Figure 5 ( a ) Sld3 / 7 recruitment reaction performed as in Fig . 2a . ( b ) Replication reaction performed as in Fig . 1g . Experiments in ( a ) and ( b ) used MCM containing wild type ( wt ) or the indicated S / T - A substitution mutant versions of Mcm4 and Mcm6 . c ) Quantification of ( b ) , performed using ImageQuant software ( GE Healthcare ) . ( d ) Representative tetrads from diploids heterozygous for mcm4 - 25A and mcm6ΔN + 11A .

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Figure 5(a) Sld3/7 recruitment reaction performed as in Fig. 2a.(b) Replication reaction performed as in Fig. 1g. Experiments in (a) and (b) used MCM containing wild type (wt) or the indicated S/T-A substitution mutant versions of Mcm4 and Mcm6.c) Quantification of (b), performed using ImageQuant software (GE Healthcare).(d) Representative tetrads from diploids heterozygous for mcm4-25A and mcm6ΔN+11A.


Figure 6 ( b ) Immunoblots of reactions performed as in ( a ) part i ( lanes 1 - 3 ) or ii ( lanes 4 - 6 ) . ( c ) Reactions in i and ii were performed as described in ( a ) parts i and ii , respectively .

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Figure 6(b) Immunoblots of reactions performed as in (a) part i (lanes 1-3) or ii (lanes 4-6).(c) Reactions in i and ii were performed as described in (a) parts i and ii, respectively.


Figure 7 ( a , b ) Recruitment reactions performed as in Fig . 2a . Sld7 was omitted in ( b ) . ( c ) Recruitment reaction conducted as in Fig . 3a , with washes performed at 0 . 3 M KCl . ( d ) Replication reaction performed as in Fig . 1g . Experiments in ( a ) - ( d ) used MCM containing wild type ( wt ) or S / T - D substitution mutants of Mcm4 and Mcm6 as indicated . In ( d ) , MCM loading was equalised across samples by using 5 - fold more mutant than wild type MCM protein , as is shown in Fig . EV6c .

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Figure 7(a, b) Recruitment reactions performed as in Fig. 2a. Sld7 was omitted in (b).(c) Recruitment reaction conducted as in Fig. 3a, with washes performed at 0.3 M KCl.(d) Replication reaction performed as in Fig. 1g. Experiments in (a)-(d) used MCM containing wild type (wt) or S/T-D substitution mutants of Mcm4 and Mcm6 as indicated. In (d), MCM loading was equalised across samples by using 5-fold more mutant than wild type MCM protein, as is shown in Fig. EV6c.


Figure 1 ( A ) UBQLN4 associates with polyubiquitinated proteasomal substrates . Flag - tagged UBQLN4 protein was affinity - purified from extracts of HeLa cells that were treated with ( + ) or without ( - ) 20 μM MG - 132 for 4 h before harvesting , and the Flag - precipitates ( IP : Flag ) were blotted with anti - polyubiquitin ( FK2 ) , anti - BAG6 , and anti - Flag antibodies . Mock indicates empty vector transfection . Note that UBQLN4 - BAG6 co - precipitation can be observed exclusively in the presence of polyubiquitinated substrates . ( B ) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide ( CHX ) . HeLa cells expressing Flag - tagged UBQLN4 were treated with 5 μM MG - 132 as well as CHX ( either 10 or 25 μg / mL ) for 4 h as indicated , and then subjected to immunoprecipitation with an anti - Flag antibody as in ( A ) . ( D ) HeLa cells were treated as in ( C ) , and Flag - precipitates were blotted as in ( B ) .

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Figure 1(A) UBQLN4 associates with polyubiquitinated proteasomal substrates. Flag-tagged UBQLN4 protein was affinity-purified from extracts of HeLa cells that were treated with (+) or without (-) 20 μM MG-132 for 4 h before harvesting, and the Flag-precipitates (IP:Flag) were blotted with anti-polyubiquitin (FK2), anti-BAG6, and anti-Flag antibodies. Mock indicates empty vector transfection. Note that UBQLN4-BAG6 co-precipitation can be observed exclusively in the presence of polyubiquitinated substrates.(B) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide (CHX). HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 μM MG-132 as well as CHX (either 10 or 25 μg/mL) for 4 h as indicated, and then subjected to immunoprecipitation with an anti-Flag antibody as in (A).(D) HeLa cells were treated as in (C), and Flag-precipitates were blotted as in (B).


Figure 2 ( A ) UBQLN4 co - localizes with cytoplasmic aggregates after puromycin treatment . HeLa cells expressing Flag - tagged UBQLN4 were treated with 5 µg / mL puromycin for 2 h and then followed by 1 % Triton X - 100 extraction and 4 % paraformaldehyde fixation . Fixed cells were stained with anti - polyubiquitin FK2 ( shown in red to detect ubiquitin - positive cytoplasmic aggregates , ALIS ) and anti - Flag antibodies ( shown in green ) . ( B , C ) Puromycin - treated cells with UBQLN4 siRNA contain increased number of ALIS . At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 and # 2 ) or control siRNA ( 5 nM each ) , the cells were treated with 5 µg / mL puromycin for 2 h , and then subjected to immunostaining with an anti - polyubiquitin FK2 antibody ( shown in green , B ) as in ( A ) . The number of ALIS was determined . The quantified data in ( C ) represent mean ± SEM . n = 76 cells for control siRNA , n = 89 cells for UBQLN4 siRNA # 1 , and n = 81 cells for UBQLN4 siRNA # 2 . P ‐ values were calculated by Welch ' s t - test between the siRNA ‐ transfected and the respective control condition ( * P < 0 . 01 ) . ( B , C ) Puromycin - treated cells with UBQLN4 siRNA contain increased number of ALIS . At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 and # 2 ) or control siRNA ( 5 nM each ) , the cells were treated with 5 µg / mL puromycin for 2 h , and then subjected to immunostaining with an anti - polyubiquitin FK2 antibody ( shown in green , B ) as in ( A ) . The number of ALIS was determined . The quantified data in ( C ) represent mean ± SEM . n = 76 cells for control siRNA , n = 89 cells for UBQLN4 siRNA # 1 , and n = 81 cells for UBQLN4 siRNA # 2 . P ‐ values were calculated by Welch ' s t - test between the siRNA ‐ transfected and the respective control condition ( * P < 0 . 01 ) . ( D ) Depletion of UBQLN4 makes HeLa cells more sensitive to puromycin - induced cell death . After 48 h of UBQLN4 siRNA treatment , the cells were treated with 5 µg / mL puromycin for 15 . 5 h and then viability was measured . The data represent mean ± SD calculated from 3 independent experiments ( n = 3 ) . P ‐ values were calculated by Student ' s t - test compared with puromycin - treated control siRNA cells ( * P < 0 . 01 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) UBQLN4 co-localizes with cytoplasmic aggregates after puromycin treatment. HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 µg/mL puromycin for 2 h and then followed by 1% Triton X-100 extraction and 4% paraformaldehyde fixation. Fixed cells were stained with anti-polyubiquitin FK2 (shown in red to detect ubiquitin-positive cytoplasmic aggregates, ALIS) and anti-Flag antibodies (shown in green).(B, C) Puromycin-treated cells with UBQLN4 siRNA contain increased number of ALIS. At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1 and #2) or control siRNA (5 nM each), the cells were treated with 5 µg/mL puromycin for 2 h, and then subjected to immunostaining with an anti-polyubiquitin FK2 antibody (shown in green, B) as in (A). The number of ALIS was determined. The quantified data in (C) represent mean ± SEM. n = 76 cells for control siRNA, n = 89 cells for UBQLN4 siRNA#1, and n = 81 cells for UBQLN4 siRNA #2. P‐values were calculated by Welch's t-test between the siRNA‐transfected and the respective control condition (*P < 0.01).(B, C) Puromycin-treated cells with UBQLN4 siRNA contain increased number of ALIS. At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1 and #2) or control siRNA (5 nM each), the cells were treated with 5 µg/mL puromycin for 2 h, and then subjected to immunostaining with an anti-polyubiquitin FK2 antibody (shown in green, B) as in (A). The number of ALIS was determined. The quantified data in (C) represent mean ± SEM. n = 76 cells for control siRNA, n = 89 cells for UBQLN4 siRNA#1, and n = 81 cells for UBQLN4 siRNA #2. P‐values were calculated by Welch's t-test between the siRNA‐transfected and the respective control condition (*P < 0.01).(D) Depletion of UBQLN4 makes HeLa cells more sensitive to puromycin-induced cell death. After 48 h of UBQLN4 siRNA treatment, the cells were treated with 5 µg/mL puromycin for 15.5 h and then viability was measured. The data represent mean ± SD calculated from 3 independent experiments (n=3). P‐values were calculated by Student's t-test compared with puromycin-treated control siRNA cells (*P < 0.01).


Figure 3 ( B ) IL - 2Rα ΔSS mislocalized to the cytosolic fraction . HeLa cells , expressing Flag - tagged IL - 2Rα WT or ΔSS , were fractionated to the cytosolic ( Cyt . ) and membrane ( Mem . ) fractions . Tubulin was used as a cytoplasmic marker , while calnexin was used for the ER membrane fraction . ( C ) IL - 2Rα ΔSS was not glycosylated . Flag - tagged IL - 2Rα WT or ΔSS proteins was expressed in HeLa cells , and immunoprecipitated with an anti - Flag antibody . The precipitates were incubated with ( + ) or without ( - ) 10 unit of the deglycosylation enzyme PNGase F for 2 h , and subjected to western blot analysis with an anti - Flag antibody . Glycosylated and non - glycosylated signals are indicated . ( D ) The glycosylated form of IL - 2Rα WT is a stable protein , while mislocalized IL - 2Rα ΔSS is degraded rapidly via its TMD . A series of IL - 2Ra deletion proteins were expressed in HeLa cells and then chased with 20 μg / mL CHX for the indicated periods . Actin was used as a loading control . The right panel is a quantified graph of the left blot signals represent mean ± SD calculated from 4 independent experiments ( n = 4 ) . ( E ) IL - 2Rα ΔSS is degraded in a proteasome - dependent manner . HeLa cells expressing Flag - tagged IL - 2Rα ΔSS were treated with 20 μg / mL CHX as well as 0 . 1 % DMSO ( DM . ) , 10 μM MG - 132 ( MG . ) , 2 μM Bortezomib ( Bor . ) , 10 μM Leupeptin ( Leu . ) , 0 . 1 μM Bafilomycin A1 ( Baf . ) , or 10 μg / ml Pepstatin A ( pep . A ) / E64d for the indicated periods . The right panel indicates that the quantified data of the left blot signals represent mean ± SD calculated from 3 independent experiments ( n = 3 ) . ( F ) The Flag - tagged IL - 2Rα ΔSS mutant and T7 - tagged ubiquitin were co - expressed in HeLa cells and the cells were treated with ( + ) or without ( - ) 10 μM MG - 132 . After 4 h , Flag - precipitates were blotted with anti - T7 and anti - Flag antibodies . ( G ) HeLa cells expressing Flag - tagged IL - 2RaWT , ΔSS , and ΔSSΔTM proteins were treated with ( + ) or without ( - ) 10 μM MG - 132 . At 4 h after MG - 132 treatment , the cells were lysed and analyzed by immunoblotting using the indicated antibodies . Note that a higher migrating form of non - glycosylated ( and thus defective ) IL - 2Ra WT ( indicated by the arrowhead ) is detected strongly in the presence of MG - 132 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) IL-2Rα ΔSS mislocalized to the cytosolic fraction. HeLa cells, expressing Flag-tagged IL-2Rα WT or ΔSS, were fractionated to the cytosolic (Cyt.) and membrane (Mem.) fractions. Tubulin was used as a cytoplasmic marker, while calnexin was used for the ER membrane fraction.(C) IL-2Rα ΔSS was not glycosylated. Flag-tagged IL-2Rα WT or ΔSS proteins was expressed in HeLa cells, and immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F for 2 h, and subjected to western blot analysis with an anti-Flag antibody. Glycosylated and non-glycosylated signals are indicated.(D) The glycosylated form of IL-2Rα WT is a stable protein, while mislocalized IL-2Rα ΔSS is degraded rapidly via its TMD. A series of IL-2Ra deletion proteins were expressed in HeLa cells and then chased with 20 μg/mL CHX for the indicated periods. Actin was used as a loading control. The right panel is a quantified graph of the left blot signals represent mean ± SD calculated from 4 independent experiments (n=4).(E) IL-2Rα ΔSS is degraded in a proteasome-dependent manner. HeLa cells expressing Flag-tagged IL-2Rα ΔSS were treated with 20 μg/mL CHX as well as 0.1% DMSO (DM.), 10 μM MG-132 (MG.), 2 μM Bortezomib (Bor.), 10 μM Leupeptin (Leu.), 0.1 μM Bafilomycin A1 (Baf.), or 10 μg/ml Pepstatin A (pep. A)/E64d for the indicated periods. The right panel indicates that the quantified data of the left blot signals represent mean ± SD calculated from 3 independent experiments (n=3).(F) The Flag-tagged IL-2Rα ΔSS mutant and T7-tagged ubiquitin were co-expressed in HeLa cells and the cells were treated with (+) or without (-) 10 μM MG-132. After 4 h, Flag-precipitates were blotted with anti-T7 and anti-Flag antibodies.(G) HeLa cells expressing Flag-tagged IL-2RaWT, ΔSS, and ΔSSΔTM proteins were treated with (+) or without (-) 10 μM MG-132. At 4 h after MG-132 treatment, the cells were lysed and analyzed by immunoblotting using the indicated antibodies. Note that a higher migrating form of non-glycosylated (and thus defective) IL-2Ra WT (indicated by the arrowhead) is detected strongly in the presence of MG-132.


Figure 4 ( A , B ) IL - 2Rα ΔSS is stabilized in UBQLN4 - knockdown cells . HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 ~ # 3 ) or control siRNA . At 48 h after siRNA transfection , Flag tagged - IL - 2Rα ΔSS was expressed in the cells . At 24 h after IL - 2RaSS transfection , the cells were chased with 20 μg / mL CHX and harvested at the indicated time after CHX addition . Anti - Flag signals in the control or UBQLN4 siRNA - treated cells ( siRNA # 1 ~ # 3 ) were quantified at the indicated time points . The data represent mean ± SEM calculated from 4 independent experiments ( n = 4 ) . The UBQLN4 signal is marked by an arrowhead , while the band just below it ( indicated by an asterisk ) is a non - specific band , since this signal was never affected by any of the UBQLN4 siRNAs . Actin was used as a loading control . ( C ) Polyubiquitin modification of IL - 2Rα ΔSS was not diminished , but rather strengthened , in UBQLN4 knockdown cells . At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6 , Flag - tagged IL - 2Rα ΔSS and T7 - tagged ubiquitin were expressed with ( + ) or without ( - ) 10 mM MG - 132 . From these cells , Flag - tagged IL - 2Rα ΔSS was affinity - purified using Hot lysis analysis , and Flag - precipitates were blotted with an anti - T7 antibody to detect polyubiquitination of IL - 2Rα ΔSS .

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Figure 4(A, B) IL-2Rα ΔSS is stabilized in UBQLN4-knockdown cells. HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1~#3) or control siRNA. At 48 h after siRNA transfection, Flag tagged-IL-2Rα ΔSS was expressed in the cells. At 24 h after IL-2RaSS transfection, the cells were chased with 20 μg/mL CHX and harvested at the indicated time after CHX addition. Anti-Flag signals in the control or UBQLN4 siRNA-treated cells (siRNA#1~#3) were quantified at the indicated time points. The data represent mean ± SEM calculated from 4 independent experiments (n=4). The UBQLN4 signal is marked by an arrowhead, while the band just below it (indicated by an asterisk) is a non-specific band, since this signal was never affected by any of the UBQLN4 siRNAs. Actin was used as a loading control.(C) Polyubiquitin modification of IL-2Rα ΔSS was not diminished, but rather strengthened, in UBQLN4 knockdown cells. At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6, Flag-tagged IL-2Rα ΔSS and T7-tagged ubiquitin were expressed with (+) or without (-) 10 mM MG-132. From these cells, Flag-tagged IL-2Rα ΔSS was affinity-purified using Hot lysis analysis, and Flag-precipitates were blotted with an anti-T7 antibody to detect polyubiquitination of IL-2Rα ΔSS.


Figure 5 ( A ) A series of Flag - tagged truncated mutants of IL - 2Ra substrates were expressed in HeLa cells with T7 - tagged UBQLN4 and treated with ( + ) or without ( - ) 10 µM MG - 132 for 4 h . Flag - IL - 2Ra substrates were immunoprecipitated and their co - precipitation with UBQLN4 was analyzed . ( B ) T7 - UBQLN4 was immunoprecipitated , and its interactions with a series of IL - 2Ra proteins were detected .

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Figure 5(A) A series of Flag-tagged truncated mutants of IL-2Ra substrates were expressed in HeLa cells with T7-tagged UBQLN4 and treated with (+) or without (-) 10 µM MG-132 for 4 h. Flag-IL-2Ra substrates were immunoprecipitated and their co-precipitation with UBQLN4 was analyzed.(B) T7-UBQLN4 was immunoprecipitated, and its interactions with a series of IL-2Ra proteins were detected.


Figure 6 ( B ) UBQLN4 dominantly co - precipitates IL - 2Rα ΔSS over UBQLN1 . Precipitates of Flag - tagged UBQLN1 or UBQLN4 from HeLa cell lysates were probed with IL - 2Rα ΔSS . ( C ) The complete amino acid sequences of human UBQLN1 and UBQLN4 were analyzed using the disorder / order structure prediction program PONDR - FIT . The STI - II region of UBQLN4 is indicated by the red box , and that of UBQLN1 is boxed blue . Note that the disorder tendency of the UBL , STI - I , and UBA regions is nearly identical between UBQLN1 and UBQLN4 ( indicated by black and green boxes ) , while that of the STI - II region is quite distinct , and the STI - II of UBQLN4 shows a high probability of an ordered structure . The numbers denote the corresponding amino acid positions in these proteins . ( E ) Substitution of the STI - II sequence of UBQLN4 with that of UBQLN1 ( designated as the 4 - STI - II - 1 mutant protein ) abolished its high affinity to the IL - 2Rα ΔSS client protein . ( F ) Substitution of the STI - II sequence of UBQLN1 with that of UBQLN4 ( designated as the 1 - STI - II - 4 ) did not enable UBQLN1 to recognize IL - 2Rα ΔSS .

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Figure 6(B) UBQLN4 dominantly co-precipitates IL-2Rα ΔSS over UBQLN1. Precipitates of Flag-tagged UBQLN1 or UBQLN4 from HeLa cell lysates were probed with IL-2Rα ΔSS.(C) The complete amino acid sequences of human UBQLN1 and UBQLN4 were analyzed using the disorder/order structure prediction program PONDR-FIT. The STI-II region of UBQLN4 is indicated by the red box, and that of UBQLN1 is boxed blue. Note that the disorder tendency of the UBL, STI-I, and UBA regions is nearly identical between UBQLN1 and UBQLN4 (indicated by black and green boxes), while that of the STI-II region is quite distinct, and the STI-II of UBQLN4 shows a high probability of an ordered structure. The numbers denote the corresponding amino acid positions in these proteins.(E) Substitution of the STI-II sequence of UBQLN4 with that of UBQLN1 (designated as the 4-STI-II-1 mutant protein) abolished its high affinity to the IL-2Rα ΔSS client protein.(F) Substitution of the STI-II sequence of UBQLN1 with that of UBQLN4 (designated as the 1-STI-II-4) did not enable UBQLN1 to recognize IL-2Rα ΔSS.


Figure 7 ( B , C and D ) The wild - type ( WT ) form of Flag - tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7 - tagged IL - 2RαΔSS ( B and C ) . At 4 h after the addition of 10 μM MG - 132 , UBQLN4 was immunoprecipitated with an anti - Flag antibody and the precipitates were blotted with an anti - T7 antibody . ( B , C and D ) The wild - type ( WT ) form of Flag - tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7 - tagged IL - 2Rα ΔSS ( B and C ) and T7 - tagged ubiquitin ( D ) . At 4 h after the addition of 10 μM MG - 132 , UBQLN4 was immunoprecipitated with an anti - Flag antibody and the precipitates were blotted with an anti - T7 antibody . ( E ) GST - tagged and purified UBQLN4 and its truncated derivatives ( ΔUBA and ΔSTI - II ) were incubated with K48 - linked polyubiquitin chains . After in vitro GST pull - down , the precipitates were probed with an anti - polyubiquitin ( FK2 ) antibody .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2RαΔSS (B and C). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2Rα ΔSS (B and C) and T7-tagged ubiquitin (D). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.(E) GST-tagged and purified UBQLN4 and its truncated derivatives (ΔUBA and ΔSTI-II) were incubated with K48-linked polyubiquitin chains. After in vitro GST pull-down, the precipitates were probed with an anti-polyubiquitin (FK2) antibody.


Figure 8 ( A ) UBQLN4 interacts with non - glycosylated ( and thus defective ) IL - 2Ra WT in SRP54 - knockdown cells . After treatment of HeLa cells with SRP54 siRNA ( 10 nM ) for 48 h , Flag - tagged IL - 2RaWT and T7 - tagged UBQLN4 proteins were expressed , and the cells were treated with ( + ) or without ( - ) 10 µM MG - 132 at 4 h before harvesting . UBQLN4 was affinity - purified from cell extracts and probed with an anti - Flag antibody . Note that the levels of non - glycosylated IL - 2RaWT ( indicated as Defective ) increased , while the glycosylated form of this protein ( indicated as Assembled ) decreased in SRP54 knockdown cells . ( B , C ) SRP54 knockdown stimulates polyubiquitinated proteins co - precipitation of UBQLN4 . Flag - tagged UBQLN4 and T7 - tagged ubiquitin were expressed in SRP54 siRNA - treated cells , and Flag precipitates were probed with an anti - T7 antibody to detect polyubiquitinated clients co - precipitation with UBQLN4 under no addition of MG - 132 ( B ) . Note that the interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown cells treated with MG - 132 for 4 h before harvesting ( C ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) UBQLN4 interacts with non-glycosylated (and thus defective) IL-2Ra WT in SRP54-knockdown cells. After treatment of HeLa cells with SRP54 siRNA (10 nM) for 48 h, Flag-tagged IL-2RaWT and T7-tagged UBQLN4 proteins were expressed, and the cells were treated with (+) or without (-) 10 µM MG-132 at 4 h before harvesting. UBQLN4 was affinity-purified from cell extracts and probed with an anti-Flag antibody. Note that the levels of non-glycosylated IL-2RaWT (indicated as Defective) increased, while the glycosylated form of this protein (indicated as Assembled) decreased in SRP54 knockdown cells.(B, C) SRP54 knockdown stimulates polyubiquitinated proteins co-precipitation of UBQLN4. Flag-tagged UBQLN4 and T7-tagged ubiquitin were expressed in SRP54 siRNA-treated cells, and Flag precipitates were probed with an anti-T7 antibody to detect polyubiquitinated clients co-precipitation with UBQLN4 under no addition of MG-132 (B). Note that the interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown cells treated with MG-132 for 4 h before harvesting (C).


Figure 1A . Cytosolicnon - heme iron levels in wild - type mice subjected to sham or I / R procedure two days after surgery . Two - tailed unpaired T - test was performed . N = 4 mice for each group . B . Mitochondrialnon - heme iron levels in wild - type mice subjected to sham or I / R procedure two days after surgery . * P = 0 . 024 with two - tailed unpaired T - test . N = 4 mice for each group . C . Mitochondrial labile iron in H9c2 cells with or without H2O2 treatment measured using RPA fluorescence . * P < 0 . 0001 with two - tailed unpaired T - test . N = 8 independent samples for PBS group and N = 10 independent samples for H2O2 group . D . Cytosoliclabile iron in H9c2 cells with or without H2O2 treatment measured using calcein fluorescence . * P = 0 . 0042 with two - tailed unpaired T - test . N = 11 independent samples in each group . E . Mitochondrialiron in humancardiac tissue sample from non - failing hearts and from hearts with ischemic cardiomyopathy ( ISCM ) . * P = 0 . 041 with two - tailed unpaired T - test . N = 4 independent samples in each group . F . Cytosoliciron in humancardiac tissue sample from non - failing hearts and from hearts with ischemic cardiomyopathy ( ISCM ) . Two - tailed unpaired T - test was performed . N = 4 independent samples in each group . All data are expressed as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Cytosolicnon-heme iron levels in wild-type mice subjected to sham or I/R procedure two days after surgery. Two-tailed unpaired T-test was performed. N=4 mice for each group.B. Mitochondrialnon-heme iron levels in wild-type mice subjected to sham or I/R procedure two days after surgery. * P=0.024 with two-tailed unpaired T-test. N=4 mice for each group.C. Mitochondrial labile iron in H9c2 cells with or without H2O2 treatment measured using RPA fluorescence. * P<0.0001 with two-tailed unpaired T-test. N=8 independent samples for PBS group and N=10 independent samples for H2O2 group.D. Cytosoliclabile iron in H9c2 cells with or without H2O2 treatment measured using calcein fluorescence. * P=0.0042 with two-tailed unpaired T-test. N=11 independent samples in each group.E. Mitochondrialiron in humancardiac tissue sample from non-failing hearts and from hearts with ischemic cardiomyopathy (ISCM). * P=0.041 with two-tailed unpaired T-test. N=4 independent samples in each group.F. Cytosoliciron in humancardiac tissue sample from non-failing hearts and from hearts with ischemic cardiomyopathy (ISCM). Two-tailed unpaired T-test was performed. N=4 independent samples in each group. All data are expressed as mean ± SEM.


Figure 2A . Cytosolic iron in H9c2 cells preloaded with radioactive 55Fe and treated with indicated iron chelators . ANOVA followed by post - hoc Tukey test was performed . * P = 7 . 9E - 8 PBS vs . DFO . * P = 9 . 4E - 6 BPD vs . DFO . * P = 0 . 003 PBS vs . BPD . N = 4 independent samples for PBS group and N = 6 independent samples for other groups . B . Nuclear iron in H9c2 cells preloaded with radioactive 55Fe and treated with indicated iron chelators . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 0003 PBS vs . BPD . * P = 5 . 58E - 5 PBS vs . DFO . N = 4 independent samples for PBS group and N = 6 independent samples for other groups . C . Representative RPA fluorescence staining for labile mitochondrial iron in H9c2 cells with indicated iron chelator treatment . Scale bar = 100μm . D . Labile mitochondrial iron measured by RPA fluorescence in H9c2 cells with indicated iron chelator treatment . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 016 PBS vs . BPD . * P = 0 . 04 DFO vs . BPD . N = 8 independent samples for each group . E . H2O2 - induced cell death in H9c2 cells with indicated treatments . ANOVA followed by post - hoc Tukey test was performed . * P = 5E - 8 PBS - PBS vs . PBS - H2O2 . * P = 4E - 6 PBS - H2O2 vs . DFO - H2O2 . * P = 5E - 8 PBS - H2O2 vs . BPD - H2O2 . * P = 1E - 7 BPD - H2O2 vs . DFO - H2O2 . N = 6 independent samples for each group . F . Labile mitochondrial iron in H9c2 cells with indicated treatment . PBS with and without H2O2 data were copied from Fig 1C . ANOVA followed by post - hoc Tukey test was performed . * P = 1E - 8 PBS - PBS vs . PBS - H2O2 . * P = 3E - 9 PBS - H2O2 vs . BPD - H2O2 . N = 8 independent samples for PBS - PBS , and N = 12 for the other groups . All data are expressed as mean ± SEM . N . S . = not signigicant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Cytosolic iron in H9c2 cells preloaded with radioactive 55Fe and treated with indicated iron chelators. ANOVA followed by post-hoc Tukey test was performed. * P=7.9E-8 PBS vs. DFO. * P=9.4E-6 BPD vs. DFO. * P=0.003 PBS vs. BPD. N=4 independent samples for PBS group and N=6 independent samples for other groups.B. Nuclear iron in H9c2 cells preloaded with radioactive 55Fe and treated with indicated iron chelators. ANOVA followed by post-hoc Tukey test was performed. * P=0.0003 PBS vs. BPD. * P=5.58E-5 PBS vs. DFO. N=4 independent samples for PBS group and N=6 independent samples for other groups.C. Representative RPA fluorescence staining for labile mitochondrial iron in H9c2 cells with indicated iron chelator treatment. Scale bar = 100μm.D. Labile mitochondrial iron measured by RPA fluorescence in H9c2 cells with indicated iron chelator treatment. ANOVA followed by post-hoc Tukey test was performed.* P=0.016 PBS vs. BPD. * P=0.04 DFO vs. BPD. N=8 independent samples for each group.E. H2O2-induced cell death in H9c2 cells with indicated treatments. ANOVA followed by post-hoc Tukey test was performed.* P=5E-8 PBS-PBS vs. PBS-H2O2. * P=4E-6 PBS-H2O2 vs. DFO-H2O2. * P=5E-8 PBS-H2O2 vs. BPD-H2O2. * P=1E-7 BPD-H2O2 vs. DFO-H2O2. N=6 independent samples for each group.F. Labile mitochondrial iron in H9c2 cells with indicated treatment. PBS with and without H2O2 data were copied from Fig 1C. ANOVA followed by post-hoc Tukey test was performed. * P=1E-8 PBS-PBS vs. PBS-H2O2. * P=3E-9 PBS-H2O2 vs. BPD-H2O2. N=8 independent samples for PBS-PBS, and N=12 for the other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.


Figure 3A . Mitochondrial non - heme iron in ABCB8 NTG and TG mice . N = 4 mice for NTG and 6 mice for TG . * P = 0 . 021 with two - tailed unpaired T - test . B . Cytosolic non - heme iron in ABCB8 NTG and TG mice . N = 4 mice for NTG and 6 mice for TG . Two - tailed unpaired T - test was performed . C . Baseline ejection fraction of littermate ABCB8 NTG and TG mice . N = 6 mice in each group . Two - tailed unpaired T - test was performed . D . Baseline fractional shortening of littermate ABCB8 NTG and TG mice . N = 6 mice in each group . Two - tailed unpaired T - test was performed . E . Lipid peroxidation products in hearts of TG and NTG mice . N = 6 mice in each group . Two - tailed unpaired T - test was performed . F . Relative expression of antioxidant genes in NTG and TG mice . N = 6 mice in each group . Two - tailed unpaired T - test was performed . All data are expressed as mean ± SEM . N . S . = not signigicant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Mitochondrial non-heme iron in ABCB8 NTG and TG mice. N=4 mice for NTG and 6 mice for TG. * P=0.021 with two-tailed unpaired T-test.B. Cytosolic non-heme iron in ABCB8 NTG and TG mice. N=4 mice for NTG and 6 mice for TG. Two-tailed unpaired T-test was performed.C. Baseline ejection fraction of littermate ABCB8 NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed.D. Baseline fractional shortening of littermate ABCB8 NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed.E. Lipid peroxidation products in hearts of TG and NTG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed.F. Relative expression of antioxidant genes in NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed. All data are expressed as mean ± SEM. N.S. = not signigicant.


Figure 4A . Cardiac function in ABCB8 NTG and TG mice undergoing sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed for each time point . * P < 0 . 05 compared with TG - sham at the same time point . $ P < 0 . 05 compared with NTG - I / R at the same time point . § P < 0 . 05 compared with NTG - sham at the same time point . N = 5 mice for NTG - sham and NTG - I / R , and N = 6 mice for TG - sham and TG - I / R . Exact P - values are included in Appendix Table S3 . B . Representative images of TUNEL staining for mice with indicated genotype undergone indicated procedure . Scale bar = 100μm . C . Quantification of apoptosis in ABCB8 TG and NTG mice with indicated procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 035 . N = 4 mice for TG - I / R , and N = 6 mice for all other groups . D . Nppa expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 3 . 24E - 4 NTG - sham vs . NTG - I / R . * P = 0 . 002 TG - sham vs . TG - I / R . * P = 8E - 6 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham and NTG - I / R , and N = 6 mice for TG - sham and TG - I / R . E . Nppb expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P < 1E - 8 NTG - sham vs . NTG - I / R . * P = 0 . 032 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham and NTG - I / R , and N = 6 mice for TG - sham and TG - I / R . F . Myh7 expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 04 NTG - sham vs . NTG - I / R . * P = 0 . 034 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham and NTG - I / R , and N = 6 mice for TG - sham and TG - I / R . All data are expressed as mean ± SEM . N . S . = not signigicant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Cardiac function in ABCB8 NTG and TG mice undergoing sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed for each time point. * P<0.05 compared with TG-sham at the same time point. $ P<0.05 compared with NTG-I/R at the same time point. § P<0.05 compared with NTG-sham at the same time point. N=5 mice for NTG-sham and NTG-I/R, and N=6 mice for TG-sham and TG-I/R. Exact P-values are included in Appendix Table S3.B. Representative images of TUNEL staining for mice with indicated genotype undergone indicated procedure. Scale bar = 100μm.C. Quantification of apoptosis in ABCB8 TG and NTG mice with indicated procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.035. N=4 mice for TG-I/R, and N=6 mice for all other groups.D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=3.24E-4 NTG-sham vs. NTG-I/R. * P=0.002 TG-sham vs. TG-I/R. * P=8E-6 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and NTG-I/R, and N=6 mice for TG-sham and TG-I/R.E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 NTG-sham vs. NTG-I/R. * P=0.032 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and NTG-I/R, and N=6 mice for TG-sham and TG-I/R.F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.04 NTG-sham vs. NTG-I/R. * P=0.034 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and NTG-I/R, and N=6 mice for TG-sham and TG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.


Figure 5A . Representative H & amp ; amp ; E staining of peri - infarct area demonstrated reduced cellular injury in ABCB8 TG mice after I / R . Scale bar = 115μm . B . Representative H & amp ; amp ; E and Masson Trichrome ( MT ) staining in mouseheart 28 days after indicated procedure . Scale bar = 1100μm . C . Quantification of tissue fibrosis in ABCB8 NTG and TG mice subjected to I / R . * P = 0 . 047 with two - tailed unpaired T - test . 2 - 3 sections from each mice were quantified ; N = 4 mice for NTG and N = 6 mice for TG . D . Nppa expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 006 NTG - sham vs . NTG - I / R . * P = 0 . 015 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham and TG - I / R , N = 6 mice for TG - sham , and N = 4 mice for NTG - I / R . E . Nppb expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 001 NTG - sham vs . NTG - I / R . * P = 0 . 004 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham and TG - I / R , N = 6 mice for TG - sham , and N = 4 mice for NTG - I / R . F . Myh7 expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 002 NTG - sham vs . NTG - I / R . * P = 0 . 011 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham and TG - I / R , N = 6 mice for TG - sham , and N = 4 mice for NTG - I / R . All data are expressed as mean ± SEM . N . S . = not signigicant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Representative H&amp;E staining of peri-infarct area demonstrated reduced cellular injury in ABCB8 TG mice after I/R. Scale bar = 115μm.B. Representative H&amp;E and Masson Trichrome (MT) staining in mouseheart 28 days after indicated procedure. Scale bar = 1100μm.C. Quantification of tissue fibrosis in ABCB8 NTG and TG mice subjected to I/R. * P=0.047 with two-tailed unpaired T-test. 2-3 sections from each mice were quantified; N=4 mice for NTG and N=6 mice for TG.D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.006 NTG-sham vs. NTG-I/R. * P=0.015 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R.E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 NTG-sham vs. NTG-I/R. * P=0.004 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R.F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.002 NTG-sham vs. NTG-I/R. * P=0.011 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.


Figure 6A . Mitochondrial non - heme iron in wild - type mice treated with vehicle control or indicated iron chelator for 7 days . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 03 . N = 4 mice for PBS and DFO , and N = 5 mice for BPD . B . Cytosolic non - heme iron in wild - type mice treated with vehicle control or indicated iron chelator for 7 days . ANOVA followed by post - hoc Tukey test was performed . * P = 5E - 4 PBS vs . DFO . * P = 0 . 01 DFO vs . BPD . P = 0 . 06 PBS vs . DFO . N = 4 mice for PBS and DFO , and N = 5 mice for BPD . C . Nuclear non - heme iron in wild - type mice treated with vehicle control or indicated iron chelator for 7 days . N = 4 mice for PBS and DFO and N = 5 mice for BPD . D . Cardiac function in wild - type mice treated with vehicle control or indicated iron chelator for 7 days . N = 5 mice for each group . E . Cardiac function of chelator - treated mice after I / R . ANOVA followed by post - hoc Tukey test was performed for each time point . * P < 0 . 0001 compared to PBS - I / R group at the same time point . # P < 0 . 0001 compared to PBS - sham group at the same time point . Exact P - values are included in Appendix Table S3 . N = 5 micePBS - sham and BPD - I / R , N = 6 mice for all other groups . F . Representative Hematoxylin Eosin ( Hamp ; amp ; E ) and Masson Trichrome ( MT ) staining of heart sections in mice with indicated chelator treatment undergone sham or I / R . Scale bar = 1100μm . Bar graph represent quantification of tissue fibrosis . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 048 . 2 - 3 sections from each mice were quantified , N = 4 mice for PBS , N = 5 mice for DFO and N = 6 mice for BPD . G . Nppa expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 002 PBS - sham vs . PBS - I / R . * P = 0 . 02 DFO - sham vs . DFO I / R . * P = 0 . 007 PBS - I / R vs . BPD I / R . N = 6 mice for PBS - sham and DFO - sham , N = 4 mice for all other groups . H . Nppb expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 0007 PBS - sham vs . PBS - I / R . * P = 0 . 014 DFO - sham vs . DFO I / R . * P = 0 . 003 PBS - I / R vs . BPD I / R . N = 6 mice for PBS - sham and DFO - sham , N = 4 mice for all other groups . I . Myh7 expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 0001 PBS - sham vs . PBS - I / R . * P = 0 . 038 DFO - sham vs . DFO I / R . * P = 0 . 006 PBS - I / R vs . BPD I / R . N = 6 mice for PBS - sham and DFO - sham , N = 4 mice for all other groups . All data are expressed as mean ± SEM . N . S . = not signigicant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Mitochondrial non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. ANOVA followed by post-hoc Tukey test was performed. * P=0.03. N=4 mice for PBS and DFO, and N=5 mice for BPD.B. Cytosolic non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. ANOVA followed by post-hoc Tukey test was performed. * P=5E-4 PBS vs. DFO. * P=0.01 DFO vs. BPD. P=0.06 PBS vs. DFO. N=4 mice for PBS and DFO, and N=5 mice for BPD.C. Nuclear non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. N=4 mice for PBS and DFO and N=5 mice for BPD.D. Cardiac function in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. N=5 mice for each group.E. Cardiac function of chelator-treated mice after I/R. ANOVA followed by post-hoc Tukey test was performed for each time point. * P<0.0001 compared to PBS-I/R group at the same time point. # P<0.0001 compared to PBS-sham group at the same time point. Exact P-values are included in Appendix Table S3. N=5 micePBS-sham and BPD-I/R, N=6 mice for all other groups.F. Representative Hematoxylin Eosin (Hamp;amp;E) and Masson Trichrome (MT) staining of heart sections in mice with indicated chelator treatment undergone sham or I/R. Scale bar = 1100μm. Bar graph represent quantification of tissue fibrosis. ANOVA followed by post-hoc Tukey test was performed. * P=0.048. 2-3 sections from each mice were quantified, N=4 mice for PBS, N=5 mice for DFO and N=6 mice for BPD.G. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.002 PBS-sham vs. PBS-I/R. * P=0.02 DFO-sham vs. DFO I/R. * P=0.007 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.H. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P= 0.0007 PBS-sham vs. PBS-I/R. * P= 0.014 DFO-sham vs. DFO I/R. * P= 0.003 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.I. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-sham vs. PBS-I/R. * P=0.038 DFO-sham vs. DFO I/R. * P=0.006 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.


Figure 7A . Cardiac mitochondrial iron in ABCB8 KO and WT mice with indicated treatment harvested 4 weeks after the tamoxifen treatment . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 02 WT - vehicle vs . WT - BPD . * P = 0 . 001 WT - vehicle vs . ABCB8 KO - vehicle . * P = 0 . 004 ABCB8 KO - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle and N = 7 for all other groups . B . Ejection fraction in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout . ANOVA followed by post - hoc Tukey test was performed . * P < 1E - 8 WT - vehicle vs . ABCB8 KO - vehicle . * P < 1E - 8 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle and N = 7 for all other groups . C . Fractional shortening in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout . ANOVA followed by post - hoc Tukey test was performed . * P < 1E - 8 WT - vehicle vs . ABCB8 KO - vehicle . * P < 1E - 8 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle and N = 7 for all other groups . D . Nppa expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 017 WT - vehicle vs . ABCB8 KO - vehicle . * P = 0 . 01 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle and N = 7 for all other groups . E . Nppb expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 001 WT - vehicle vs . ABCB8 KO - vehicle . * P = 0 . 0003 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle and N = 7 for all other groups . F . Myh7 expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 011 WT - vehicle vs . ABCB8 KO - vehicle . * P = 0 . 045 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle and N = 7 for all other groups . All data are expressed as mean ± SEM . N . S . = not signigicant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Cardiac mitochondrial iron in ABCB8 KO and WT mice with indicated treatment harvested 4 weeks after the tamoxifen treatment. ANOVA followed by post-hoc Tukey test was performed. * P=0.02 WT-vehicle vs. WT-BPD. * P= 0.001 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.004 ABCB8 KO-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.B. Ejection fraction in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 WT-vehicle vs. ABCB8 KO-vehicle. * P<1E-8 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.C. Fractional shortening in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 WT-vehicle vs. ABCB8 KO-vehicle. * P<1E-8 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.017 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.01 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.0003 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.011 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.045 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.


Figure 8A . Representative western blot demonstrating ABCB8 overexpression in H9c2 cells . EV = empty vector . N = 3 independent samples for each group . B . Complex I ROS production in mitochondria with ABCB8 downregulation with or without rotenone . ANOVA followed by post - hoc Tukey test was performed . N = 6 independent samples for each group . C . Complex III ROS production in mitochondria with ABCB8 downregulation with or without antimycin A . ANOVA followed by post - hoc Tukey test was performed . N = 6 independent samples for each group . D . Complex I ROS production in mitochondria with ABCB8 overexpression with or without rotenone . EV = empty vector . ANOVA followed by post - hoc Tukey test was performed . N = 9 independent samples for EV - baseline and N = 8 independent samples for the other groups . E . Complex IIIROS production in mitochondria with ABCB8 overexpression with or without antimycin A . EV = empty vector . ANOVA followed by post - hoc Tukey test was performed . N = 9 independent samples for EV - baseline and N = 8 independent samples for the other groups . F . Mitochondrial ROS in H9c2 cells with various treatments with or without oxidative stress . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 2E - 8 PBS - PBS vs . PBS - H2O2 . * P = 2E - 8 DFO - H2O2 vs . BPD - H2O2 . G . Mitochondrial membrane potential as measured by TMRE intensity in H9c2 cells treated with indicated iron chelator with or without oxidative stress . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 1E - 8 PBS - PBS vs . PBS - H2O2 . * P = 1 . 8E - 8 DFO - PBS vs . DFO - H2O2 . H . The levels of lipid peroxidation products in ABCB8 KO mice with or without chelator treatment . N = 4 mice for WT - vehicle , N = 6 mice for ABCB8 KO - vehicle , and N = 5 mice for all other groups . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 004 WT - vehicle vs . ABCB8 KO - vehicle . * P = 1E - 6 ABCB8 KO - vehicle vs . ABCB8 KO - BPD . I . Lipid peroxidation products in ABCB8 TG and NTG mice two days after I / R . N = 6 mice for NTG - sham , and N = 4 mice for the other groups . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 024 NTG - sham vs . NTG - I / R . * P = 0 . 016 NTG - I / R vs . TG - I / R . All data are expressed as mean ± SEM . N . S . = not signigicant .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. Representative western blot demonstrating ABCB8 overexpression in H9c2 cells. EV = empty vector. N=3 independent samples for each group.B. Complex I ROS production in mitochondria with ABCB8 downregulation with or without rotenone. ANOVA followed by post-hoc Tukey test was performed. N= 6 independent samples for each group.C. Complex III ROS production in mitochondria with ABCB8 downregulation with or without antimycin A. ANOVA followed by post-hoc Tukey test was performed. N= 6 independent samples for each group.D. Complex I ROS production in mitochondria with ABCB8 overexpression with or without rotenone. EV= empty vector. ANOVA followed by post-hoc Tukey test was performed. N=9 independent samples for EV-baseline and N=8 independent samples for the other groups.E. Complex IIIROS production in mitochondria with ABCB8 overexpression with or without antimycin A. EV= empty vector. ANOVA followed by post-hoc Tukey test was performed. N=9 independent samples for EV-baseline and N=8 independent samples for the other groups.F. Mitochondrial ROS in H9c2 cells with various treatments with or without oxidative stress. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=2E-8 PBS-PBS vs. PBS-H2O2. * P=2E-8 DFO- H2O2 vs. BPD-H2O2.G. Mitochondrial membrane potential as measured by TMRE intensity in H9c2 cells treated with indicated iron chelator with or without oxidative stress. N= 6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=1E-8 PBS-PBS vs. PBS-H2O2. * P=1.8E-8 DFO-PBS vs. DFO-H2O2.H. The levels of lipid peroxidation products in ABCB8 KO mice with or without chelator treatment. N=4 mice for WT-vehicle, N=6 mice for ABCB8 KO-vehicle, and N=5 mice for all other groups. ANOVA followed by post-hoc Tukey test was performed. * P=0.004 WT-vehicle vs. ABCB8 KO-vehicle. * P=1E-6 ABCB8 KO-vehicle vs. ABCB8 KO-BPD.I. Lipid peroxidation products in ABCB8 TG and NTG mice two days after I/R. N=6 mice for NTG-sham, and N=4 mice for the other groups. ANOVA followed by post-hoc Tukey test was performed. * P=0.024 NTG-sham vs. NTG-I/R. * P=0.016 NTG-I/R vs. TG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.


Figure 9A . Mitochondrial aconitase activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2 . N = 4 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 0004 EV - PBS vs . EV - H2O2 . * P = 0 . 0001 EV - H2O2 vs . ABCB8 - H2O2 . B . Complex I activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 0005 EV - PBS vs . EV - H2O2 . * P = 0 . 00001 EV - H2O2 vs . ABCB8 - H2O2 . C . Complex II activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 001 EV - PBS vs . EV - H2O2 . * P = 0 . 001 EV - H2O2 vs . ABCB8 - H2O2 . D . Complex IV activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 0008 EV - PBS vs . EV - H2O2 . * P = 0 . 0006 EV - H2O2 vs . ABCB8 - H2O2 . E . Mitochondrial aconitase activity in H9c2 cells treated with indicated iron chelators with or without H2O2 . N = 4 independent samples in each group . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 036 PBS - PBS vs . PBS - H2O2 . * P = 0 . 0047 DFO - PBS vs . DFO - H2O2 . * P = 0 . 035 PBS - H2O2 vs . BPD - H2O2 . F . Complex I activity in H9c2 cells treated with indicated iron chelators with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 0004 PBS - PBS vs . PBS - H2O2 . * P = 0 . 0003 DFO - PBS vs . DFO - H2O2 . * P < 0 . 0001 PBS - H2O2 vs . BPD - H2O2 . G . Complex II activity in H9c2 cells treated with indicated iron chelators with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 0001 PBS - PBS vs . PBS - H2O2 . * P < 0 . 0001 DFO - PBS vs . DFO - H2O2 . * P < 0 . 0001 PBS - H2O2 vs . BPD - H2O2 . H . Complex IV activity in H9c2 cells treated with indicated iron chelators with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey test was performed . * P = 0 . 02 PBS - PBS vs . PBS - H2O2 . * P = 0 . 0009 DFO - PBS vs . DFO - H2O2 . * P = 0 . 006 PBS - H2O2 vs . BPD - H2O2 . All data are expressed as mean ± SEM .

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Figure 9A. Mitochondrial aconitase activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=4 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0004 EV-PBS vs. EV-H2O2. * P=0.0001 EV-H2O2 vs. ABCB8-H2O2.B. Complex I activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0005 EV-PBS vs. EV-H2O2. * P=0.00001 EV-H2O2 vs. ABCB8-H2O2.C. Complex II activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 EV-PBS vs. EV-H2O2. * P=0.001 EV-H2O2 vs. ABCB8-H2O2.D. Complex IV activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0008 EV-PBS vs. EV-H2O2. * P=0.0006 EV-H2O2 vs. ABCB8-H2O2.E. Mitochondrial aconitase activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=4 independent samples in each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.036 PBS-PBS vs. PBS-H2O2. * P=0.0047 DFO-PBS vs. DFO-H2O2. * P=0.035 PBS- H2O2 vs. BPD-H2O2.F. Complex I activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0004 PBS-PBS vs. PBS-H2O2. * P=0.0003 DFO-PBS vs. DFO-H2O2. * P<0.0001 PBS- H2O2 vs. BPD-H2O2.G. Complex II activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-PBS vs. PBS-H2O2. * P<0.0001 DFO-PBS vs. DFO-H2O2. * P<0.0001 PBS- H2O2 vs. BPD-H2O2.H. Complex IV activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.02 PBS-PBS vs. PBS-H2O2. * P=0.0009 DFO-PBS vs. DFO-H2O2. * P=0.006 PBS- H2O2 vs. BPD-H2O2. All data are expressed as mean ± SEM.


Figure 1Restriction enzyme diagnostic test for the presence of the R749H mutation . qRT - PCR with primers specific to both wild - type and mutant RNAPII ( left panel ) or to the mutant form of RNAPII ( right panel ) , confirming that only the slow version of RNAPII is expressed in homozygous slow / slow ESCs . The sequences of the respective forward primers are shown . The " WT / slow allele " primer is complementary to the sequence in exon 14 upstream of the mutation . The " slow allele only " primer has its 3 ' end matching the mutated codon 749 and does not anneal to the WT DNA sequence . The mean ± SEM is plotted , n = 2 .

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Figure 1Restriction enzyme diagnostic test for the presence of the R749H mutation.qRT-PCR with primers specific to both wild-type and mutant RNAPII (left panel) or to the mutant form of RNAPII (right panel), confirming that only the slow version of RNAPII is expressed in homozygous slow/slow ESCs. The sequences of the respective forward primers are shown. The "WT/slow allele" primer is complementary to the sequence in exon 14 upstream of the mutation. The "slow allele only" primer has its 3' end matching the mutated codon 749 and does not anneal to the WT DNA sequence. The mean ± SEM is plotted, n=2.


Figure 3Meta - gene profile of normalized 4sU - DRB - seq reads in WT / WT and slow / slow ESCs . Density and violin plot of elongation rate ( bases / min ) calculated for genes common in all genotypes in WT / WT , WT / slow and slow / slow ESCs .

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Figure 3Meta-gene profile of normalized 4sU-DRB-seq reads in WT/WT and slow/slow ESCs.Density and violin plot of elongation rate (bases/min) calculated for genes common in all genotypes in WT/WT, WT/slow and slow/slow ESCs. Box


Figure 4Brightfield images and analysis of NPC markers by immunofluorescence staining ( Sox1 and Nestin ) or RT - qPCR ( Nestin and Pax6 ) ( n = 3 , mean ± SEM ) . Brightfield images of aggregates . Brightfield images and immunofluorescence staining for Nestin and neuronal marker Tuj1 in NSC cultures grown in EGF / FGF proliferating conditions . White arrows indicate NSCs and green arrows differentiated cells . Two small panels on the right are examples of Tuj1 + neuronal cells in slow / slow NSCs cultures . Immunofluorescence staining for neuronal marker Tuj1 in neuronal cultures grown on poly - ornithine / laminin at 21 days of differentiation .

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Figure 4Brightfield images and analysis of NPC markers by immunofluorescence staining (Sox1 and Nestin) or RT-qPCR (Nestin and Pax6) (n=3, mean ±SEM).Brightfield images of aggregates.Brightfield images and immunofluorescence staining for Nestin and neuronal marker Tuj1 in NSC cultures grown in EGF/FGF proliferating conditions. White arrows indicate NSCs and green arrows differentiated cells. Two small panels on the right are examples of Tuj1+ neuronal cells in slow/slow NSCs cultures.Immunofluorescence staining for neuronal marker Tuj1 in neuronal cultures grown on poly-ornithine/laminin at 21 days of differentiation.


Figure 5RT - PCR analysis validation of selected alternatively spliced exons . RT - PCR was performed on total RNA from WT or slow / slow ESCs , NPCs or neurons . PCR products were visualized and quantified by Bioanalyzer ( Agilent ) . Images are representative of experiments performed in triplicate . Themean ± SEMis plotted with * p - value < 0 . 05 , * * p < 0 . 005 , * * * < 0 . 0001 as determined by t - test .

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Figure 5RT-PCR analysis validation of selected alternatively spliced exons. RT-PCR was performed on total RNA from WT or slow/slow ESCs, NPCs or neurons. PCR products were visualized and quantified by Bioanalyzer (Agilent). Images are representative of experiments performed in triplicate. Themean ± SEMis plotted with *p-value<0.05, **p<0.005, ***<0.0001 as determined by t-test.


Figure 6Box plot showing the length of upregulated ( UP ) , downregulated ( DOWN ) , not affected genes ( ALL ) , and pre - mRNAs affected by alternative splicing ( AE , for Alternative exons ) for ESCs ( left panel ) ; length of upregulated ( UP ) , downregulated ( DOWN ) , not affected genes ( ALL ) , pre - mRNAs affected by alternative splicing ( AE , for Alternative exons ) , and not affected genes that are only expressed in neurons ( but not in ESCs ) ( ALL _ notESC ) , for Neurons ( right panel ) . n . s . , p - value > 0 . 05 , * * p < 0 . 005 ; * * * * p < 0 . 0001 as determined by Mann - Whitney t - test . Boxes delimit the first and third quartiles . The horizontal lines represent the data medians . Whiskers are drawn down to the 1st and 99th percentile . Percentage of genes that are downregulated in ESCs and neurons ; plotted as a sliding window of 100 genes by length .

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Figure 6Box plot showing the length of upregulated (UP), downregulated (DOWN), not affected genes (ALL), and pre-mRNAs affected by alternative splicing (AE, for Alternative exons) for ESCs (left panel); length of upregulated (UP), downregulated (DOWN), not affected genes (ALL), pre-mRNAs affected by alternative splicing (AE, for Alternative exons), and not affected genes that are only expressed in neurons (but not in ESCs) (ALL_notESC), for Neurons (right panel). n.s., p-value>0.05, **p<0.005; ****p<0.0001 as determined by Mann-Whitney t-test. Boxes delimit the first and third quartiles. The horizontal lines represent the data medians. Whiskers are drawn down to the 1st and 99th percentile. Percentage of genes that are downregulated in ESCs and neurons; plotted as a sliding window of 100 genes by length.


Figure 1 ( B ) Immunofluorescence ( IF ) validation of mintbody specificity . Mouse MC12 cells , which stably express H3K27me3 - mintbody ( sfGFP ) , are labelled with antibodies specific for H3K27me3 ( Cy5 ) and H3K9me3 ( Cy3 ) . DNA is stained with Hoechst33342 . Single confocal sections are shown . Arrows mark Xi . Scale bar = 10 μm .

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Figure 1(B) Immunofluorescence (IF) validation of mintbody specificity. Mouse MC12 cells, which stably express H3K27me3-mintbody (sfGFP), are labelled with antibodies specific for H3K27me3 (Cy5) and H3K9me3 (Cy3). DNA is stained with Hoechst33342. Single confocal sections are shown. Arrows mark Xi. Scale bar = 10 μm.


Figure 2 ( D ) ESCs as in ( B ) were engineered to express H3K27me3 - mintbody ( SNAP / JF646 ) and H4K20me1 - mintbody ( mCherry ) . Cells were cultured in the absence of LIF for 2 days . Time - lapse images for 12 z - plane confocal stack were acquired every 1 h . Maximum intensity projection images are shown with elapse time ( hh : mm ) . Arrowheads mark Xi . Scale bar = 10

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Figure 2(D) ESCs as in (B) were engineered to express H3K27me3-mintbody (SNAP/JF646) and H4K20me1-mintbody (mCherry). Cells were cultured in the absence of LIF for 2 days. Time-lapse images for 12 z-plane confocal stack were acquired every 1 h. Maximum intensity projection images are shown with elapse time (hh:mm). Arrowheads mark Xi. Scale bar = 10 μm


Figure 4 ( E ) H4K20me1 ( blue ) and H3K27me3 ( red ) accumulation across the Xi after 12 h of DOX treatment . The black line is a locally estimated scatterplot smoothing ( LOESS ) regression on all 10 - kb windows ( dots ) . Below each plot shown is the Xist locus ( green bar ) and Xist entry sites ( black bars ) .

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Figure 4(E) H4K20me1 (blue) and H3K27me3 (red) accumulation across the Xi after 12 h of DOX treatment. The black line is a locally estimated scatterplot smoothing (LOESS) regression on all 10-kb windows (dots). Below each plot shown is the Xist locus (green bar) and Xist entry sites (black bars).


Figure 5 ( A ) Genome browser tracks showing H3K27me3 ( top ) and H4K20me1 ( bottom ) accumulation at a gene silenced rapidly ( Rnf12 ) or more slowly ( Pgk1 ) . Allele - specific tracks were overlaid ( B6 in red and Cast in blue ) . Note strong H4K20me1 bi - allelic pre - marking at gene bodies . ( B ) Average H3K27me3 ( top ) and H4K20me1 ( bottom ) enrichment at the B6 allele over initially active or inactive genes ± 30kb at the X chromosome . Shown is data for all time points . Data information : TSS : transcription start site ; TES : transcript end site .

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Figure 5(A) Genome browser tracks showing H3K27me3 (top) and H4K20me1 (bottom) accumulation at a gene silenced rapidly (Rnf12) or more slowly (Pgk1). Allele-specific tracks were overlaid (B6 in red and Cast in blue). Note strong H4K20me1 bi-allelic pre-marking at gene bodies.(B) Average H3K27me3 (top) and H4K20me1 (bottom) enrichment at the B6 allele over initially active or inactive genes ± 30kb at the X chromosome. Shown is data for all time points. Data information: TSS : transcription start site ; TES : transcript end site.


Figure 6 ( B ) Representative image of IF / RNA FISH for Xist RNA and H4K20me1 in cells expressing XistFL or XistΔBC . Arrowheads point to the Xist RNA domains . Scale bar = 5 μm . ( C ) Graph represents the mean % + / - StDev of Xist RNA domains enriched for H4K20me1 ( left ) or H3K27me3 ( right ) in cells expressing XistWT or XistΔBC . Shown are averages from at least 2 independent experiments ; minimum of 50 Xist RNA domains were counted per experiment ; only P - values corresponding to significant differences from unpaired Student ' s t - test comparing mutants to Xist FL are indicated as * ( P - value < 0 . 05 ) . Data for H3K27me3 was extracted from published data ( Bousard et al . , 2019 ) .

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Figure 6(B) Representative image of IF/RNA FISH for Xist RNA and H4K20me1 in cells expressing XistFL or XistΔBC. Arrowheads point to the Xist RNA domains. Scale bar = 5 μm.(C) Graph represents the mean % +/- StDev of Xist RNA domains enriched for H4K20me1 (left) or H3K27me3 (right) in cells expressing XistWT or XistΔBC. Shown are averages from at least 2 independent experiments; minimum of 50 Xist RNA domains were counted per experiment; only P-values corresponding to significant differences from unpaired Student's t-test comparing mutants to Xist FL are indicated as * (P-value < 0.05). Data for H3K27me3 was extracted from published data (Bousard et al., 2019).


Figure 1 ( A ) Differential interference contrast micrograph of live T . agilis cell . The arrow points to the cell anterior , where the rostrum is located ; arrowheads point to some of the flagella . ( B ) Transmission electron micrographs of T . agilis centriole embedded in resin - longitudinal view ; the hub ( arrowhead ) is visible in the cartwheel - bearing region ( pink line ) , but not in the distal region ( white line ) . ( C ) Transmission electron micrographs of T . agilis centriole embedded in resin in transverse view from distal end ( left ) and corresponding image circularized and symmetrized with the CentrioleJ plugin ( middle ) , with schematic indicating principal architectural elements ( right ) .

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Figure 1(A) Differential interference contrast micrograph of live T. agilis cell. The arrow points to the cell anterior, where the rostrum is located; arrowheads point to some of the flagella.(B) Transmission electron micrographs of T. agilis centriole embedded in resin - longitudinal view; the hub (arrowhead) is visible in the cartwheel-bearing region (pink line), but not in the distal region (white line).(C) Transmission electron micrographs of T. agilis centriole embedded in resin in transverse view from distal end (left) and corresponding image circularized and symmetrized with the CentrioleJ plugin (middle), with schematic indicating principal architectural elements (right).


Figure 2 ( A ) Transverse 2D slices through T . spp . central cartwheel STA at indicated height from proximal ( 0 nm ) to distal ( 8 . 7 nm ) . The dashed box indicates corresponding region shown in ( C ) . ( B ) Transverse view of T . spp . central cartwheel STA 3D map ; 9 spoke densities emanate from the hub and the CID is present within the hub . The diameter of the hub is 23 . 0 ± 0 nm ( N = 3 ; here and thereafter in the figure legends , N corresponds to 2D measurements from STA ) . An electron dense structure is present inside the CID ( grey ) , which is also visible to some extent before symmetrizing . Note that the coloring of elements in this and other figure panels is merely illustrative and not meant to reflect underlying molecular boundaries . ( C ) 2D longitudinal view of T . spp . central cartwheel STA delineated by a dashed box in ( A ) . Arrowheads denote position of line scans along the vertical axis at the level of the CID ( purple ) and hub ( blue ) , with corresponding pixel intensities in arbitrary units ( AU ) . Plot profiles are shifted horizontally relative to each other for better visibility . Some maxima are highlighted with dashed lines ; the average distance between two CID elements is 8 . 5 ± 0 . 2 nm ( N = 4 ) . Note that hub densities are elongated in the vertical direction , resulting in broad peak profiles where two maxima can be discerned ( dashed blue lines ) . ( D ) Longitudinal view of T . spp . central cartwheel STA . The average distance between emanating spokes is 8 . 0 ± 1 . 5 nm ( N = 2 ) ; these are measurements on STA and are within the error of the CID periodicities reported ( C ) ; the same applies for other measurements hereafter . Discontinuous densities in the center of the CID ( grey ) are visible , as well as densities that vertically bridge successive hub elements ( arrows ) . Boxed area is shown magnified in ( E ) . Proximal is down in this and all other figure panels showing STA longitudinal views . ( E ) The CID axis is located distal relative to the axis of spoke densities , corresponding to an average shift of 0 . 9 ± 0 . 7 nm ( N = 3 ) . ( F ) Transverse 2D slice through T . agilis central cartwheel STA at indicated height from proximal ( 0 nm ) to distal ( 12 nm ) . The dashed box indicates corresponding region shown in ( H ) . ( G ) Transverse view of T . agilis central cartwheel STA 3D map ; 9 spoke densities emanate from the hub and the CID is present within the hub , which has a diameter of 22 . 7 ± 0 . 2 nm ( N = 3 ) . ( H ) 2D longitudinal view of T . agilis central cartwheel STA delineated by a dashed box in ( F ) . Arrowheads denote position of line scans along the vertical axis at the level of the CID ( dark pink ) and hub ( light pink ) , with corresponding pixel intensities in arbitrary units ( AU ) . Plot profiles are shifted horizontally relative to each other for better visibility . Some maxima are highlighted with dashed lines ; the average distance between two CID elements is 8 . 3 ± 0 . 5 nm ( N = 9 ) . Note that hub densities are elongated in the vertical direction , resulting in broad peak profiles where two maxima can be discerned ( dashed light pink lines ) . ( I ) Longitudinal view of T . agilis central cartwheel STA . The average distance between emanating spokes is 8 . 2 ± 0 . 5 nm ( N = 9 ) . Discontinuous densities in the center of the CID ( grey ) are visible at this lower contour level compared to ( G ) . Note densities vertically bridging successive hub elements ( arrows ) . Boxed area is shown magnified in ( J ) . ( J ) The CID axis is located distal relative to axis of spoke densities , corresponding to an average shift of 0 . 8 ± 0 . 3 nm ( N = 9 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Transverse 2D slices through T. spp. central cartwheel STA at indicated height from proximal (0 nm) to distal (8.7 nm). The dashed box indicates corresponding region shown in (C). (B) Transverse view of T. spp. central cartwheel STA 3D map; 9 spoke densities emanate from the hub and the CID is present within the hub. The diameter of the hub is 23.0 ± 0 nm (N=3; here and thereafter in the figure legends, N corresponds to 2D measurements from STA). An electron dense structure is present inside the CID (grey), which is also visible to some extent before symmetrizing. Note that the coloring of elements in this and other figure panels is merely illustrative and not meant to reflect underlying molecular boundaries. (C) 2D longitudinal view of T. spp. central cartwheel STA delineated by a dashed box in (A). Arrowheads denote position of line scans along the vertical axis at the level of the CID (purple) and hub (blue), with corresponding pixel intensities in arbitrary units (AU). Plot profiles are shifted horizontally relative to each other for better visibility. Some maxima are highlighted with dashed lines; the average distance between two CID elements is 8.5 ± 0.2 nm (N=4). Note that hub densities are elongated in the vertical direction, resulting in broad peak profiles where two maxima can be discerned (dashed blue lines). (D) Longitudinal view of T. spp. central cartwheel STA. The average distance between emanating spokes is 8.0 ± 1.5 nm (N=2); these are measurements on STA and are within the error of the CID periodicities reported (C); the same applies for other measurements hereafter. Discontinuous densities in the center of the CID (grey) are visible, as well as densities that vertically bridge successive hub elements (arrows). Boxed area is shown magnified in (E). Proximal is down in this and all other figure panels showing STA longitudinal views. (E) The CID axis is located distal relative to the axis of spoke densities, corresponding to an average shift of 0.9 ± 0.7 nm (N=3) . (F) Transverse 2D slice through T. agilis central cartwheel STA at indicated height from proximal (0 nm) to distal (12 nm). The dashed box indicates corresponding region shown in (H). (G) Transverse view of T. agilis central cartwheel STA 3D map; 9 spoke densities emanate from the hub and the CID is present within the hub, which has a diameter of 22.7 ± 0.2 nm (N=3). (H) 2D longitudinal view of T. agilis central cartwheel STA delineated by a dashed box in (F). Arrowheads denote position of line scans along the vertical axis at the level of the CID (dark pink) and hub (light pink), with corresponding pixel intensities in arbitrary units (AU). Plot profiles are shifted horizontally relative to each other for better visibility. Some maxima are highlighted with dashed lines; the average distance between two CID elements is 8.3 ± 0.5 nm (N=9). Note that hub densities are elongated in the vertical direction, resulting in broad peak profiles where two maxima can be discerned (dashed light pink lines). (I) Longitudinal view of T. agilis central cartwheel STA. The average distance between emanating spokes is 8.2 ± 0.5 nm (N=9). Discontinuous densities in the center of the CID (grey) are visible at this lower contour level compared to (G). Note densities vertically bridging successive hub elements (arrows). Boxed area is shown magnified in (J). (J) The CID axis is located distal relative to axis of spoke densities, corresponding to an average shift of 0.8 ± 0.3 nm (N=9).