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 .

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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=0.006 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.


Adult Swiss mice ( n = 11 Veh ; 15 AβOs ) received a single i . c . v . injection of vehicle or 10 pmol AβOs and were assessed in a glucose tolerance test ( 2 g glucose / kg body weight , i . p . ) 7 days after injection . Blood levels of glucose were measured at several time points following glucose administration . Bar graph represents areas under the curves in the time course plot . Data are representative of three independent experiments with similar results . Left panel : * * * P = 0 . 0006 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * P = 0 . 0207 , Student ' s t - test .

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Adult Swiss mice (n = 11 Veh; 15 AβOs) received a single i.c.v. injection of vehicle or 10 pmol AβOs and were assessed in a glucose tolerance test (2 g glucose/kg body weight, i.p.) 7 days after injection. Blood levels of glucose were measured at several time points following glucose administration. Bar graph represents areas under the curves in the time course plot. Data are representative of three independent experiments with similar results. Left panel: ***P = 0.0006, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0207, Student's t-test.


Insulin tolerance test ( 1 IU insulin / kg body weight , i . p . ) ( n = 7 Veh ; 8 AβOs ) . Blood levels of glucose were measured at several time points following insulin administration . Bar graph represents the kinetic constants for glucose disappearance ( Kitt ) calculated from the time course plot . Data are representative of two independent experiments with similar results . Left panel : * P = 0 . 0456 and * * * P = 0 . 0007 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * * P = 0 . 0033 , Student ' s t - test .

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Insulin tolerance test (1 IU insulin/kg body weight, i.p.) (n = 7 Veh; 8 AβOs). Blood levels of glucose were measured at several time points following insulin administration. Bar graph represents the kinetic constants for glucose disappearance (Kitt) calculated from the time course plot. Data are representative of two independent experiments with similar results. Left panel: *P = 0.0456 and ***P = 0.0007, two-way ANOVA followed by Bonferroni post hoc test; right panel: **P = 0.0033, Student's t-test.


Glucose tolerance test ( 2 g glucose / kg body weight , i . p . ) in mice 7 days after a single intracaudal ( C ; n = 8 animals / group ) or intraperitoneal ( D ; n = 13 animals / group ) injection of AβOs ( 10 pmol ) or vehicle .

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Glucose tolerance test (2 g glucose/kg body weight, i.p.) in mice 7 days after a single intracaudal (C; n = 8 animals/group) or intraperitoneal (D; n = 13 animals/group) injection of AβOs (10 pmol) or vehicle.


Glucose tolerance test ( 2 g glucose / kg body weight , i . p . ) in 8 - to 13 - month - old APP / PS1 mice ( E ; n = 9 animals / group ) or 6 - month - old 3xTg - AD male mice ( F ; n = 10 WT ; 9 3xTg ) , or their corresponding wild - type littermates . Bar graph represents areas under the curves in the time course plots . In ( E ) , left panel : * P = 0 . 0466 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : & amp ; amp ; amp ; P = 0 . 072 , Student ' s t - test . In ( F ) , left panel : * P = 0 . 0171 and # P = 0 . 0781 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * P = 0 . 0101 , Student ' s t - test .

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Glucose tolerance test (2 g glucose/kg body weight, i.p.) in 8- to 13-month-old APP/PS1 mice (E; n = 9 animals/group) or 6-month-old 3xTg-AD male mice (F; n = 10 WT; 9 3xTg), or their corresponding wild-type littermates. Bar graph represents areas under the curves in the time course plots. In (E), left panel: *P = 0.0466, two-way ANOVA followed by Bonferroni post hoc test; right panel: &amp;amp;P = 0.072, Student's t-test. In (F), left panel: *P = 0.0171 and #P = 0.0781, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0101, Student's t-test.


CD68 immunoreactivity in white adipose tissue ( scale bar = 25 μm , images representative of one animal each from a total of four animals per experimental group ) . Arrow points to a region stained with CD68 antibody . * P = 0 . 0109 , Student ' s t - test .

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CD68 immunoreactivity in white adipose tissue (scale bar = 25 μm, images representative of one animal each from a total of four animals per experimental group). Arrow points to a region stained with CD68 antibody. *P = 0.0109, Student's t-test.


Epididymal fat mass was analyzed in mice ( n = 6 animals / group ) 7 days after i . c . v . injection of vehicle or AβOs . Data are representative of three independent experiments with similar results . * P = 0 . 0255 .

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Epididymal fat mass was analyzed in mice (n = 6 animals/group) 7 days after i.c.v. injection of vehicle or AβOs. Data are representative of three independent experiments with similar results. *P = 0.0255.


Relative expression of leptin ( C ) , TNF - α ( D ) and IL - 6 ( E ) , respectively , in white adipose tissue of mice ( n = 7 Veh ; 9 AβOs ) 7 days after i . c . v . injection of vehicle or AβOs . In ( C ) , * P = 0 . 0394 ; in ( D ) , * * P = 0 . 0038 ; in ( E ) , * P = 0 . 0305 ; Student ' s t - test .

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Relative expression of leptin (C), TNF-α (D) and IL-6 (E), respectively, in white adipose tissue of mice (n = 7 Veh; 9 AβOs) 7 days after i.c.v. injection of vehicle or AβOs. In (C), *P = 0.0394; in (D), **P = 0.0038; in (E), *P = 0.0305; Student's t-test.


p - JNK ( F ; n = 5 animals / group ) and IRS - 1pSer312 ( G ; n = 6 animals / group ) levels ( normalized by total JNK and total IRS - 1 , respectively ) in skeletal muscle of mice 7 days after i . c . v . injection of vehicle or AβOs . In ( F ) , * P = 0 . 0464 ; in ( G ) , * P = 0 . 0081 ; Student ' s t - test .

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p-JNK (F; n = 5 animals/group) and IRS-1pSer312 (G; n = 6 animals/group) levels (normalized by total JNK and total IRS-1, respectively) in skeletal muscle of mice 7 days after i.c.v. injection of vehicle or AβOs. In (F), *P = 0.0464; in (G), *P = 0.0081; Student's t-test.


Representative images of GLUT - 4 immunofluorescence in insulin - stimulated skeletal muscle from mice that were i . c . v . - injected with vehicle ( Veh ) or 10 pmol AβOs . Bar graphs show quantification of GLUT - 4 surface immunoreactivity in skeletal muscle of mice that received intraperitoneal injections of PBS or insulin ( 1 IU / kg body weight ) 7 days after i . c . v . injection of vehicle or AβOs , as indicated ( n = 5 animals / group ) . Scale bar = 25 μm . * P = 0 . 0144 , one - way ANOVA followed by Bonferroni post hoc test .

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Representative images of GLUT-4 immunofluorescence in insulin-stimulated skeletal muscle from mice that were i.c.v.-injected with vehicle (Veh) or 10 pmol AβOs. Bar graphs show quantification of GLUT-4 surface immunoreactivity in skeletal muscle of mice that received intraperitoneal injections of PBS or insulin (1 IU/kg body weight) 7 days after i.c.v. injection of vehicle or AβOs, as indicated (n = 5 animals/group). Scale bar = 25 μm. *P = 0.0144, one-way ANOVA followed by Bonferroni post hoc test.


GLUT - 4 mRNA ( n = 4 animals / group ) and total protein levels ( normalized to actin levels ; n = 5 Veh ; 6 AβOs ) were unchanged in skeletal muscle of Swiss mice injected with vehicle ( Veh ) or 10 pmol AβOs .

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GLUT-4 mRNA (n = 4 animals/group) and total protein levels (normalized to actin levels; n = 5 Veh; 6 AβOs) were unchanged in skeletal muscle of Swiss mice injected with vehicle (Veh) or 10 pmol AβOs.


Plasma levels of insulin ( K ; n = 12 animals / group ) , leptin ( L ; n = 11 Veh ; 12 AβOs ) , cholesterol ( M ; n = 8 Veh ; 6 AβOs ) , triglycerides ( N ; n = 8 Veh ; 6 AβOs ) or noradrenaline ( O ; n = 7 Veh ; 8 AβOs ) measured 7 days after i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs . In ( O ) , * P = 0 . 0361 , Student ' s t - test .

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Plasma levels of insulin (K; n = 12 animals/group), leptin (L; n = 11 Veh; 12 AβOs), cholesterol (M; n = 8 Veh; 6 AβOs), triglycerides (N; n = 8 Veh; 6 AβOs) or noradrenaline (O; n = 7 Veh; 8 AβOs) measured 7 days after i.c.v. injection of vehicle (Veh) or 10 pmol AβOs. In (O), *P = 0.0361, Student's t-test.


Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle ( Veh ) or AβOs ( 500 nM ) for 3 h . Binding of AβOs was detected using anti - oligomer monoclonal antibody NU4 ( red ) . Neurons were double - labeled using MAP - 2 antibody ( green ) . Images represent typical results from experiments with three independent hypothalamic cultures ( three coverslips / experimental condition per independent experiment ) . Scale bar = 30 and 10 μm for main panels and insets , respectively .

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Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle (Veh) or AβOs (500 nM) for 3 h. Binding of AβOs was detected using anti-oligomer monoclonal antibody NU4 (red). Neurons were double-labeled using MAP-2 antibody (green). Images represent typical results from experiments with three independent hypothalamic cultures (three coverslips/experimental condition per independent experiment). Scale bar = 30 and 10 μm for main panels and insets, respectively.


Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs ( 500 nM ) and immunolabeled with anti - GFAP ( green ) and NU4 ( red ) antibodies . Insets show AβOs binding to neuronal dendrites , whereas no binding was detected to GFAP - positive cells .

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Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs (500 nM) and immunolabeled with anti-GFAP (green) and NU4 (red) antibodies. Insets show AβOs binding to neuronal dendrites, whereas no binding was detected to GFAP-positive cells.


Representative DCFfluorescence images from hypothalamic neuronalcultures exposed to vehicle or AβOs ( 500 nM , 4 h ) . Insets show optical zoom images of the indicated areas . Scale bars = 100 and 50 μm for main panels and insets , respectively . Graph shows integrated DCFfluorescence intensities ( relative units ; see Materials and Methods ) ( n = 3 independent hypothalamic cultures ; three wells / experimental condition per experiment ; three images acquired per well ) . Bars represent means ± SEM . # P = 0 . 0604 , one - sample t - test compared with a fixed value of 100 RUs .

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Representative DCFfluorescence images from hypothalamic neuronalcultures exposed to vehicle or AβOs (500 nM, 4 h). Insets show optical zoom images of the indicated areas. Scale bars = 100 and 50 μm for main panels and insets, respectively. Graph shows integrated DCFfluorescence intensities (relative units; see Materials and Methods) (n = 3 independent hypothalamic cultures; three wells/experimental condition per experiment; three images acquired per well). Bars represent means ± SEM. #P = 0.0604, one-sample t-test compared with a fixed value of 100 RUs.


LDH activity ( IU / l ) in culture media of hypothalamic cultures exposed to vehicle or AβOs ( 500 nM , 3 h ) .

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LDH activity (IU/l) in culture media of hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h).


Representative immunofluorescence images of eIF2α - P in hypothalamic cultures exposed to vehicle or AβOs ( 500 nM , 3 h ) in the absence or presence of infliximab ( 1 μg / ml ) . Scale bar = 30 μm . Graph represents integrated immunofluorescence intensities of eIF2α - P levels from three independent hypothalamic cultures ( three coverslips / experimental condition per experiment , 20 images per coverslip ) . Bars represent means ± SEM . * P = 0 . 0489 , one - way ANOVA followed by Bonferroni post hoc test comparing AβO - treated versus vehicle - treated cultures .

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Representative immunofluorescence images of eIF2α-P in hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Scale bar = 30 μm. Graph represents integrated immunofluorescence intensities of eIF2α-P levels from three independent hypothalamic cultures (three coverslips/experimental condition per experiment, 20 images per coverslip). Bars represent means ± SEM. *P = 0.0489, one-way ANOVA followed by Bonferroni post hoc test comparing AβO-treated versus vehicle-treated cultures.


Representative images of hypothalamic cultures exposed to AβOs ( 500 nM , 3 h ) and double - labeled with NU4 ( oligomer - sensitive ) and eIF2α - P antibodies . Arrow points to a neuron presenting high levels of eIF2α - P in the absence of AβO binding . Nuclear staining ( DAPI ) is shown in blue . Scale bar = 30 μm .

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Representative images of hypothalamic cultures exposed to AβOs (500 nM, 3 h) and double-labeled with NU4 (oligomer-sensitive) and eIF2α-P antibodies. Arrow points to a neuron presenting high levels of eIF2α-P in the absence of AβO binding. Nuclear staining (DAPI) is shown in blue. Scale bar = 30 μm.


Representative images of hypothalamic neurons labeled with NU4 antibody exposed to AβOs ( 500 nM , 3 h ) in the absence or presence of infliximab ( 1 μg / ml ) . Similar patterns of AβO binding were observed in both conditions . Scale bar = 20 μm .

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Representative images of hypothalamic neurons labeled with NU4 antibody exposed to AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Similar patterns of AβO binding were observed in both conditions. Scale bar = 20 μm.


Western blot analysis of eIF2α - P levels in the hypothalamus of mice 4 h ( A ; n = 4 animals / group ) or 7 days ( B ; n = 6 Veh ; 5 AβOs ) after a single i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs . Graphs show densitometric data normalized by total eIF2α levels . * P = 0 . 0213 .

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Western blot analysis of eIF2α-P levels in the hypothalamus of mice 4 h (A; n = 4 animals/group) or 7 days (B; n = 6 Veh; 5 AβOs) after a single i.c.v. injection of vehicle (Veh) or 10 pmol AβOs. Graphs show densitometric data normalized by total eIF2α levels. *P = 0.0213.


Western blot analysis of ATF4 levels in the hypothalamus of mice 4 h after i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs ; graph shows densitometric data normalized by β - actin ( n = 7 Veh ; 8 AβOs ) . # P = 0 . 0731 ; Student ' s t - test .

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Western blot analysis of ATF4 levels in the hypothalamus of mice 4 h after i.c.v. injection of vehicle (Veh) or 10 pmol AβOs; graph shows densitometric data normalized by β-actin (n = 7 Veh; 8 AβOs). #P = 0.0731; Student's t-test.


Western blot analysis of hypothalamic phospho - IKKβ levels in the hypothalamus of mice 4 h ( C ; n = 6 animals / group ) or 7 days ( D ; n = 4 Veh ; 5 AβOs ) after i . c . v . injection of vehicle or 10 pmol AβOs . Graphs show densitometric data normalized by total IKKβ levels . In ( D ) , * P = 0 . 0437 ; in ( E ) , * P = 0 . 0444 ; Student ' s t - test .

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Western blot analysis of hypothalamic phospho-IKKβ levels in the hypothalamus of mice 4 h (C; n = 6 animals/group) or 7 days (D; n = 4 Veh; 5 AβOs) after i.c.v. injection of vehicle or 10 pmol AβOs. Graphs show densitometric data normalized by total IKKβ levels. In (D), *P = 0.0437; in (E), *P = 0.0444; Student's t-test.


Western blot analysis of IκBα ( F ; n = 6 animals / group ) and cytoplasmic phospho - p65 - NF - κB ( G ; n = 4 Veh ; 5 AβOs ) in the hypothalamus of mice 4 h after i . c . v . injection of vehicle or 10 pmol AβOs . Graphs show densitometric data normalized by actin ( F ) or total NF - κB levels ( G ) . * P = 0 . 0207 .

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Western blot analysis of IκBα (F; n = 6 animals/group) and cytoplasmic phospho-p65-NF-κB (G; n = 4 Veh; 5 AβOs) in the hypothalamus of mice 4 h after i.c.v. injection of vehicle or 10 pmol AβOs. Graphs show densitometric data normalized by actin (F) or total NF-κB levels (G). *P = 0.0207.


Nuclear NF - κB levels in the hypothalamus 6 h after i . c . v . injection of vehicle or 10 pmol AβOs in mice . Graphs show NF - κB levels normalized by nuclear marker , lamin B ( n = 6 animals / group ) . * * P = 0 . 0024 ; Student ' s t - test .

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Nuclear NF-κB levels in the hypothalamus 6 h after i.c.v. injection of vehicle or 10 pmol AβOs in mice. Graphs show NF-κB levels normalized by nuclear marker, lamin B (n = 6 animals/group). **P = 0.0024; Student's t-test.


IRS - 1pSer636 ( I ; n = 4 animals / group ) and pTyr465 ( J ; n = 6 Veh ; 4 AβOs ) levels in the hypothalamus 7 days after i . c . v . injection of vehicle or AβOs in mice . Graphs show IRS - 1pSer or IRS - 1pTyr levels normalized by total IRS - 1 . In ( I ) , * P = 0 . 0043 ; in ( J ) , * P = 0 . 0275 ; Student ' s t - test .

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IRS-1pSer636 (I; n = 4 animals/group) and pTyr465 (J; n = 6 Veh; 4 AβOs) levels in the hypothalamus 7 days after i.c.v. injection of vehicle or AβOs in mice. Graphs show IRS-1pSer or IRS-1pTyr levels normalized by total IRS-1. In (I), *P = 0.0043; in (J), *P = 0.0275; Student's t-test.


Twelve - hour food intake after a single i . c . v . infusion of insulin ( 200 mU ) in mice . Experiment was performed 7 days after i . c . v . injection of vehicle or AβOs ( n = 5 PBS ; 5 Veh + Insulin ; 9 AβOs + Insulin ) , data are representative of two independent experiments with similar results . * * * P < 0 . 0001 , one - way ANOVA followed by Bonferroni post hoc test comparing Veh - Insulin versus PBS groups .

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Twelve-hour food intake after a single i.c.v. infusion of insulin (200 mU) in mice. Experiment was performed 7 days after i.c.v. injection of vehicle or AβOs (n = 5 PBS; 5 Veh + Insulin; 9 AβOs + Insulin), data are representative of two independent experiments with similar results. ***P < 0.0001, one-way ANOVA followed by Bonferroni post hoc test comparing Veh-Insulin versus PBS groups.


Representative images of AβO immunoreactivity ( detected using anti - oligomer monoclonal antibody NU4 ) in the hypothalamus of control , sham - operated adult cynomolgus macaques ( Sh ; n = 3 ) or macaques that received i . c . v . injections of AβOs ( n = 3 ; see Materials and Methods ) . Nuclear staining ( DAPI ) is shown in blue . Insets show optical zoom images of the areas indicated by white dashed rectangles in the main panels . Scale bars = 100 and 20 μm for main panels and insets , respectively .

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Representative images of AβO immunoreactivity (detected using anti-oligomer monoclonal antibody NU4) in the hypothalamus of control, sham-operated adult cynomolgus macaques (Sh; n = 3) or macaques that received i.c.v. injections of AβOs (n = 3; see Materials and Methods). Nuclear staining (DAPI) is shown in blue. Insets show optical zoom images of the areas indicated by white dashed rectangles in the main panels. Scale bars = 100 and 20 μm for main panels and insets, respectively.


Representative images showing eIF2α - P ( B ) , phospho - IKKβ ( C ) and IκBα ( D ) immunoreactivities in the hypothalamus of cynomolgus macaques that received i . c . v . injections of AβOs or control ( sham - operated ; Sh ) macaques ( n = 3 animals / group ) . Graphs show immunolabeling optical density analysis from three images acquired in the hypothalamus of each macaque ( three control versus three AβO - injected animals ) . In ( B ) , # P = 0 . 0523 ; in ( C ) # P = 0 . 1123 ; unpaired Student ' s t - test with Welch ' s correction for unequal variances ; AβO - injected monkeys compared to sham - operated monkeys . Scale bars = 50

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

Representative images showing eIF2α-P (B), phospho-IKKβ (C) and IκBα (D) immunoreactivities in the hypothalamus of cynomolgus macaques that received i.c.v. injections of AβOs or control (sham-operated; Sh) macaques (n = 3 animals/group). Graphs show immunolabeling optical density analysis from three images acquired in the hypothalamus of each macaque (three control versus three AβO-injected animals). In (B), #P = 0.0523; in (C) #P = 0.1123; unpaired Student's t-test with Welch's correction for unequal variances; AβO-injected monkeys compared to sham-operated monkeys. Scale bars = 50 μm


Accumulated chow intake ( normalized by body weight ) measured during 7 days following a single i . c . v . injection of vehicle or 10 pmol AβOs in mice ( n = 13 Veh ; 10 AβOs ; data are representative of two independent experiments with similar results ) . * * * P < 0 . 0001 ; Student ' s t - test .

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Accumulated chow intake (normalized by body weight) measured during 7 days following a single i.c.v. injection of vehicle or 10 pmol AβOs in mice (n = 13 Veh; 10 AβOs; data are representative of two independent experiments with similar results). ***P < 0.0001; Student's t-test.


Daily body weight measured during 7 days after i . c . v . injection of vehicle or AβOs ( n = 7 animals / group ; data are representative of two independent experiments with similar results ) .

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Daily body weight measured during 7 days after i.c.v. injection of vehicle or AβOs (n = 7 animals/group; data are representative of two independent experiments with similar results).


Adult Swiss mice received a single i . c . v . injection of vehicle or 10 pmol AβOs , and hypothalamic levels of mRNA for AgRP ( C ; n = 6 Veh ; 5 AβOs ) , NPY ( D ; n = 6 Veh ; 5 AβOs ) and POMC ( E ; n = 7 animals / group ) were analyzed 7 days after injection . In ( C ) , * P = 0 . 0191 ; in ( D ) , * P = 0 . 0115 ; Student ' s t - test .

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Adult Swiss mice received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (C; n = 6 Veh; 5 AβOs), NPY (D; n = 6 Veh; 5 AβOs) and POMC (E; n = 7 animals/group) were analyzed 7 days after injection. In (C), *P = 0.0191; in (D), *P = 0.0115; Student's t-test.


Swiss mice received a single i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs , and their brains were analyzed by Fluorojade staining of degenerating cells 7 days after the injection . Representative images of Fluorojade staining in the hypothalamus of vehicle - or AβO - injected mice ( n = 4 / group ) . Scale bar = 100 μm in left panels ( top and bottom ) and 20 μm in right panels ( top and bottom ) . Positive control ( bottom left panel ) was the hippocampus of a mouse that received one i . c . v . injection of quinolinic acid ( 36 . 8 nmol ) and was analyzed 24 h after .

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

Swiss mice received a single i.c.v. injection of vehicle (Veh) or 10 pmol AβOs, and their brains were analyzed by Fluorojade staining of degenerating cells 7 days after the injection. Representative images of Fluorojade staining in the hypothalamus of vehicle- or AβO-injected mice (n = 4/group). Scale bar = 100 μm in left panels (top and bottom) and 20 μm in right panels (top and bottom). Positive control (bottom left panel) was the hippocampus of a mouse that received one i.c.v. injection of quinolinic acid (36.8 nmol) and was analyzed 24 h after.


Glucose tolerance test ( 2 g glucose / kg body weight , i . p . ) in mice that received i . c . v . injections of vehicle , vehicle + TUDCA , AβOs or AβOs + TUDCA ( when used , TUDCA was administered in 5 i . c . v . injections of 5 μg TUDCA each , before and after oligomer injection ; see Materials and Methods . Control groups received injections of saline ) . Glucose tolerance test ( GTT ) was performed 7 days after i . c . v . injection of vehicle or AβOs . Bar graph represents areas under the curves ( AUC ) in the time course plots ( n = 15 Veh ; 15 AβOs ; 10 Veh + TUDCA ; 16 AβOs + TUDCA ) . Data are representative of two independent experiments with similar results . Left panel : * * P = 0 . 0048 , * * P = 0 . 003 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * P = 0 . 0384 , one - way ANOVA followed by Bonferroni post hoc test .

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Glucose tolerance test (2 g glucose/kg body weight, i.p.) in mice that received i.c.v. injections of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA (when used, TUDCA was administered in 5 i.c.v. injections of 5 μg TUDCA each, before and after oligomer injection; see Materials and Methods. Control groups received injections of saline). Glucose tolerance test (GTT) was performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 15 Veh; 15 AβOs; 10 Veh + TUDCA; 16 AβOs + TUDCA). Data are representative of two independent experiments with similar results. Left panel: **P = 0.0048, **P = 0.003, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0384, one-way ANOVA followed by Bonferroni post hoc test.


Plasma noradrenaline ( NA ) levels measured 7 days after i . c . v . injection of vehicle , vehicle + TUDCA , AβOs or AβOs + TUDCA in mice ( n = 7 animals / group ) . Data are representative of two independent experiments with similar results . * P = 0 . 0071 , one - way ANOVA followed by Bonferroni post hoc test .

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Plasma noradrenaline (NA) levels measured 7 days after i.c.v. injection of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA in mice (n = 7 animals/group). Data are representative of two independent experiments with similar results. *P = 0.0071, one-way ANOVA followed by Bonferroni post hoc test.


Glucose tolerance test ( 2 g glucose / kg body weight , i . p . ) in TNFR1 − / − mice or wild - type littermates performed 7 days after i . c . v . injection of vehicle or AβOs . Bar graph represents areas under the curves ( AUC ) in the time course plots ( n = 8 WT + Veh ; 7 WT + AβOs ; 7 TNFR − / − + Veh ; 8 TNFR − / − + AβOs ) . Left panel : * * P = 0 . 0049 , * * * P < 0 . 0001 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * P = 0 . 0001 , one - way ANOVA followed by Bonferroni post hoc test .

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Glucose tolerance test (2 g glucose/kg body weight, i.p.) in TNFR1−/− mice or wild-type littermates performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 8 WT + Veh; 7 WT + AβOs; 7 TNFR−/− + Veh; 8 TNFR−/− + AβOs). Left panel: **P = 0.0049, ***P < 0.0001, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0001, one-way ANOVA followed by Bonferroni post hoc test.


Western blot analysis of phospho - IKKβ ( D ; n = 6 WT + Veh ; 6 WT + AβOs ; 4 TNFR − / − + Veh ; 6 TNFR − / − + AβOs ) and IRS - 1pSer636 levels ( E ; n = 6 WT + Veh ; 5 WT + AβOs ; 6 TNFR − / − + Veh ; 5 TNFR − / − + AβOs ) in the hypothalamus of wild - type ( WT ) or TNFR1 − / − mice 10 days after i . c . v . injection of vehicle or AβOs . Representative images from Western blot experiments were always run on the same gels but represent noncontiguous lanes . In ( D ) , * P = 0 . 0088 , Student ' s t - test ; in ( E ) , * P = 0 . 0428 , one - way ANOVA followed by Bonferroni post hoc test .

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

Western blot analysis of phospho-IKKβ (D; n = 6 WT + Veh; 6 WT + AβOs; 4 TNFR−/− + Veh; 6 TNFR−/− + AβOs) and IRS-1pSer636 levels (E; n = 6 WT + Veh; 5 WT + AβOs; 6 TNFR−/− + Veh; 5 TNFR−/− + AβOs) in the hypothalamus of wild-type (WT) or TNFR1−/− mice 10 days after i.c.v. injection of vehicle or AβOs. Representative images from Western blot experiments were always run on the same gels but represent noncontiguous lanes. In (D), *P = 0.0088, Student's t-test; in (E), *P = 0.0428, one-way ANOVA followed by Bonferroni post hoc test.


Adult Swiss mice pre - treated with minocycline or PBS received a single i . c . v . injection of vehicle or 10 pmol AβOs , and hypothalamic levels of mRNA for AgRP ( F ; n = 5 Veh ; 6 AβOs ; 5 Veh + Mino ; 4 AβOs + Mino ) and NPY ( G ; n = 14 Veh ; 13 AβOs ; 8 Veh + Mino ; 9 AβOs + Mino ) were analyzed 7 days after injection . In ( F ) , * P = 0 . 0097 , one - way ANOVA followed by Bonferroni post hoc test ; in ( G ) , * P = 0 . 0219 , one - way ANOVA followed by Bonferroni post hoc test .

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

Adult Swiss mice pre-treated with minocycline or PBS received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (F; n = 5 Veh; 6 AβOs; 5 Veh + Mino; 4 AβOs + Mino) and NPY (G; n = 14 Veh; 13 AβOs; 8 Veh + Mino; 9 AβOs + Mino) were analyzed 7 days after injection. In (F), *P = 0.0097, one-way ANOVA followed by Bonferroni post hoc test; in (G), *P = 0.0219, one-way ANOVA followed by Bonferroni post hoc test.


Glucose tolerance test ( GTT ) in APP / PS1 mice before and after i . c . v . injection of infliximab ( 0 . 2 μg daily for 4 days ) . Bar graph represents areas under the curves ( AUC ) in the time course plots ( n = 9 animals / group ) . Left panel : * P = 0 . 0177 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * P = 0 . 0327 , paired t - test .

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Glucose tolerance test (GTT) in APP/PS1 mice before and after i.c.v. injection of infliximab (0.2 μg daily for 4 days). Bar graph represents areas under the curves (AUC) in the time course plots (n = 9 animals/group). Left panel: *P = 0.0177, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0327, paired t-test.


B : Scatter Plot of mass spectrometry assay . Dots depict protein intensity ( IBAQ ) and relative significance obtained from mass spectrometry assay . X - axis : Difference of Che - 1 intensity versus IP control ; on the y - axis are the corresponding − log10 P - values . Significant Che - 1 interacting proteins relevant in this study are represented in blue .

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B: Scatter Plot of mass spectrometry assay. Dots depict protein intensity (IBAQ) and relative significance obtained from mass spectrometry assay. X-axis: Difference of Che-1 intensity versus IP control; on the y-axis are the corresponding −log10 P-values. Significant Che-1 interacting proteins relevant in this study are represented in blue.


C : Nuclear extracts from Kms27 MM cells treated or not with RNase A were subjected to immunoprecipitation with the indicated antibodies ( abs ) and analyzed by western blot ( WB ) with the indicated abs . Input corresponds to 5 % of the total extract used for immunoprecipitation .

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C: Nuclear extracts from Kms27 MM cells treated or not with RNase A were subjected to immunoprecipitation with the indicated antibodies (abs) and analyzed by western blot (WB) with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.


D : ( Top ) qRT - PCR approach described in the schematic representation . ( Bottom ) RIP - qPCR analysis with anti - Che - 1 specific antibody or control rabbit IgG of Kms27 MM cells . Data are expressed as percent of Input . Error bars represent the standard error of three different biological experiments performed in technical duplicate ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 , n . s . = not significant ) .

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D: (Top) qRT-PCR approach described in the schematic representation. (Bottom) RIP-qPCR analysis with anti-Che-1 specific antibody or control rabbit IgG of Kms27 MM cells. Data are expressed as percent of Input. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ***P<0.005, ****P<0.001, n.s. = not significant).


E : Colocalization assays in Kms27 and RPMI8226 MM cell lines by combining In Situ Hybridization ( FISH ) staining of NEAT1 ( Stellaris ) with immunofluorescence of Che - 1 . Scale bar 10 μm .

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E: Colocalization assays in Kms27 and RPMI8226 MM cell lines by combining In Situ Hybridization (FISH) staining of NEAT1 (Stellaris) with immunofluorescence of Che-1. Scale bar 10 μm.


F : ( Left ) RT - qPCR analysis of NEAT1 expression levels in Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in technical duplicate ( Two Tailed T test * * * * P < 0 . 001 ) . ( Right ) Nuclear extracts from KMS27 MM cells were immunoprecipitated with the indicated abs and analyzed by WB with the indicated abs .

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F: (Left) RT-qPCR analysis of NEAT1 expression levels in Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ****P<0.001). (Right) Nuclear extracts from KMS27 MM cells were immunoprecipitated with the indicated abs and analyzed by WB with the indicated abs.


A : Quantitative RT - PCR ( RT - qPCR ) of Kms27 MM cells transiently transfected with two different LNA GapmeR NEAT1 or GapmeR Control oligonucleotides . Values were normalized to Actin expression . Error bars represent the standard error of three different experiments performed in duplicate ( Two Tailed T test * * * * P < 0 . 001 ) .

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A: Quantitative RT-PCR (RT-qPCR) of Kms27 MM cells transiently transfected with two different LNA GapmeR NEAT1 or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments performed in duplicate (Two Tailed T test ****P<0.001).


B : WB analysis with indicated abs of total extracts from Kms27 MM cells transiently transfected as in A .

B: WB analysis with indicated abs of total extracts from Kms27 MM cells transiently transfected as in A.


C : Representative confocal images of Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A , GapmeR NEAT1B or GapmeR Control oligonucleotides and immunostained with anti - Che - 1 antibody . Scale bar 10 μm .

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C: Representative confocal images of Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A, GapmeR NEAT1B or GapmeR Control oligonucleotides and immunostained with anti-Che-1 antibody. Scale bar 10 μm.


D : Isolated cell compartments from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides , were subjected to WB with the indicated abs .

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D: Isolated cell compartments from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides, were subjected to WB with the indicated abs.


E : Che - 1 ChIP - seq signal intensity of LNA GapmeR NEAT1A ( 2 replicates on right ) and GapmeR Control ( 2 replicates on left ) and at co - localizing Che - 1 / NEAT1 sites ( N = 14654 sites ) . Signal intensity from 1 ( weak ) to 12 ( strongest ) .

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E: Che-1 ChIP-seq signal intensity of LNA GapmeR NEAT1A (2 replicates on right) and GapmeR Control (2 replicates on left) and at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 12 (strongest).


F : FISH images of Kms27 MM cells depleted or not for Che - 1 expression ( siChe - 1A ) labeled with Hoechst ( cell nuclei ) and Quasar 670 ( NEAT1 RNA ) . Scale bar 10 μm .

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F: FISH images of Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) labeled with Hoechst (cell nuclei) and Quasar 670 (NEAT1 RNA). Scale bar 10 μm.


G : NEAT1 ChIRP - seq signal intensity of Kms27 MM cells transiently transfected with siChe - 1A or siControl at co - localizing Che - 1 / NEAT1 sites ( N = 14654 sites ) . Signal intensity from 1 ( weak ) to 14 ( strongest ) .

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G: NEAT1 ChIRP-seq signal intensity of Kms27 MM cells transiently transfected with siChe-1A or siControl at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 14 (strongest).


B : Chromatin extracts from Kms27 and RPMI8226 MM cells treated or not with RNase H , were subjected to immunoprecipitation with S9 . 6 antibody and analyzed by WB with the indicated abs . Input corresponds to 5 % of the total extract used for immunoprecipitation .

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B: Chromatin extracts from Kms27 and RPMI8226 MM cells treated or not with RNase H, were subjected to immunoprecipitation with S9.6 antibody and analyzed by WB with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.


C : Representative confocal images showing the interaction between Che - 1 and S9 . 6 revealed by PLA in Kms27 MM cells . Negative controls were performed by omitting Che - 1 or S9 . 6 primary antibody . Scale bar 10 μm .

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C: Representative confocal images showing the interaction between Che-1 and S9.6 revealed by PLA in Kms27 MM cells. Negative controls were performed by omitting Che-1 or S9.6 primary antibody. Scale bar 10 μm.


D : Chromatin extracts from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides were immunoprecipitated with S9 . 6 antibody and analyzed by WB with the indicated antibodies . Input corresponds to 5 % of the total extract used for immunoprecipitation .

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D: Chromatin extracts from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides were immunoprecipitated with S9.6 antibody and analyzed by WB with the indicated antibodies. Input corresponds to 5% of the total extract used for immunoprecipitation.


E : ( Left ) Representative confocal images of Kms27 MM cells treated or not with RNase H , transiently transfected with siChe - 1A or siControl and immunostained with S9 . 6 antibody and . Scale bar 10 μm . ( Right ) Box plot showing S9 . 6 signal nuclear intensity per cell ( Arbitrary Unit - AU ) . siChe - 1A cells are described in green color , while siControl in grey color . 100 cells were counted in each replicate . Each dot represents a cell . Median scores per sample are written at the top of the median red line . P value : < 1 . 02 - 18 . ANOVA test was performed to evaluate statistical significance . The red center line denotes the median value ( 50th percentile ) while the black box contains the 25th to 75th percentiles of data . The whiskers mark the 5th and 95th percentiles .

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

E: (Left) Representative confocal images of Kms27 MM cells treated or not with RNase H, transiently transfected with siChe-1A or siControl and immunostained with S9.6 antibody and. Scale bar 10 μm. (Right) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in green color, while siControl in grey color. 100 cells were counted in each replicate. Each dot represents a cell. Median scores per sample are written at the top of the median red line. P value: <1.02-18. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


F : ( Top ) S9 . 6 antibody was used for dot blot analysis to evaluate RNA : DNA hybrids formation in Kms27 MM cells by serial dilutions of genomic DNA starting at 1 . 5 micrograms . ( Bottom ) Quantification of S9 . 6 levels by densitometric analysis . Values were normalized to a Methylene blue loading control . Error bars represent the standard error of three different biological experiments ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 ) .

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

F: (Top) S9.6 antibody was used for dot blot analysis to evaluate RNA:DNA hybrids formation in Kms27 MM cells by serial dilutions of genomic DNA starting at 1.5 micrograms. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ***P<0.005, ****P<0.001).


G : Enrichment density of DRIP - seq signals at NEAT1 / Che1 co - localizing sites in Kms27 MM cells identified in siRNA control ( siControl ) ( grey ) and siRNA Che - 1 ( siChe - 1A ) ( dark green ) samples . Enrichment density was calculated on the relative size of each peak ( start to end of each fragment ) extended 2000 bp upstream and downstream . Right frame represents the signal obtained from 14654 randomly selected sites from the same experiments .

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

G: Enrichment density of DRIP-seq signals at NEAT1/Che1 co-localizing sites in Kms27 MM cells identified in siRNA control (siControl) (grey) and siRNA Che-1 (siChe-1A) (dark green) samples. Enrichment density was calculated on the relative size of each peak (start to end of each fragment) extended 2000 bp upstream and downstream. Right frame represents the signal obtained from 14654 randomly selected sites from the same experiments.


D : ( Top ) Representative confocal images of Kms27 MM cells transfected and treated as in C and immunostained with S9 . 6 antibody . Scale bar 10 μm . ( Bottom ) Box plot showing S9 . 6 signal nuclear intensity per cell ( Arbitrary Unit - AU ) . siChe - 1A cells are described in dark grey , siChe1A plus CX5461 in green color , while siControl in grey color . Median scores per sample are written at the top of the median red line . P value : < 1 . 30 - 54 . ANOVA test was performed to evaluate statistical significance . The red center line denotes the median value ( 50th percentile ) while the black box contains the 25th to 75th percentiles of data . The whiskers mark the 5th and 95th percentiles .

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

D: (Top) Representative confocal images of Kms27 MM cells transfected and treated as in C and immunostained with S9.6 antibody. Scale bar 10 μm. (Bottom) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in dark grey, siChe1A plus CX5461 in green color, while siControl in grey color. Median scores per sample are written at the top of the median red line. P value: <1.30-54. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


E : ( Top ) Dot blot analysis of decreasing amounts of genomic DNA extracts from Kms27 MM cells depleted or not for Che - 1 expression ( siChe - 1A ) and treated with CX5461 , were probed using abs directed against RNA : DNA hybrids . ( Bottom ) Quantification of S9 . 6 levels by densitometric analysis . Values were normalized to a Methylene blue loading control . Error bars represent the standard error of three different biological experiments ( Two Tailed T test * * * * P < 0 . 001 ) .

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

E: (Top) Dot blot analysis of decreasing amounts of genomic DNA extracts from Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) and treated with CX5461, were probed using abs directed against RNA:DNA hybrids. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ****P<0.001).