( 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 .

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(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.


( 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 .

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

(A, 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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(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.


( 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 .

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(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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(B) T7-UBQLN4 was immunoprecipitated, and its interactions with a series of IL-2Ra proteins were detected.


( 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 .

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(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 .

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(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 .

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(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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(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.


( 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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 ) .

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(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).


A . 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 .

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A. 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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

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 .

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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.


A . Representative western blot demonstrating ABCB8 overexpression in H9c2 cells . EV = empty vector . N = 3 independent samples for each group .

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A. 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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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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.


Restriction enzyme diagnostic test for the presence of the R749H mutation .

Restriction 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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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.


Meta - gene profile of normalized 4sU - DRB - seq reads in WT / WT and slow / slow ESCs .

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Meta-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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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


Brightfield images and analysis of NPC markers by immunofluorescence staining ( Sox1 and Nestin ) or RT - qPCR ( Nestin and Pax6 ) ( n = 3 , mean ± SEM ) .

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Brightfield 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 .

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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 .

Immunofluorescence staining for neuronal marker Tuj1 in neuronal cultures grown on poly-ornithine/laminin at 21 days of differentiation.


RT - 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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RT-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.


Box 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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Box 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.


( 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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(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.


( 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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(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


( 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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(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).


( 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 .

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(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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(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.


( 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 .

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(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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(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).


( 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 .

(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 ) .

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(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 ) .

(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).


( 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 ) .

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

(A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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).


( A ) ( Top ) 2D slice through STA transverse view of T . spp . microtubule triplet , with insets showing position of pinhead ( dashed green box ) and A - C linker ( dashed red box ) . ( Bottom ) Longitudinal 2D slice of STA centered on the pinhead ( left ) or A - C linker ( right ) . ( B ) Transverse view of T . spp . microtubule triplet STA . Microtubule protofilament numbers are indicated , as are the pinhead and A - C linker ( only the C - link is visible ; the A - link lies on the edge of the volume and is thus less well resolved in this STA - for better view see STA centered on A - C linker in ( D ) ) . Prominent microtubule inner densities within the A - microtubule are highlighted ( empty arrowhead next to A9 , chevron next to A5 ) , as are additional external densities at the A - B and B - C inner junctions ( black arrowheads ) . Double arrowheads point to viewing direction in indicated panels . ( C ) Longitudinal view of T . spp . STA centered on the pinhead from the viewing point indicated in ( B ) . The pinfeet ( PinF1 and PinF2 ) and pinbody ( PinB ) are indicated , as are microtubule protofilaments A3 and A4 . The average distance between pinfeet elements is 8 . 6 ± 0 . 4 nm and 7 . 9 ± 0 . 4 nm ( N = 3 each ) . Corresponding transverse views are shown below , illustrating the connection of PinF2 with protofilament A3 . ( D ) Longitudinal view of T . spp . STA centered on the A - C linker from the viewing point indicated in ( B ) . Microtubule protofilaments A8 / 9 and C9 / C10 of two adjacent triplets are indicated , as are the connected A - and C - links . The average distance between A - and C - links is 8 . 4 ± 0 . 4 nm and 8 . 4 ± 0 . 3 nm ( both N = 6 ) . Corresponding transverse views are shown below , chevrons point to connection .

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

(A) (Top) 2D slice through STA transverse view of T. spp. microtubule triplet, with insets showing position of pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker (right). (B) Transverse view of T. spp. microtubule triplet STA. Microtubule protofilament numbers are indicated, as are the pinhead and A-C linker (only the C-link is visible; the A-link lies on the edge of the volume and is thus less well resolved in this STA -for better view see STA centered on A-C linker in (D)). Prominent microtubule inner densities within the A-microtubule are highlighted (empty arrowhead next to A9, chevron next to A5), as are additional external densities at the A-B and B-C inner junctions (black arrowheads). Double arrowheads point to viewing direction in indicated panels. (C) Longitudinal view of T. spp. STA centered on the pinhead from the viewing point indicated in (B). The pinfeet (PinF1 and PinF2) and pinbody (PinB) are indicated, as are microtubule protofilaments A3 and A4. The average distance between pinfeet elements is 8.6 ± 0.4 nm and 7.9 ± 0.4 nm (N=3 each). Corresponding transverse views are shown below, illustrating the connection of PinF2 with protofilament A3. (D) Longitudinal view of T. spp. STA centered on the A-C linker from the viewing point indicated in (B). Microtubule protofilaments A8/9 and C9/C10 of two adjacent triplets are indicated, as are the connected A- and C-links. The average distance between A- and C-links is 8.4 ± 0.4 nm and 8.4 ± 0.3 nm (both N=6). Corresponding transverse views are shown below, chevrons point to connection.


( E ) ( Top ) 2D slice through STA transverse view of T . agilis microtubule triplet , with insets showing position of pinhead ( dashed green box ) and A - C linker ( dashed red box ) . ( Bottom ) Longitudinal 2D slice of STA centered on the pinhead ( left ) or A - C linker ( right ) . ( F ) Transverse view of T . agilis microtubule triplet STA . Microtubule protofilament numbers are indicated , as are the pinhead and A - C linker ( only the C - link is visible ; the A - link lies on the edge of the volume and is thus less well resolved in this STA - for better view see STA centered on A - C linker in ( H ) ) . Prominent microtubule inner densities ( MIPs ) within the A - microtubule are highlighted ( empty arrowhead next to A9 ) , as are additional external densities at the A - B and B - C inner junctions ( black arrowheads ) . Double arrowheads point to viewing direction in indicated panels . ( G ) Longitudinal view of T . agilis STA centered on the pinhead from the viewing point indicated in ( F ) . The pinfeet ( PinF1 and PinF2 ) and pinbody ( PinB ) are indicated , as are microtubule protofilaments A3 / A4 . The average distance between pinfeet elements is 8 . 4 ± 0 nm in each case ( both N = 3 ) . Corresponding transverse views are shown below , illustrating the connection of PinF2 with protofilament A3 . ( H ) Longitudinal view of T . agilis STA centered on the A - C linker from the viewing point indicated in ( F ) . Microtubule protofilaments A8 / 9 and C9 / C10 of two adjacent triplets are indicated , as are the connected A - and C - links . The average distance between A - and C - links is 8 . 4 ± 0 . 3 nm and 8 . 4 ± 0 . 9 nm ( N = 6 and N = 5 , respectively ) . Corresponding transverse views are shown below , chevrons point to connection ; the connection of the A - link with A9 is only partially visible in the transverse view at this height , as indicated by the dashed chevron .

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

(E) (Top) 2D slice through STA transverse view of T. agilis microtubule triplet, with insets showing position of pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker (right). (F) Transverse view of T. agilis microtubule triplet STA. Microtubule protofilament numbers are indicated, as are the pinhead and A-C linker (only the C-link is visible; the A-link lies on the edge of the volume and is thus less well resolved in this STA -for better view see STA centered on A-C linker in (H)). Prominent microtubule inner densities (MIPs) within the A-microtubule are highlighted (empty arrowhead next to A9), as are additional external densities at the A-B and B-C inner junctions (black arrowheads). Double arrowheads point to viewing direction in indicated panels. (G) Longitudinal view of T. agilis STA centered on the pinhead from the viewing point indicated in (F). The pinfeet (PinF1 and PinF2) and pinbody (PinB) are indicated, as are microtubule protofilaments A3/A4. The average distance between pinfeet elements is 8.4 ± 0 nm in each case (both N=3). Corresponding transverse views are shown below, illustrating the connection of PinF2 with protofilament A3. (H) Longitudinal view of T. agilis STA centered on the A-C linker from the viewing point indicated in (F). Microtubule protofilaments A8/9 and C9/C10 of two adjacent triplets are indicated, as are the connected A- and C-links. The average distance between A- and C-links is 8.4 ± 0.3 nm and 8.4 ± 0.9 nm (N=6 and N=5, respectively). Corresponding transverse views are shown below, chevrons point to connection; the connection of the A-link with A9 is only partially visible in the transverse view at this height, as indicated by the dashed chevron.


( A ) Transverse 2D slices through STA comprising 64 % and 36 % of sub - volumes of T . mirabilis central cartwheel at indicated height from proximal ( 0 nm ) to distal ( 8 . 0 nm ) . Note filamentous cartwheel inner densities ( fCID ) in the center of the hub .

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

(A) Transverse 2D slices through STA comprising 64 % and 36 % of sub-volumes of T. mirabilis central cartwheel at indicated height from proximal (0 nm) to distal (8.0 nm). Note filamentous cartwheel inner densities (fCID) in the center of the hub.


( D , E ) 2D longitudinal view of T . mirabilis central region of the cartwheel STA 64 % ( D ) and 36 % ( E ) classes in the region delineated by dashed boxes in ( A ) . Arrowheads denote position of line scans along vertical axis at the hub level , with corresponding normalized pixel intensities ( in arbitrary units ) . Some maxima are highlighted by dashed lines . The average distance between hub units in the 64 % class is 8 . 4 ± 0 . 6 nm ( N = 8 ; D ) ; in the 36 % class , the distance between hub densities alternates between 3 . 2 ± 0 . 3 nm and 5 . 2 ± 0 . 3 nm and ( both N = 9 ; E ) . Dashed white line in ( E ) indicates the offset between two superimposed hub units at the level of spoke densities , which occurs every other hub unit pair .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 2D longitudinal view of T. mirabilis central region of the cartwheel STA 64% (D) and 36% (E) classes in the region delineated by dashed boxes in (A). Arrowheads denote position of line scans along vertical axis at the hub level, with corresponding normalized pixel intensities (in arbitrary units). Some maxima are highlighted by dashed lines. The average distance between hub units in the 64 % class is 8.4 ± 0.6 nm (N=8; D); in the 36 % class, the distance between hub densities alternates between 3.2 ± 0.3 nm and 5.2 ± 0.3 nm and (both N=9; E). Dashed white line in (E) indicates the offset between two superimposed hub units at the level of spoke densities, which occurs every other hub unit pair.


( H ) ( Top ) 2D slice through STA transverse view of microtubule triplet in T . mirabilis , with insets showing pinhead ( dashed green box ) and A - C linker ( dashed red box ) . ( Bottom ) Longitudinal 2D slice of STA centered on the pinhead ( left ) or A - C

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

(H) (Top) 2D slice through STA transverse view of microtubule triplet in T. mirabilis, with insets showing pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker


( E ) Purified CrSAS - 6 [ NL ] protein self - organized into stacks and analyzed by cryo - ET ; one longitudinal and one transverse view are highlighted by a dashed rectangle and a dashed circle , respectively . ( F , G ) 2D views through STA of in vitro self - assembled CrSAS - 6 [ NL ] proteins . Transverse sections at the indicated heights through one assembly unit ( F ) , as well as longitudinal view ( G ) of the area delineated by a dashed box in ( F ) ; white arrowhead denotes position of line scan along the vertical axis at the level of the hub , with corresponding pixel intensities in arbitrary units ( AU ) . The average distance between two hub elements is 4 . 5 ± 0 . 3 nm ( N = 7 ; dashed lines ) . ( H ) Longitudinal view of in vitro self - assembled CrSAS - 6 [ NL ] proteins STA 3D map . Note densities bridging successive hubs vertically ( arrows ) . ( I ) Two CrSAS - 6 [ HR ] single rings fitted into the 3D map of in vitro self - assembled CrSAS - 6 [ NL ] proteins . Dashed box indicates longitudinal section through hub element ( left ) , double chevron indicates viewing point of longitudinal external view ( right ) . Note that the 3D map can accommodate rings stacked 4 . 4 nm apart ( measured on fitted ring models ) , and that vertical densities linking hubs are partially occupied .

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

(E) Purified CrSAS-6[NL] protein self-organized into stacks and analyzed by cryo-ET; one longitudinal and one transverse view are highlighted by a dashed rectangle and a dashed circle, respectively. (F, G) 2D views through STA of in vitro self-assembled CrSAS-6[NL] proteins. Transverse sections at the indicated heights through one assembly unit (F), as well as longitudinal view (G) of the area delineated by a dashed box in (F); white arrowhead denotes position of line scan along the vertical axis at the level of the hub, with corresponding pixel intensities in arbitrary units (AU). The average distance between two hub elements is 4.5 ± 0.3 nm (N=7; dashed lines). (H) Longitudinal view of in vitro self-assembled CrSAS-6[NL] proteins STA 3D map. Note densities bridging successive hubs vertically (arrows). (I) Two CrSAS-6[HR] single rings fitted into the 3D map of in vitro self-assembled CrSAS-6[NL] proteins. Dashed box indicates longitudinal section through hub element (left), double chevron indicates viewing point of longitudinal external view (right). Note that the 3D map can accommodate rings stacked 4.4 nm apart (measured on fitted ring models), and that vertical densities linking hubs are partially occupied.


( c ) Representative micrographs of a clustering assay with the proteins indicated on the left . GFP and mCherry fluorescence are consistently displayed in cyan and magenta ,

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

(c) Representative micrographs of a clustering assay with the proteins indicated on the left. GFP and mCherry fluorescence are consistently displayed in cyan and magenta, respectively


( a ) Top : schematic representation of the STG - p62 construct . Bottom : characterization of several STG - p62 clones in comparison to parental Hap1

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

(a) Top: schematic representation of the STG-p62 construct. Bottom: characterization of several STG-p62 clones in comparison to parental Hap1 cells


( b ) Wild type or STG - p62 Hap1 cells were mock or puromycin treated for 2 hours and stained for ubiquitin . The endogenous p62 was stained with an anti - p62 antibody in the parental Hap1 cells , while the fluorescence of the GFP tag was recorded in STG - p62 cells . Arrowheads indicate co - localizing

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Wild type or STG-p62 Hap1 cells were mock or puromycin treated for 2 hours and stained for ubiquitin. The endogenous p62 was stained with an anti-p62 antibody in the parental Hap1 cells, while the fluorescence of the GFP tag was recorded in STG-p62 cells. Arrowheads indicate co-localizing puncta


( c ) Purified STG - p62 was tested for cluster formation in presence of 20µM GST - 4xUb . Averages and SDs of three independent replicates are shown for the + GST - 4xUb sample and of two for STG - p62

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

(c) Purified STG-p62 was tested for cluster formation in presence of 20µM GST-4xUb. Averages and SDs of three independent replicates are shown for the +GST-4xUb sample and of two for STG-p62 alone


( d , e ) FCS analysis of diffusion ( d ) and brightness ( e ) of the GFP1 - 5 ruler and of STG - p62 in the cytoplasm of Hap1 cells . Dashed lines represent the linear regression of the GFP1 - 5 ruler data . Regression coefficients are indicated . For all data points averages and SDs ( on both axes for STG - p62 ) are shown ( see methods for a list of the number of measurements performed for each construct ) . MW : molecular weight . D : diffusion coefficient ( µm2 / s )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FCS analysis of diffusion (d) and brightness (e) of the GFP1-5 ruler and of STG-p62 in the cytoplasm of Hap1 cells. Dashed lines represent the linear regression of the GFP1-5 ruler data. Regression coefficients are indicated. For all data points averages and SDs (on both axes for STG-p62) are shown (see methods for a list of the number of measurements performed for each construct). MW: molecular weight. D: diffusion coefficient (µm2/s)


( a ) Representative micrographs of cluster formation assays conducted with the indicated proteins

(a) Representative micrographs of cluster formation assays conducted with the indicated proteins


( c ) Quantification of cluster formation assays conducted with the indicated proteins . mCherry - p62 was pre - incubated with the indicated 4xUb variants , followed by the addition of streptavidin immediately before

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

(c) Quantification of cluster formation assays conducted with the indicated proteins. mCherry-p62 was pre-incubated with the indicated 4xUb variants, followed by the addition of streptavidin immediately before imaging


( d ) Representative micrographs of the indicated samples taken 60 minutes after addition of

(d) Representative micrographs of the indicated samples taken 60 minutes after addition of streptavidin


( e ) Quantification of cluster formation assays conducted with the indicated

(e) Quantification of cluster formation assays conducted with the indicated proteins


( f ) Quantification of cluster formation assays conducted with 2µM mCherry - p62 incubated with the indicated concentration of GST - GFP - 4xUb . Box : the highest particle number per field detected in each sample was plotted against the relative GST - GFP - 4xUb concentration . Data points were fitted to a sigmoidal curve . The regression coefficient is

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

(f) Quantification of cluster formation assays conducted with 2µM mCherry-p62 incubated with the indicated concentration of GST-GFP-4xUb. Box: the highest particle number per field detected in each sample was plotted against the relative GST-GFP-4xUb concentration. Data points were fitted to a sigmoidal curve. The regression coefficient is indicated


( g ) Quantification of cluster formation assays conducted with GFP - p62 and the indicated GST - tagged ubiquitin

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR

(g) Quantification of cluster formation assays conducted with GFP-p62 and the indicated GST-tagged ubiquitin chains