containing autophagic bodies in the vacuole. The Δapg8Δpep4 cells expressing 3 × HA-Apg8p, TK116 cells, were shifted to SD(−N) medium for two hours. Cells were immunolabeled with anti-N mAb, 16B12, followed by 10-nm gold-conjugated goat anti-mouse IgG. AB, vacuole; N, nucleus; V, vacuole.


Immuno - EM of autophagosomes . A and B , Mature autophagosome . C and D , Premature autophagosome . Arrows show expanded regions of the intramembrane space of the premature autophagosomes . TK116 cells were incubated in SD ( − N ) medium for one hour ( D ) , two hours ( B and C ) , or three hours ( A ) . The localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by the incubation with 5 - nm gold - ( A and D ) or 10 - nm gold - ( B and C ) conjugated goat anti - mouse IgG . AP , autophagosome ; N , nucleus ; V , vacuole .

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Immuno-EM of autophagosomes. A and B, Mature autophagosome. C and D, Premature autophagosome. Arrows show expanded regions of the intramembrane space of the premature autophagosomes. TK116 cells were incubated in SD(−N) medium for one hour (D), two hours (B and C), or three hours (A). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A and D) or 10-nm gold- (B and C) conjugated goat anti-mouse IgG. AP, autophagosome; N, nucleus; V, vacuole.


Possible intermediate structures of autophagosome . A , A membrane sac under construction . Arrows show gold particles . B and C , Isolation membranes . B , Isolation membrane is detected along the arrows . C , The small arrow shows a semicircular isolation membrane and a large arrow marks its open area . D , Apg8p - residing structures gathered in the area close to the vacuole . TK116 cells were incubated in SD ( − N ) medium for one hour ( A and C ) , or two hours ( B and D ) . The localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by the incubation with 5 - nm gold - ( A ) or 10 - nm gold - ( B - D ) conjugated goat anti - mouse IgG . V , vacuole .

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Possible intermediate structures of autophagosome. A, A membrane sac under construction. Arrows show gold particles. B and C, Isolation membranes. B, Isolation membrane is detected along the arrows. C, The small arrow shows a semicircular isolation membrane and a large arrow marks its open area. D, Apg8p-residing structures gathered in the area close to the vacuole. TK116 cells were incubated in SD(−N) medium for one hour (A and C), or two hours (B and D). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A) or 10-nm gold- (B-D) conjugated goat anti-mouse IgG. V, vacuole.


Fine morphology of Δypt7 and Δapg8 cells and proteinase - K accession to proAPI in these mutants . A , EM image of Δypt7 cell starved in SD ( − N ) medium for four hours . Autophagosomes ( AP , arrows ) were accumulated . B , EM image of the starved Δapg8 cell . C - E , The representatives of the membrane structures detected in the starved Δapg8 cells . C , Autophagosome - like structure indistinguishable from autophagosome . D and E , Aberrant multivesicular structures . N , nucleus ; V , vacuole . F , Proteinase K - sensitivity of proAPI . Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD ( − N ) medium for 4 . 5 h . The lysates were treated with 100 μg / ml proteinase K in the presence or absence of Triton X - 100 . Asterisk shows mature API and digestion product of proAPI by proteinase K .

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Fine morphology of Δypt7 and Δapg8 cells and proteinase-K accession to proAPI in these mutants. A, EM image of Δypt7 cell starved in SD(−N) medium for four hours. Autophagosomes (AP, arrows) were accumulated. B, EM image of the starved Δapg8 cell. C-E, The representatives of the membrane structures detected in the starved Δapg8 cells. C, Autophagosome-like structure indistinguishable from autophagosome. D and E, Aberrant multivesicular structures. N, nucleus; V, vacuole. F, Proteinase K-sensitivity of proAPI. Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD(−N) medium for 4.5 h. The lysates were treated with 100 μg/ml proteinase K in the presence or absence of Triton X-100. Asterisk shows mature API and digestion product of proAPI by proteinase K.


A Mpc1 and Mpc2 are present under fermentative conditions , while Mpc1 and Mpc3 are present under respiratory conditions . MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose ( YPD ) or glycerol ( YPG ) . Mitochondrial matrix protein aconitase ( Aco1 ) , 23 ‐ kDa translocase of the inner membrane subunit ( Tim23 ) , and 70 ‐ kDa translocase of the outer membrane subunit ( Tom70 ) were used as loading controls .

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A Mpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions. MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose (YPD) or glycerol (YPG). Mitochondrial matrix protein aconitase (Aco1), 23‐kDa translocase of the inner membrane subunit (Tim23), and 70‐kDa translocase of the outer membrane subunit (Tom70) were used as loading controls.


B Kinetics of the switch from Mpc2 to Mpc3 . Yeast cells were grown in YPD until mid ‐ log phase and then shifted to YPG medium . Whole cell extracts were taken at the indicated time points after the shift , and HA ‐ tagged Mpc2 and Mpc3 detected by Western blotting . Mpc3 is induced very rapidly by switch from YPD to YPG , while Mpc2 levels decrease only gradually .

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B Kinetics of the switch from Mpc2 to Mpc3. Yeast cells were grown in YPD until mid‐log phase and then shifted to YPG medium. Whole cell extracts were taken at the indicated time points after the shift, and HA‐tagged Mpc2 and Mpc3 detected by Western blotting. Mpc3 is induced very rapidly by switch from YPD to YPG, while Mpc2 levels decrease only gradually.


C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN ‐ PAGE . Isolated mitochondria expressing HA ‐ tagged MPC proteins were run on blue native gels , and MPC complexes detected with an HA ‐ specific antibody . MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions , and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions . In addition , a complex of ˜ 300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3 . Non ‐ specific reactivity of the anti ‐ HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk ( * ) .

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C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN‐PAGE. Isolated mitochondria expressing HA‐tagged MPC proteins were run on blue native gels, and MPC complexes detected with an HA‐specific antibody. MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions, and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions. In addition, a complex of ˜300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3. Non‐specific reactivity of the anti‐HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk (*).


A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant . Growth tests were performed with cells transformed with MPC1 , MPC2 , or MPC3 expression plasmids or with empty vectors in indicated combinations . A serial dilution of yeast cells was spotted on agar plates with glucose ‐ containing synthetic minimal medium with ( SD + AA ) or without ( SD − AA ) amino acids .

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A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant. Growth tests were performed with cells transformed with MPC1, MPC2, or MPC3 expression plasmids or with empty vectors in indicated combinations. A serial dilution of yeast cells was spotted on agar plates with glucose‐containing synthetic minimal medium with (SD+AA) or without (SD−AA) amino acids.


B Plasmid ‐ encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co ‐ expressed with either Mpc2 or Mpc3 . Cells were grown in selective medium containing glycerol as a carbon source . MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti ‐ HA or anti ‐ Flag antibodies . Mitochondrial enzymes aconitase ( Aco1 ) and malate dehydrogenase ( Mdh1 ) were used as loading controls .

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B Plasmid‐encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co‐expressed with either Mpc2 or Mpc3. Cells were grown in selective medium containing glycerol as a carbon source. MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti‐HA or anti‐Flag antibodies. Mitochondrial enzymes aconitase (Aco1) and malate dehydrogenase (Mdh1) were used as loading controls.


C BN ‐ PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid ‐ based protein expression . Non ‐ specific reactivity of the antibody is denoted by an asterisk ( * ) . It has to be noted that all complexes migrate slightly faster than in Fig C , most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA ‐ tag in Fig C .

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C BN‐PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid‐based protein expression. Non‐specific reactivity of the antibody is denoted by an asterisk (*). It has to be noted that all complexes migrate slightly faster than in Fig C, most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA‐tag in Fig C.


D Mpc1 is co ‐ immunoprecipitated with Mpc2 or Mpc3 . Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol ‐ containing medium were lysed in 1 % digitonin , and either Mpc2 ‐ Flag or Mpc3 ‐ Flag was immunoprecipitated . Mpc1 ‐ HA was detected by Western blot with an antibody directed against the epitope tag . The matrix protein aconitase ( Aco1 ) and 40 ‐ kDa translocase of the outer membrane subunit ( Tom40 ) were not co ‐ immunoprecipitated .

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D Mpc1 is co‐immunoprecipitated with Mpc2 or Mpc3. Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol‐containing medium were lysed in 1% digitonin, and either Mpc2‐Flag or Mpc3‐Flag was immunoprecipitated. Mpc1‐HA was detected by Western blot with an antibody directed against the epitope tag. The matrix protein aconitase (Aco1) and 40‐kDa translocase of the outer membrane subunit (Tom40) were not co‐immunoprecipitated.


E , F Stoichiometry of Mpc1 complexes . Chemical cross ‐ linking indicates that Mpc1 / Mpc2 and Mpc1 / Mpc3 form heterodimers of the predicted molecular weight . DSG , disuccimidyl glutarate . Cross ‐ linking products with unknown partners are labeled with a question mark ( ? ) .

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E, F Stoichiometry of Mpc1 complexes. Chemical cross‐linking indicates that Mpc1/Mpc2 and Mpc1/Mpc3 form heterodimers of the predicted molecular weight. DSG, disuccimidyl glutarate. Cross‐linking products with unknown partners are labeled with a question mark (?).


A , B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 ( A ) and Mpc3 ( B ) . Mitochondria were treated with IASD as indicated . Negative [ quenching by pretreatment with DTT ( pre ‐ DTT ) ] and positive [ lysis with 0 . 5 % Triton X ‐ 100 ( T ‐ X100 ) ] controls for the labeling reaction are shown . As a further control , also translocase of the inner membrane subunit Tim23 is detected by Western blot , which contains three endogenous cysteines , all of which are located in membrane ‐ embedded regions . Labeling of an IMS ‐ exposed cysteine with IASD is indicated by a mobility shift on SDS - PAGE corresponding to the molecular weight of IASD ( ˜ 500 Da ) .

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A, B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 (A) and Mpc3 (B). Mitochondria were treated with IASD as indicated. Negative [quenching by pretreatment with DTT (pre‐DTT)] and positive [lysis with 0.5% Triton X‐100 (T‐X100)] controls for the labeling reaction are shown. As a further control, also translocase of the inner membrane subunit Tim23 is detected by Western blot, which contains three endogenous cysteines, all of which are located in membrane‐embedded regions. Labeling of an IMS‐exposed cysteine with IASD is indicated by a mobility shift on SDS-PAGE corresponding to the molecular weight of IASD (˜500 Da).


C Protease protection assay in mitochondria expressing Mpc1 ‐ GFP , Mpc2 ‐ GFP , or Mpc3 ‐ GFP . Intact mitochondria ( M ) , mitoplasts with a ruptured outer membrane after hypo ‐ osmotic swelling ( Sw ) , or mitochondrial lysates with 0 . 5 % Triton X ‐ 100 ( Tr ) were treated with proteinase K ( PK ) . Loading controls are Tom70 ( outer membrane ) , Tim23 ( inner membrane ) , and Mdh1 ( matrix ) . GFP ‐ fused Mpc2 and Mpc3 are degraded in mitoplasts , whereas Mpc1 ‐ GFP is not .

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C Protease protection assay in mitochondria expressing Mpc1‐GFP, Mpc2‐GFP, or Mpc3‐GFP. Intact mitochondria (M), mitoplasts with a ruptured outer membrane after hypo‐osmotic swelling (Sw), or mitochondrial lysates with 0.5% Triton X‐100 (Tr) were treated with proteinase K (PK). Loading controls are Tom70 (outer membrane), Tim23 (inner membrane), and Mdh1 (matrix). GFP‐fused Mpc2 and Mpc3 are degraded in mitoplasts, whereas Mpc1‐GFP is not.


A MPCOX has higher transport activity than MPCFERM . The uptake of 14C ‐ labeled pyruvate into intact mitochondria was measured in vitro . Mitochondria had been isolated from cells grown in glycerol ‐ containing medium and expressing no subunit ( vectors ) , MPCFERM , MPCOX , Mpc2 , or Mpc3 . Imported pyruvate was quantified by re ‐ isolation of mitochondria and subsequent scintillation counting after 1 , 2 , or 5 min of incubation with [ 14C ] ‐ pyruvate . B Imported [ 14C ] ‐ pyruvate as in ( A ) after 5 ‐ min incubation . The difference between MPCOX and MPCFERM ( * * P = 0 . 0037 ) was significant ( unpaired t ‐ test ) .

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A MPCOX has higher transport activity than MPCFERM. The uptake of 14C‐labeled pyruvate into intact mitochondria was measured in vitro. Mitochondria had been isolated from cells grown in glycerol‐containing medium and expressing no subunit (vectors), MPCFERM, MPCOX, Mpc2, or Mpc3. Imported pyruvate was quantified by re‐isolation of mitochondria and subsequent scintillation counting after 1, 2, or 5 min of incubation with [14C]‐pyruvate.B Imported [14C]‐pyruvate as in (A) after 5‐min incubation. The difference between MPCOX and MPCFERM (**P = 0.0037) was significant (unpaired t‐test).


C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit ( vectors ) , MPCFERM , or MPCOX .

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C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit (vectors), MPCFERM, or MPCOX.


The chimeric construct Mpc2C3 is expressed in isolated mitochondria , while Mpc3C2 is not . Mpc1 ‐ HA and the Flag ‐ tagged chimeric proteins were detected by Western blotting in isolated mitochondria .

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The chimeric construct Mpc2C3 is expressed in isolated mitochondria, while Mpc3C2 is not. Mpc1‐HA and the Flag‐tagged chimeric proteins were detected by Western blotting in isolated mitochondria.


Mpc2C3 forms a carrier complex with Mpc1 , as analyzed by BN ‐ PAGE . When co ‐ expressed with Mpc1 , Mpc2C3 shows the same complex pattern as Mpc3 , including the 300K complex .

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Mpc2C3 forms a carrier complex with Mpc1, as analyzed by BN‐PAGE. When co‐expressed with Mpc1, Mpc2C3 shows the same complex pattern as Mpc3, including the 300K complex.


Pyruvate transport activity of the Mpc1 / Mpc2C3 complex reaches ˜ 85 % of MPCOX activity . Uptake of [ 14C ] ‐ pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro . Difference between MPCFERM and Mpc1 / Mpc2C3 is significant ( unpaired t ‐ test ; * P = 0 . 02 ) . Means of n = 4 experiments are shown , with error bars representing SEM .

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Pyruvate transport activity of the Mpc1/Mpc2C3 complex reaches ˜85% of MPCOX activity. Uptake of [14C]‐pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro. Difference between MPCFERM and Mpc1/Mpc2C3 is significant (unpaired t‐test; *P = 0.02). Means of n = 4 experiments are shown, with error bars representing SEM.


( C ) Interaction analysis between hCAP - G and hCAP - H . Bacterial cell lysates co - expressing hCAP - G ( residues 1 - 478 , 554 - 900 ) and hCAP - H ( residues 394 - 515 ) , either wild type ( WT ; lanes 2 , 10 and 13 ) , 3Q ( F463Q , F469Q and F473Q ; lane 3 ) , 5Q ( F463Q , F469Q , F473Q , F501Q and Y503Q ; lane 4 ) , 3A ( F463A , F469A and F473A ; lane 5 ) , 5A ( F463A , F469A , F473A , F501A and Y503A ; lane 6 ) or 2A ( F501A and Y503A ; lane 11 ) , or a C - terminal deletion mutant ( 506 - 514 residues were deleted from 394 - 515 ; lane 14 ) were applied to Ni - NTA agarose resin , and the bound fraction was analyzed by SDS - PAGE . Alternatively , a cell lysate co - expressing mutant hCAP - G ( D647K ) and wild - type hCAP - H was examined ( lane 7 ) . The uninduced cell lysate was also used as a negative control ( lane 9 ) .

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(C) Interaction analysis between hCAP-G and hCAP-H. Bacterial cell lysates co-expressing hCAP-G (residues 1-478, 554-900) and hCAP-H (residues 394-515), either wild type (WT; lanes 2, 10 and 13), 3Q (F463Q, F469Q and F473Q; lane 3), 5Q (F463Q, F469Q, F473Q, F501Q and Y503Q; lane 4), 3A (F463A, F469A and F473A; lane 5), 5A (F463A, F469A, F473A, F501A and Y503A; lane 6) or 2A (F501A and Y503A; lane 11), or a C-terminal deletion mutant (506-514 residues were deleted from 394-515; lane 14) were applied to Ni-NTA agarose resin, and the bound fraction was analyzed by SDS-PAGE. Alternatively, a cell lysate co-expressing mutant hCAP-G (D647K) and wild-type hCAP-H was examined (lane 7). The uninduced cell lysate was also used as a negative control (lane 9).


( A ) Expression of condensin I subunits in insect cells . The wild - type ( WT ) or IV - 5Q mutant CAP - H subunit was co - expressed with the other four subunits ( GST - SMC4 , SMC2 , CAP - D2 and CAP - G ) in insect cells . Cell lysates were prepared and subjected to SDS - PAGE , followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 ( left panel ) or against CAP - D2 , - G , and - H ( right panel ) .

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(A) Expression of condensin I subunits in insect cells. The wild-type (WT) or IV-5Q mutant CAP-H subunit was co-expressed with the other four subunits (GST-SMC4, SMC2, CAP-D2 and CAP-G) in insect cells. Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 (left panel) or against CAP-D2, -G, and -H (right panel).


( B ) Purification of the WT and IV - 5Q mutant condensin I complexes . Protein samples purified through glutathione - affinity chromatography were subjected to SDS - PAGE and analyzed by CBB staining ( left panel ) or immunoblotting with a mixture of antibodies as described above ( middle and right panels ) .

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(B) Purification of the WT and IV-5Q mutant condensin I complexes. Protein samples purified through glutathione-affinity chromatography were subjected to SDS-PAGE and analyzed by CBB staining (left panel) or immunoblotting with a mixture of antibodies as described above (middle and right panels).


( C ) Add - back assay using the WT and mutant condensin I complexes . Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes ( from top to bottom ; WT , IV - 5Q , ∆ G , ∆ G [ IV - 5Q ] ) . The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes . The samples were fixed and labeled with an antibody against mSMC4 ( red ) . DNA was counterstained with DAPI ( blue ) . The data from a single representative experiment out of two repeats are shown . In the experiment shown here , multiple images were collected for condensin - depleted extracts supplemented with the WT ( n = 17 ) , IV - 5Q ( n = 22 ) , ∆ G ( n = 20 ) and ∆ G ( IV - 5Q ) ( n = 25 ) complexes . The scale bar represents 10 μm .

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(C) Add-back assay using the WT and mutant condensin I complexes. Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes (from top to bottom; WT, IV-5Q, ∆G, ∆G[IV-5Q]). The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes. The samples were fixed and labeled with an antibody against mSMC4 (red). DNA was counterstained with DAPI (blue). The data from a single representative experiment out of two repeats are shown. In the experiment shown here, multiple images were collected for condensin-depleted extracts supplemented with the WT (n = 17), IV-5Q (n = 22), ∆G (n = 20) and ∆G(IV-5Q) (n = 25) complexes. The scale bar represents 10 μm.


( A ) Purification of hCAP - G and hCAP - G - H subcomplexes : wild type ( WT ) , CAP - G K60D / R848E double mutant ( K60D / R848E ) and CAP - G R168E mutant ( R168E ) . Purified protein samples were subjected to SDS - PAGE and analyzed by CBB staining .

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(A) Purification of hCAP-G and hCAP-G-H subcomplexes: wild type (WT), CAP-G K60D/R848E double mutant (K60D/R848E) and CAP-G R168E mutant (R168E). Purified protein samples were subjected to SDS-PAGE and analyzed by CBB staining.


( B ) Double - stranded DNA ( dsDNA ) and single - stranded DNA binding assay of the hCAP - G . 30 - bp dsDNA was incubated with no protein ( lanes 1 ) or increasing amounts of WT hCAP - G ( WT ; lanes 2 - 4 ) . 30 - mer ssDNA was incubated with no protein ( lanes 5 ) or increasing amounts of WT hCAP - G ( WT ; lanes 6 - 8 ) .

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(B) Double-stranded DNA (dsDNA) and single-stranded DNA binding assay of the hCAP-G. 30-bp dsDNA was incubated with no protein (lanes 1) or increasing amounts of WT hCAP-G (WT; lanes 2-4). 30-mer ssDNA was incubated with no protein (lanes 5) or increasing amounts of WT hCAP-G (WT; lanes 6-8).


( C ) The dsDNA binding assay for the hCAP - G - H subcomplexes . 30 - bp dsDNA was incubated with no protein ( lanes 1 , 5 and 9 ) , increasing amounts of WT hCAP - G - H subcomplex ( WT ; lanes 2 - 4 ) , CAP - G K60D / R848E double mutant ( K60D / R848E ; lanes 6 - 8 ) or CAP - G R168E mutant ( R168E ; lanes 10 - 12 ) .

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(C) The dsDNA binding assay for the hCAP-G-H subcomplexes. 30-bp dsDNA was incubated with no protein (lanes 1, 5 and 9), increasing amounts of WT hCAP-G-H subcomplex (WT; lanes 2-4), CAP-G K60D/R848E double mutant (K60D/R848E; lanes 6-8) or CAP-G R168E mutant (R168E; lanes 10-12).


( D ) The ssDNA binding assay for the hCAP - G - H

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(D) The ssDNA binding assay for the hCAP-G-H subcomplexes


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 ) .

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

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 .

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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 )

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

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)


B - C ' ) Immunofluorescence analysis of active - caspase 3 in the ONL of P347S and P347S / miR - 181a / b + / - . D , E ) TUNEL analysis in the ONL of P347S and P347S / miR - 181a / b + / - . F ) Quantification of immunofluorescence analysis in ( B - C ' ) ( N = 4 eyes P347S and N = 6 eyes P347S / miR - 181a / b + / ) . G ) Quantification of TUNEL analysis in ( D , E ) ( N = 3 eyes / genotype ) .

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B-C') Immunofluorescence analysis of active-caspase 3 in the ONL of P347S and P347S/miR-181a/b+/-. D, E) TUNEL analysis in the ONL of P347S and P347S/miR-181a/b+/-. F) Quantification of immunofluorescence analysis in (B-C') (N=4 eyes P347S and N=6 eyes P347S/ miR-181a/b+/). G) Quantification of TUNEL analysis in (D, E) (N=3 eyes/genotype).


D - P ) Immunofluorescence analysis showed amelioration of Rhodopsin localization ( D - F ' ; white arrowheads ) ; C - arrestin expression ( G - I ' ) and PR outer segment ( OS ) and inner segment ( IS ) structures , as determined , respectively , by PNA ( J - L ' ) ; and Phalloidin staining ( M - O ' ' ) in P347S / miR - 181a / b + / - vs P347S eyes at p30 . M ' ' - O ' ' show higher magnification of M - O . Scale bars 25 μm in D - O ' ; 5μm in M ' ' - O ' ' . Fluorescence densitometry quantification of each staining is reported in ( P ) , N = 3 eye / genotype for each staining . WT vs P347S p values are reported in red , P347S vs P347S / miR - 181a / b + / - p values are reported in black .

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

D-P) Immunofluorescence analysis showed amelioration of Rhodopsin localization (D-F'; white arrowheads); C-arrestin expression (G-I') and PR outer segment (OS) and inner segment (IS) structures, as determined, respectively, by PNA (J-L'); and Phalloidin staining (M-O'') in P347S/miR-181a/b+/- vs P347S eyes at p30. M''-O'' show higher magnification of M-O. Scale bars 25 μm in D-O'; 5μm in M''-O''. Fluorescence densitometry quantification of each staining is reported in (P), N=3 eye/genotype for each staining. WT vs P347S p values are reported in red, P347S vs P347S/miR-181a/b+/- p values are reported in black.


D - L ) Immunofluorescence analysis of Rhodopsin ( D - E ' ) , C - arrestin ( F - G ' ) , PNA ( H - I ' ) ; and Phalloidin staining ( J - K ' ' ) in P347S and P347S / miR - 181a / b + / - retinas at p90 . J ' ' - K ' ' show higher magnification of J - K . Scale bars 25 μm in D - K ' ; 10μm in J ' ' - K ' ' .

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D-L) Immunofluorescence analysis of Rhodopsin (D-E'), C-arrestin (F-G'), PNA (H-I'); and Phalloidin staining (J-K'') in P347S and P347S/miR-181a/b+/- retinas at p90. J''-K'' show higher magnification of J-K. Scale bars 25 μm in D-K'; 10μm in J''-K''.


Immunofluorescence analysis of Rhodopsin C - arrestin PNA and Phalloidin staining in P347S and P347S / miR - 181a / b + / - retinas at p90 . Fluorescence densitometry quantification of each staining is reported in ( L ) , N = 3 eye / genotype / staining .

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Immunofluorescence analysis of Rhodopsin C-arrestin PNA and Phalloidin staining in P347S and P347S/miR-181a/b+/- retinas at p90. Fluorescence densitometry quantification of each staining is reported in (L), N=3 eye/genotype/staining.


M ) Measurement of P347S and P347S / miR - 181a / b + / - ONL thickness ( N ≥ 3 eyes / genotype ;

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M) Measurement of P347S and P347S/miR-181a/b+/- ONL thickness (N≥3 eyes/genotype;


N ) Graphical representation of the results of OKR analysis by the optokinetic tracking assays reported as cycles / degree . Visual acuity is preserved in P347S / miR - 181a / b + / - animals with respect to P347S ( N = 4 animals / genotype ) .

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N) Graphical representation of the results of OKR analysis by the optokinetic tracking assays reported as cycles/degree. Visual acuity is preserved in P347S/miR-181a/b+/- animals with respect to P347S (N=4 animals/genotype).


B - F ) Electron microscopy analysis shows an increase of mitochondrial fragmentation in P347S PRs with respect to WT and amelioration of the mitochondrial phenotype in P347S / miR - 181a / b + / - vs P347S PRs at p12 and p30 ( N = 2 animals / genotype ) . M , mitochondria . G - I ) The quantitative analysis of mitochondrial phenotype is expressed as measurement of the mitochondria perimeter ( G ) , mitochondria number ( H ) and as the ratio of mitochondria area per the analyzed field area ( I ) . N = 2 animals / genotype .

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B-F) Electron microscopy analysis shows an increase of mitochondrial fragmentation in P347S PRs with respect to WT and amelioration of the mitochondrial phenotype in P347S/miR-181a/b+/- vs P347S PRs at p12 and p30 (N=2 animals/genotype). M, mitochondria. G-I) The quantitative analysis of mitochondrial phenotype is expressed as measurement of the mitochondria perimeter (G), mitochondria number (H) and as the ratio of mitochondria area per the analyzed field area (I). N=2 animals/genotype.


A ) WB analysis of Drp1 , one of the key proteins involved in the mitochondrial fission pathway , in the optic cup of WT , P347S DMSO - treated , P347S / miR - 181a / b + / - DMSO - treated and of P347S / miR - 181a / b + / - FEDRATINIB - treated ( an inhibitor of the JAK2 / STAT3 pathway ) mice at p30 .

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A) WB analysis of Drp1, one of the key proteins involved in the mitochondrial fission pathway, in the optic cup of WT, P347S DMSO-treated, P347S/miR-181a/b+/- DMSO-treated and of P347S/miR-181a/b+/- FEDRATINIB-treated (an inhibitor of the JAK2/STAT3 pathway) mice at p30.


B ) Quantification revealed that Drp1 protein levels are increased in P347S with respect to WT , and partially rescued in P347S / miR - 181a / b + / - . Data are normalized to Gapdh . N ≥ 4 eye / genotype . Please note that all compared bands from WT , P347S , P347S / miR - 181a / b + / - and P347S / miR - 181a / b + / - with FEDRATINIB samples are from the same blots , which were cropped and shown organized in the panel for the sake of data presentation clarity ( see source data ) .

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B) Quantification revealed that Drp1 protein levels are increased in P347S with respect to WT, and partially rescued in P347S/miR-181a/b+/-. Data are normalized to Gapdh. N ≥ 4 eye/genotype. Please note that all compared bands from WT, P347S, P347S/miR-181a/b+/- and P347S/miR-181a/b+/- with FEDRATINIB samples are from the same blots, which were cropped and shown organized in the panel for the sake of data presentation clarity (see source data).


C - D ) qRT - PCR analysis reveals decreased levels of the Drp1 transcript and increased levels of Stat3 and Jak2 transcripts in the eyes of P347S / miR - 181a / b + / - vs those of P347S animals . N ≥ 4 eyes / genotype .

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C-D) qRT-PCR analysis reveals decreased levels of the Drp1 transcript and increased levels of Stat3 and Jak2 transcripts in the eyes of P347S/miR-181a/b+/- vs those of P347S animals. N≥4 eyes/genotype.


F ) Quantification of WB in A reveals decreased levels of Drp1 in P347S / miR - 181a / b + / - DMSO - treated vs P347S and increased levels in P347S / miR - 181a / b + / - FEDRATINIB - treated ex - vivo retinas . N ≥ 4 eyes / genotype .

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F) Quantification of WB in A reveals decreased levels of Drp1 in P347S/miR-181a/b+/- DMSO-treated vs P347S and increased levels in P347S/miR-181a/b+/- FEDRATINIB-treated ex-vivo retinas. N≥4 eyes/genotype.


( D - U ) Immunofluorescence analysis showed amelioration of Rhodopsin localization ( p30 D - G ' , p70 L - O ' ; white arrowheads ) and C - arrestin ( p30 H - K ' , p70 P - S ' ; white arrowheads ) expression in Sponge - miR - 181a / b - injected eyes vs the corresponding controls also highlighting improvement of the OS structure . At both time points the effect of amelioration was more significant and evident when the AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b was used . Scale bars are 25 μm . Fluorescence densitometry quantification of each staining is reported in T for p30 and in U for p70 , N = 3 eye / treatment / staining .

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

(D-U) Immunofluorescence analysis showed amelioration of Rhodopsin localization (p30 D-G', p70 L-O'; white arrowheads) and C-arrestin (p30 H-K', p70 P-S'; white arrowheads) expression in Sponge-miR-181a/b-injected eyes vs the corresponding controls also highlighting improvement of the OS structure. At both time points the effect of amelioration was more significant and evident when the AAV2/8.CMV.GFP-Sponge-miR-181a/b was used. Scale bars are 25 μm. Fluorescence densitometry quantification of each staining is reported in T for p30 and in U for p70, N=3 eye/treatment/staining.


A - I ' ) Immunofluorescence analysis on central retinal sections at p30 showed ONL thickness increase , amelioration of Rhodopsin localization ( A - D ' ) and C - arrestin expression ( E - H ' ) in AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b - injected vs AAV2 / 8 . CMV . GFP - injected rd10 eyes . Scale bars are 25 μm . Fluorescence densitometry quantification of each staining is reported in I , N = 3 eye / treatment / staining .

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A-I') Immunofluorescence analysis on central retinal sections at p30 showed ONL thickness increase, amelioration of Rhodopsin localization (A-D') and C-arrestin expression (E-H') in AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected vs AAV2/8.CMV.GFP-injected rd10 eyes. Scale bars are 25 μm. Fluorescence densitometry quantification of each staining is reported in I, N=3 eye/treatment/staining.


Analysis of retinal function based on pupillary light responses ( PLR ) Water Maze tests . ( J ) PLR showed a significantly higher pupil constriction in rd10 mice injected with AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b than in AAV2 / 8 . CMV . GFP - injected rd10 eyes , at both 10 lux and 100 lux intensity stimuli ( N ≥ 6 eyes / treatment and N = 9 eyes / treatment , respectively ) . Data are presented as mean ± SEM . Student ' s t - test , unpaired .

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Analysis of retinal function based on pupillary light responses (PLR) Water Maze tests. (J) PLR showed a significantly higher pupil constriction in rd10 mice injected with AAV2/8.CMV.GFP-Sponge-miR-181a/b than in AAV2/8.CMV.GFP-injected rd10 eyes, at both 10 lux and 100 lux intensity stimuli (N≥6 eyes/treatment and N=9 eyes/treatment, respectively). Data are presented as mean ± SEM. Student's t-test, unpaired.


Analysis of retinal function based on pupillary light responses ( PLR ) Water Maze tests . ( K ) Water Maze test highlighted a shorter latency in reaching the platform in AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b - injected animals respect to control - injected ones ( N ≥ 6 eyes / treatment ) .

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Analysis of retinal function based on pupillary light responses (PLR) Water Maze tests. (K) Water Maze test highlighted a shorter latency in reaching the platform in AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected animals respect to control-injected ones (N≥6 eyes/treatment).


Detection of phosphorylated 17K in BYDV - infected cells using Phos - tag SDS - PAGE - immunoblotting assays with an anti - 17K antibody . dpi , days after BYDV inoculation ; P , phosphorylated 17K bands ; λ - PP , λ - phosphatase .

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Detection of phosphorylated 17K in BYDV-infected cells using Phos-tag SDS-PAGE-immunoblotting assays with an anti-17K antibody. dpi, days after BYDV inoculation; P, phosphorylated 17K bands; λ-PP, λ-phosphatase.


Active HvSnRK1 , but not its total amount , was increased in BYDV - infected barley plants as compared to the mock controls . P - SnRK1 or SnRK1 , enzymatically active SnRK1 ( with T172 phosphorylation ) or unphosphorylated SnRK1 protein .

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Active HvSnRK1, but not its total amount, was increased in BYDV-infected barley plants as compared to the mock controls. P-SnRK1 or SnRK1, enzymatically active SnRK1 (with T172 phosphorylation) or unphosphorylated SnRK1 protein.


Analysis of 17K phosphorylation by HvGRIK1 - HvSnRK1 kinases in vitro . The kinase reactions were set up by mixing the desired GST fusions of 17K , 17K3A , 17K5A , HvGRIK1 , and HvSnRK1 kinase domain ( KD ) , with the reaction products analyzed using Phos - tag SDS - PAGE - immunoblotting assays with an anti - 17K antibody . P , phosphorylated 17K bands ; arrowhead , unphosphorylated 17K .

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Analysis of 17K phosphorylation by HvGRIK1-HvSnRK1 kinases in vitro. The kinase reactions were set up by mixing the desired GST fusions of 17K, 17K3A, 17K5A, HvGRIK1, and HvSnRK1 kinase domain (KD), with the reaction products analyzed using Phos-tag SDS-PAGE-immunoblotting assays with an anti-17K antibody. P, phosphorylated 17K bands; arrowhead, unphosphorylated 17K.


Confirmation of the expression of SnRK1K139R - YFP or SnRK1 - YFP fusion protein each in two transgenic lines , SnRK1K139R - YFP - L13 and - L22 or SnRK1 - YFP - L4 and - L17 , by immunoblotting . The protein samples analyzed here or in ( B ) and ( C ) were all prepared from BYDV - infected barley plants at 21 dpi .

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Confirmation of the expression of SnRK1K139R-YFP or SnRK1-YFP fusion protein each in two transgenic lines, SnRK1K139R-YFP-L13 and -L22 or SnRK1-YFP-L4 and -L17, by immunoblotting. The protein samples analyzed here or in (B) and (C) were all prepared from BYDV-infected barley plants at 21 dpi.


Phosphorylation of BYDV 17K was increased in SnRK1 - YFP - L4 plants overexpressing SnRK1 - YFP but reduced in SnRK1K139R - YFP - L13 ectopically expressing SnRK1K139R - YFP compared with WT control . P , phosphorylated 17K bands .

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Phosphorylation of BYDV 17K was increased in SnRK1-YFP-L4 plants overexpressing SnRK1-YFP but reduced in SnRK1K139R-YFP-L13 ectopically expressing SnRK1K139R-YFP compared with WT control. P, phosphorylated 17K bands.


Quantitative comparisons of plant height ( D ) , maximum root length ( E ) among the set of BYDV - infected barley materials shown in ( C ) ( using the samples collected at 21 dpi ) .

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Quantitative comparisons of plant height (D), maximum root length (E) among the set of BYDV-infected barley materials shown in (C) (using the samples collected at 21 dpi).


protein ( H ) levels of viral CP gene in the set of BYDV - infected barley materials shown in ( C ) ( using the samples collected at 21 dpi ) .

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protein (H) levels of viral CP gene in the set of BYDV-infected barley materials shown in (C) (using the samples collected at 21 dpi).


Levels of GFP siRNA detected by sRNA blotting for the panel of tobacco materials shown in ( A ) , with arrowhead indicating the main band of GFP siRNA .

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Levels of GFP siRNA detected by sRNA blotting for the panel of tobacco materials shown in (A), with arrowhead indicating the main band of GFP siRNA.


Protein levels of PEBV CP and corrected expression of 17K , 17K5A or 17K5D in the panel of tobacco plants shown in ( D ) , revealed using immunoblotting with anti - PEBV CP or anti - 17K antibody .

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Protein levels of PEBV CP and corrected expression of 17K, 17K5A or 17K5D in the panel of tobacco plants shown in (D), revealed using immunoblotting with anti-PEBV CP or anti-17K antibody.


17K5D , but not BYDV 17K , interacted with HvSDN1 in LC ( B ) assays . Expression of 17K and 17K5D in the LC assays were verified by immunoblotting using anti - 17K antibody .

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17K5D, but not BYDV 17K, interacted with HvSDN1 in LC (B) assays. Expression of 17K and 17K5D in the LC assays were verified by immunoblotting using anti-17K antibody.


C 17K5D , but not 17K or 17K5A , showed binding to BYDV vsiRNA . The GST fusions of 17K , 17K5D , and 17K5A , as well as free GST , were used in the RNA EMSA assays with biotin - labeled vsiRNA899 or vsiRNA899 duplex as probes .

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C 17K5D, but not 17K or 17K5A, showed binding to BYDV vsiRNA. The GST fusions of 17K, 17K5D, and 17K5A, as well as free GST, were used in the RNA EMSA assays with biotin-labeled vsiRNA899 or vsiRNA899 duplex as probes.


Cleavage of BYDV vsiRNA899 by HvSDN1 ( His - HvSDN1 ) , with AtSDN1 ( His - AtSDN1 ) serving as a positive control . Asterisk denotes cleavage products . The GST fusions of 17K and 17K5D did not show vsiRNA cleavage activity .

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Cleavage of BYDV vsiRNA899 by HvSDN1 (His-HvSDN1), with AtSDN1 (His-AtSDN1) serving as a positive control. Asterisk denotes cleavage products. The GST fusions of 17K and 17K5D did not show vsiRNA cleavage activity.


Inhibition of HvSDN1 - catalyzed vsiRNA cleavage by GST - 17K5D but not GST - 17K . Free GST protein served as a negative control . Asterisk denotes cleavage products .

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Inhibition of HvSDN1-catalyzed vsiRNA cleavage by GST-17K5D but not GST-17K. Free GST protein served as a negative control. Asterisk denotes cleavage products.


The three mutants of 17K5D ( 17K5Dm4 , 17K5Dm2a , and 17K5Dm2b ) retained the ability to interact with HvSDN1 but their GST fusions failed to bind vsiRNA899 ( D ) . Neither GST nor GST - 17K showed vsiRNA899 binding .

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The three mutants of 17K5D (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) retained the ability to interact with HvSDN1 but their GST fusions failed to bind vsiRNA899 (D). Neither GST nor GST-17K showed vsiRNA899 binding.


Quantitative analysis of HvSDN1 cleavage activities , with the fluorescence recorded using Typhoon FLA9500 ( F and G ) Compared with free GST or GST - 17K , the GST fusions of 17K5D or three derivative mutants ( 17K5Dm4 , 17K5Dm2a , and 17K5Dm2b ) inhibited HvSDN1 - catalzyed cleavage of fluorogenic vsiRNA899 , which was revealed by a fluorospot assay ( F ) , relative comparison of fluorescence values ( G )

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Quantitative analysis of HvSDN1 cleavage activities, with the fluorescence recorded using Typhoon FLA9500 (F and G) Compared with free GST or GST-17K, the GST fusions of 17K5D or three derivative mutants (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) inhibited HvSDN1-catalzyed cleavage of fluorogenic vsiRNA899, which was revealed by a fluorospot assay (F), relative comparison of fluorescence values (G)


Co - IP assays revealing the presence of vsiRNA - containing HvAGO1 - HvSDN1 - P17K complex in BYDV - infected WT , SnRK1K139R - YFP - L13 , and SnRK1 - YFP - L4 plants , with mock - inoculated SnRK1 - YFP - L4 individuals as controls . The immunoprecipitates , prepared using anti - HvAGO1 antibody , were analyzed for the presence of HvSDN1 ( by immunoblotting with anti - HvSDN1 antibody ) , P17K ( asterisk ) and 17K ( arrowhead ) ( using Phos - tag SDS - PAGE coupled immunoblotting ) , and BYDV vsiRNA ( by sRNA blotting ) . Compared with WT control , the HvAGO1 immunoprecipitates derived from SnRK1 - YFP - L4 contained higher levels of P17K and vsiRNA but less amount of HvSDN1 , whereas the reverse was observed for SnRK1K139R - YFP - L13 .

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Co-IP assays revealing the presence of vsiRNA-containing HvAGO1-HvSDN1-P17K complex in BYDV-infected WT, SnRK1K139R-YFP-L13, and SnRK1-YFP-L4 plants, with mock-inoculated SnRK1-YFP-L4 individuals as controls. The immunoprecipitates, prepared using anti-HvAGO1 antibody, were analyzed for the presence of HvSDN1 (by immunoblotting with anti-HvSDN1 antibody), P17K (asterisk) and 17K (arrowhead) (using Phos-tag SDS-PAGE coupled immunoblotting), and BYDV vsiRNA (by sRNA blotting). Compared with WT control, the HvAGO1 immunoprecipitates derived from SnRK1-YFP-L4 contained higher levels of P17K and vsiRNA but less amount of HvSDN1, whereas the reverse was observed for SnRK1K139R-YFP-L13.


Co - IP assays demonstrating that the interaction between HvAGO1 and HvSDN1 was weakened by P17K . The immunoprecipitates were prepared using anti - HvSDN1 antibody from the panel of plants shown in ( B ) . Relative to WT control , the HvSDN1 immunoprecipitates derived from SnRK1 - YFP - L4 contained much less HvAGO1 , whereas the opposite was observed for SnRK1K139R - YFP - L13 .

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Co-IP assays demonstrating that the interaction between HvAGO1 and HvSDN1 was weakened by P17K. The immunoprecipitates were prepared using anti-HvSDN1 antibody from the panel of plants shown in (B). Relative to WT control, the HvSDN1 immunoprecipitates derived from SnRK1-YFP-L4 contained much less HvAGO1, whereas the opposite was observed for SnRK1K139R-YFP-L13.


17K5D compromised the interaction between HvSDN1 and the PAZ domain of HvAGO1 . The LUC signal resulted from the interaction between HvSDN1 - HvAGO1 PAZ domain was significantly decreased in the presence of 17K5D but not that of 17K .

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17K5D compromised the interaction between HvSDN1 and the PAZ domain of HvAGO1. The LUC signal resulted from the interaction between HvSDN1-HvAGO1 PAZ domain was significantly decreased in the presence of 17K5D but not that of 17K.


Verification of 17K5D expression in the transgenic lines 17K5D - L3 and - L7 by immunoblotting with anti - 17K antibody .

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Verification of 17K5D expression in the transgenic lines 17K5D-L3 and -L7 by immunoblotting with anti-17K antibody.


B - D Comparison of BYDV disease symptoms including plant growth ( B ) , plant height ( C ) , and maximum root length ( D ) between two transgenic lines and WT control at 21 dpi .

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B-D Comparison of BYDV disease symptoms including plant growth (B), plant height (C), and maximum root length (D) between two transgenic lines and WT control at 21 dpi.


protein ( F ) levels of viral CP gene in the BYDV - infected samples shown in ( B ) .

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protein (F) levels of viral CP gene in the BYDV-infected samples shown in (B).


BYDV vsiRNA abundance in the set of samples shown in ( B ) , as detected by sRNA blotting at 14 dpi .

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BYDV vsiRNA abundance in the set of samples shown in (B), as detected by sRNA blotting at 14 dpi.


Relative to TaSDN1 - DD line , TaSDN1 protein was decreased in TaSDN1 - Dd and TaSDN1 - dd mutants , with a stronger reduction found for TaSDN1 - dd . The reactive TaSDN1 protein detected in TaSDN1 - dd was caused by the un - mutated TaSDN1 - A and - B homoeologs .

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Relative to TaSDN1-DD line, TaSDN1 protein was decreased in TaSDN1-Dd and TaSDN1-dd mutants, with a stronger reduction found for TaSDN1-dd. The reactive TaSDN1 protein detected in TaSDN1-dd was caused by the un-mutated TaSDN1-A and -B homoeologs.


protein ( M ) levels of BYDV CP gene in TaSDN1 - DD , TaSDN1 - Dd and TaSDN1 - dd plants at 21 dpi .

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protein (M) levels of BYDV CP gene in TaSDN1-DD, TaSDN1-Dd and TaSDN1-dd plants at 21 dpi.


BYDV vsiRNA abundance in TaSDN1 - DD , TaSDN1 - Dd , and TaSDN1 - dd plants detected by sRNA blotting at 21 dpi of BYDV .

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BYDV vsiRNA abundance in TaSDN1-DD, TaSDN1-Dd, and TaSDN1-dd plants detected by sRNA blotting at 21 dpi of BYDV.


The graphs depict the recovery of CAR1 - mcherry ( left ) and tPH - CynA - KikGR ( right ) . Mean ± SEM is shown for n = 2 cells .

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The graphs depict the recovery of CAR1-mcherry (left) and tPH-CynA-KikGR (right). Mean ± SEM is shown for n = 2 cells.


Time - lapse confocal images of tPH - CynA - KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell . Front and back of the cell are shown .

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Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell. Front and back of the cell are shown.


Time - lapse confocal images of AX3 cell co - expressing PH - Crac - GFP ( green ) and another established PI ( 3 , 4 ) P2 biosensor tPH - TAPP1 - RFP ( red ) . Box shows the vesicle .

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Time-lapse confocal images of AX3 cell co-expressing PH-Crac-GFP (green) and another established PI(3,4)P2 biosensor tPH-TAPP1-RFP (red). Box shows the vesicle.


Time - lapse confocal images of tPH - CynA - KikGR ( green ) expressing wild - type AX3 cells . FM4 - 64 ( red ) was added at time 0 during live cell imaging . Front and back of the cell are shown . Box shows the vesicle .

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Time-lapse confocal images of tPH-CynA-KikGR (green) expressing wild-type AX3 cells. FM4-64 (red) was added at time 0 during live cell imaging. Front and back of the cell are shown. Box shows the vesicle.


Time - lapse confocal images of wild - type AX3 cells expressing tPH - CynA - KikGR ( green ) and PH - crac ( red ) before and after PI3K inhibition by 5 µM Wortmannin .

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Time-lapse confocal images of wild-type AX3 cells expressing tPH-CynA-KikGR (green) and PH-crac (red) before and after PI3K inhibition by 5 µM Wortmannin.


Confocal images of tPH - CynA - KikGR ( green ) and PH - Crac ( red ) expressing wild - type Dictyostelium AX3 cells ( left ) and PI3K1 - 2 - Dictyostelium cells ( right ) treated with 5 µM Latrunculin A .

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Confocal images of tPH-CynA-KikGR (green) and PH-Crac (red) expressing wild-type Dictyostelium AX3 cells (left) and PI3K1-2- Dictyostelium cells (right) treated with 5 µM Latrunculin A.


Time - lapse confocal images showing dynamic localization of PI ( 3 , 4 ) P2 . Nocodazole was added at time 0 during the live cell imaging . Quantification of vesicle number , vesicle number / min , and vesicle lifetime ( s ) from experiment in E . T - test was carried out by GraphPad Prism , * * P ≤ 0 . 01 versus After Nocodazole group ; mean ± SEM ( n = 10 ) .

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

Time-lapse confocal images showing dynamic localization of PI(3,4)P2. Nocodazole was added at time 0 during the live cell imaging.   Quantification of vesicle number, vesicle number/min, and vesicle lifetime (s) from experiment in E. T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus After Nocodazole group; mean±SEM (n = 10).


( L ) Representative confocal images of tPH - CynA - KikGR in wild - type Dictyostelium AX2 or RacH − cells treated with 5 μM latrunculin A Scale bar : 5 μm . ( M ) Ratio of membrane to cytosol intensity of tPH - CynA - KikGR in wild - type Dictyostelium AX2 and RacH − cells . T - test was carried out by GraphPad Prism , * * * * P ≤ 0 . 0001 versus AX2 group ; mean ± SEM ( n = 33 ) .

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

(L) Representative confocal images of tPH-CynA-KikGR in wild-type Dictyostelium AX2 or RacH− cells treated with 5 μM latrunculin A Scale bar: 5 μm. (M) Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wild-type Dictyostelium AX2 and RacH− cells. T-test was carried out by GraphPad Prism, ****P ≤ 0.0001 versus AX2 group; mean ± SEM (n = 33).


Time - lapse confocal images of tPH - CynA - KikGR expressing Dictyostelium AX3 cells . The macropinosome at the front of the cell was photoconverted at time 0 . White arrow points to PI ( 3 , 4 ) P2 localization at the back of the membrane . Cell front and back are shown , box showing the photoconverted region .

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

Time-lapse confocal images of tPH-CynA-KikGR expressing Dictyostelium AX3 cells. The macropinosome at the front of the cell was photoconverted at time 0. White arrow points to PI(3,4)P2 localization at the back of the membrane. Cell front and back are shown, box showing the photoconverted region.


B , C MCF - 7 cells expressing GFP - INPP4B or GFP - vector were cultured in growth media or EBSS for the indicated times , then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control ( B ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to growth media - treated GFP - vector cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( C ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc test in C ,

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

B, C MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (B). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (C). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in C,


D , E MCF - 7 cells expressing NT , INPP4B # 1 or INPP4B # 2 shRNA were cultured in growth media or EBSS for the indicated times , then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control ( D ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to growth media - treated NT shRNA cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( E ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in E .

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

D, E MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (D). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (E). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in E.


F , G MCF - 7 cells expressing GFP - INPP4B or GFP - vector were cultured in growth media or EBSS for 4 hours , then fixed and immunostained with p62 antibodies , and co - stained with DAPI and phalloidin ( F ) . Data represent the number of p62 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 50 cells / experiment ) ( G ) . Data information : Data is presented as mean ± SD . The insets at the lower right of each image are higher power regions of the boxed areas . Scale bar is 10 µm in F p values determined by one - way ANOVA with Tukey post hoc test in G ,

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O 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-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 O O O O O O O O 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, G MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (F). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (G). Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F p values determined by one-way ANOVA with Tukey post hoc test in G,