Figure 1 ( B ) RNA samples extracted from normal breast epithelial cell , MCF10A , and different subtypes of breast cancer cell lines as indicated were subjected to RT - qPCR analysis to examine the expression of circPVT1 ( n = 3 biological replicates , ± s . e . m . , ns : non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . ( C ) RNA samples extracted from 28 pairs of ER - positive ( ER + ) breast tumor and adjacent normal tissues and 14 pairs of ER - negative ( ER - ) breast tumor and adjacent normal tissues were subjected to RT - qPCR analysis to examine the expression of circPVT1 ( ± s . e . m . , * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test ) . MCF7 cells were infected with control shRNA ( sh - CTL ) or two independent shRNAs specifically targeting circPVT1 ( sh - circPVT1 # 1 and sh - circPVT1 # 2 ) were subjected to RNA extraction and RT - qPCR analysis to examine the expression of circPVT1 ( H ) ( n = 3 biological replicates , ± s . e . m . , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . MCF7 cells were infected with control shRNA ( sh - CTL ) or two independent shRNAs specifically targeting circPVT1 ( sh - circPVT1 # 1 and sh - circPVT1 # 2 ) were subjected to colony formation assay ( I ) ( n = 3 biological replicates , ± s . e . m . , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . ( J ) Quantification of the crystal violet dye as shown in ( I ) ( n = 3 biological replicates , ± s . e . m . , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . ( O ) Tamoxifen - resistant MCF7 cells transfected with si - CTL , si - circPVT1 # 1 , or si - circPVT1 # 2 were treated with or without tamoxifen ( Tam , 5 μM , 72 h ) followed by cell proliferation

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

Figure 1(B) RNA samples extracted from normal breast epithelial cell, MCF10A, and different subtypes of breast cancer cell lines as indicated were subjected to RT-qPCR analysis to examine the expression of circPVT1 (n = 3 biological replicates, ±s.e.m., ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).(C) RNA samples extracted from 28 pairs of ER-positive (ER+) breast tumor and adjacent normal tissues and 14 pairs of ER-negative (ER-) breast tumor and adjacent normal tissues were subjected to RT-qPCR analysis to examine the expression of circPVT1 (±s.e.m., *P < 0.05, **P < 0.01 by two-tailed Student's t-test).MCF7 cells were infected with control shRNA (sh-CTL) or two independent shRNAs specifically targeting circPVT1 (sh-circPVT1#1 and sh-circPVT1#2) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of circPVT1 (H) (n = 3 biological replicates, ± s.e.m., ***P < 0. 001 by two-tailed Student's t-test).MCF7 cells were infected with control shRNA (sh-CTL) or two independent shRNAs specifically targeting circPVT1 (sh-circPVT1#1 and sh-circPVT1#2) were subjected to colony formation assay (I) (n = 3 biological replicates, ± s.e.m., ***P < 0. 001 by two-tailed Student's t-test). (J) Quantification of the crystal violet dye as shown in (I) (n = 3 biological replicates, ± s.e.m., ***P < 0.001 by two-tailed Student's t-test).(O) Tamoxifen-resistant MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were treated with or without tamoxifen (Tam, 5 μM, 72 h) followed by cell proliferation assay


Figure 2 ( B ) Total RNAs extracted from MCF7 cells were incubated with or without RNase R ( 10 units / μg RNA ) at 37 ℃ for duration as indicated , followed by RT - qPCR analysis to examine the expression of PVT1 or circPVT1 ( C ) MCF7 cells were treated with Actinomycin D ( 10 g / ml ) for duration as indicated , followed by RT - qPCR analysis to examine the expression of PVT1 or circPVT1 ( F ) MCF7 cells were subjected to cellular fractionation followed by RNA extraction and RT - qPCR analysis to quantify the amount of circRNA in both nucleus and cytosol of the cells . ACTIN and U6 snoRNA were served as purity control for cytosolic and nuclear fractions , respectively ( n = 3 biological replicates , ± s . e . m . ) . ( G ) MCF7 cells transfected with si - CTL , si - circPVT1 # 1 , or si - circPVT1 # 2 were subjected to RNA - FISH analysis using probe specifically targeting circPVT1 . Red : circPVT1 ; Blue : DAPI .

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

Figure 2(B) Total RNAs extracted from MCF7 cells were incubated with or without RNase R (10 units/μg RNA) at 37℃ for duration as indicated, followed by RT-qPCR analysis to examine the expression of PVT1 or circPVT1 (C) MCF7 cells were treated with Actinomycin D (10 g/ml) for duration as indicated, followed by RT-qPCR analysis to examine the expression of PVT1 or circPVT1(F) MCF7 cells were subjected to cellular fractionation followed by RNA extraction and RT-qPCR analysis to quantify the amount of circRNA in both nucleus and cytosol of the cells. ACTIN and U6 snoRNA were served as purity control for cytosolic and nuclear fractions, respectively (n = 3 biological replicates, ± s.e.m.).(G) MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were subjected to RNA-FISH analysis using probe specifically targeting circPVT1. Red: circPVT1; Blue: DAPI.


Figure 3 ( D , E ) UCSC genome browser views of RNA - seq as described in ( A ) for TFF1 ( D ) and GREB1 ( E ) are shown . ( F ) MCF7 cells transfected with si - CTL , si - circPVT1 # 1 , or si - circPVT1 # 2 were treated with or without estrogen ( E2 , 10 - 7 M , 6 h ) followed by RNA extraction and RT - qPCR analysis to examine the expression of genes as indicated ( G ) MCF7 cells infected with sh - CTL , sh - circPVT1 # 1 , or sh - circPVT1 # 2 were treated with or without estrogen ( E2 , 10 - 7 M , 6 h ) followed by RNA extraction and RT - qPCR analysis to examine the expression of genes as

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

Figure 3(D, E) UCSC genome browser views of RNA-seq as described in (A) for TFF1 (D) and GREB1 (E) are shown.(F) MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were treated with or without estrogen (E2, 10-7 M, 6 h) followed by RNA extraction and RT-qPCR analysis to examine the expression of genes as indicated (G) MCF7 cells infected with sh-CTL, sh-circPVT1#1, or sh-circPVT1#2 were treated with or without estrogen (E2, 10-7 M, 6 h) followed by RNA extraction and RT-qPCR analysis to examine the expression of genes as indicated


Figure 4MCF7 cells transfected with control siRNA ( si - CTL ) or siRNAs specifically targeting circPVT1 ( si - circPVT1 # 1 and si - circPVT1 # 2 ) were treated with or without estrogen ( E2 , 10 - 7 M , 6 h ) followed by immunoblotting analysis ( D ) to examine the expression of ERα ( E ) Tumor samples as described in Fig 1K were subjected to RNA extraction and RT - qPCR analysis to examine the expression of ESR1 ( n = 6 biological replicates , ± s . e . m . , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . ( H ) Wild - type circPVT1 - luc ( circPVT1 - luc ( WT ) ) and its mutant form with the potential miR - 181a - 2 - 3p or miR - 6715b - 5p binding site mutated ( circPVT1 ( MT ) - luc ) were transfected into HEK293T cells with or without CTL , miR - 181a - 2 - 3p , or miR - 6715b - 5p mimic followed by luciferase activity measurement ( n = 3 biological replicates , ± s . e . m . , ns : non - significant , * * P < 0 . 01 by two - tailed Student ' s t - test ) . MCF7 cells transfected with si - CTL or si - circPVT1 in the presence or absence of miR - 181a - 2 - 3p inhibitor were subjected to RT - qPCR analysis using antibodies as indicated ( n = 3 biological replicates , ± s . e . m . , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . MCF7 cells transfected with si - CTL or si - circPVT1 in the presence or absence of miR - 181a - 2 - 3p inhibitor to examine the expression of genes as indicated and immunoblotting analysis ( M ) using antibodies as indicatedMCF7 cells transfected with si - CTL or si - circPVT1 in the presence or absence of miR - 181a - 2 - 3p inhibitor were subjected to RT - qPCR analysis using antibodies as indicated ( n = 3 biological replicates , ± s . e . m . , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . ( P ) MCF7 cells were infected with control shRNA ( sh - CTL ) or shRNAs specifically targeting circPVT1 ( sh - circPVT1 ) in the presence or absence of miR - 181a - 2 - 3p inhibitor were subjected to colony formation assay . ( Q ) Quantification of the crystal violet dye as shown in ( P ) ( n = 3 biological replicates , ± s . e . m . , * * P < 0 . 01 by two - tailed Student ' s t - test ) .

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

Figure 4MCF7 cells transfected with control siRNA (si-CTL) or siRNAs specifically targeting circPVT1 (si-circPVT1#1 and si-circPVT1#2) were treated with or without estrogen (E2, 10-7 M, 6 h) followed by immunoblotting analysis (D) to examine the expression of ERα(E) Tumor samples as described in Fig 1K were subjected to RNA extraction and RT-qPCR analysis to examine the expression of ESR1 (n = 6 biological replicates, ±s.e.m., ***P < 0.001 by two-tailed Student's t-test).(H) Wild-type circPVT1-luc (circPVT1-luc (WT)) and its mutant form with the potential miR-181a-2-3p or miR-6715b-5p binding site mutated (circPVT1 (MT)-luc) were transfected into HEK293T cells with or without CTL, miR-181a-2-3p, or miR-6715b-5p mimic followed by luciferase activity measurement (n = 3 biological replicates, ±s.e.m., ns: non-significant, **P < 0.01 by two-tailed Student's t-test).MCF7 cells transfected with si-CTL or si-circPVT1 in the presence or absence of miR-181a-2-3p inhibitor were subjected to RT-qPCR analysis using antibodies as indicated (n = 3 biological replicates, ±s.e.m., **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).MCF7 cells transfected with si-CTL or si-circPVT1 in the presence or absence of miR-181a-2-3p inhibitor to examine the expression of genes as indicated and immunoblotting analysis (M) using antibodies as indicatedMCF7 cells transfected with si-CTL or si-circPVT1 in the presence or absence of miR-181a-2-3p inhibitor were subjected to RT-qPCR analysis using antibodies as indicated (n = 3 biological replicates, ±s.e.m., **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).(P) MCF7 cells were infected with control shRNA (sh-CTL) or shRNAs specifically targeting circPVT1 (sh-circPVT1) in the presence or absence of miR-181a-2-3p inhibitor were subjected to colony formation assay. (Q) Quantification of the crystal violet dye as shown in (P) (n = 3 biological replicates, ± s.e.m., **P < 0.01 by two-tailed Student's t-test).


Figure 5 ( F ) Tumor samples as described in Fig 1K were subjected RT - qPCR analysis to examine the expression of genes as indicatedTumor samples as described in Fig 1M were subjected to RNA extraction and RT - qPCR analysis ( G ) Tumor samples as described in Fig 1M were subjected to examine the immunohistochemistry analysis ( H ) using anti - CD8 antibody . Dark brown staining indicates CD8 - positive cell .

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

Figure 5(F) Tumor samples as described in Fig 1K were subjected RT-qPCR analysis to examine the expression of genes as indicatedTumor samples as described in Fig 1M were subjected to RNA extraction and RT-qPCR analysis (G)Tumor samples as described in Fig 1M were subjected to examine the immunohistochemistry analysis (H) using anti-CD8 antibody. Dark brown staining indicates CD8-positive cell.


Figure 6 ( A ) MCF7 cells infected with control shRNA ( sh - CTL ) or shRNAs specifically targeting MAVS ( sh - MAVS ) or STING ( sh - MAVS ) were transfected with control siRNA ( si - CTL ) or siRNA specifically targeting circPVT1 ( si - circPVT1 ) followed by RT - qPCR analysis to examine the expression of genes as indicated ( n = 3 biological replicates , ± s . e . m . , ns : non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . MCF7 cells infected with sh - CTL , sh - MAVS , sh - MDA5 , or sh - RIGI were transfected with si - CTL or si - circPVT1 followed by using antibodies as indicated and RT - qPCR analysis ( C ) to examine the expression of genes as indicated ( n = 3 biological replicates , ± s . e . m . , ns : non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed Student ' s t - test ) . HEK293T cells transfected with HA - tagged MAVS - FL , MAVS - NTD , or MAVS - CTD were subjected to RIP using anti - HA antibody followed by immunoblotting analysis ( K ) using antibodies as indicated ( L ) HEK293T cells transfected with HA - tagged MAVS and Flag - tagged RIGI in the presence or absence of circPVT1 were subjected to immunoprecipitation ( IP ) with anti - Flag M2 agarose followed by immunoblotting analysis using antibodies as indicated .

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

Figure 6(A) MCF7 cells infected with control shRNA (sh-CTL) or shRNAs specifically targeting MAVS (sh-MAVS) or STING (sh-MAVS) were transfected with control siRNA (si-CTL) or siRNA specifically targeting circPVT1 (si-circPVT1) followed by RT-qPCR analysis to examine the expression of genes as indicated (n = 3 biological replicates, ±s.e.m., ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).MCF7 cells infected with sh-CTL, sh-MAVS, sh-MDA5, or sh-RIGI were transfected with si-CTL or si-circPVT1 followed by using antibodies as indicated and RT-qPCR analysis (C) to examine the expression of genes as indicated (n = 3 biological replicates, ±s.e.m., ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student's t-test).HEK293T cells transfected with HA-tagged MAVS-FL, MAVS-NTD, or MAVS-CTD were subjected to RIP using anti-HA antibody followed by immunoblotting analysis(K) using antibodies as indicated(L) HEK293T cells transfected with HA-tagged MAVS and Flag-tagged RIGI in the presence or absence of circPVT1 were subjected to immunoprecipitation (IP) with anti-Flag M2 agarose followed by immunoblotting analysis using antibodies as indicated.


Figure 7MCF7 cells transfected with control ASO ( ASO - CTL ) or ASO specifically targeting circPVT1 ( ASO - circPVT1 ) were subjected to RT - qPCR analysis to examine the expression of genes as indicatedMCF7 cells transfected with control ASO ( ASO - CTL ) or ASO specifically targeting circPVT1 ( ASO - circPVT1 ) were subjected immunoblotting analysis E ) using antibodies as indicatedMCF7 cells transfected with control ASO ( ASO - CTL ) or ASO specifically targeting circPVT1 ( ASO - circPVT1 ) were subjected to RT - qPCR analysis to examine the expression of genes as indicated ( I , J ) MCF7 cells were treated with ASO - CTL , ASO - circPVT1 ( 50 nM ) , or fulvestrant ( ICI , 1 μM ) for duration as indicated followed by cell proliferation assay ( I ) and colony formation ( J ) ( n = 3 biological replicates , ± s . e . m . , * * p < 0 . 01 , * * * p < 0 . 001 , day 4 by two - tailed Student ' s t - test ) . Tumor samples as described in ( K ) were subjected to RNA extraction and RT - qPCR analysis to examine the expression of genes as indicatedTumor samples as described in ( K ) were subjected to RNA extraction and RT - qPCR analysis to examine the expression of genes as

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

Figure 7MCF7 cells transfected with control ASO (ASO-CTL) or ASO specifically targeting circPVT1 (ASO-circPVT1) were subjected to RT-qPCR analysis to examine the expression of genes as indicatedMCF7 cells transfected with control ASO (ASO-CTL) or ASO specifically targeting circPVT1 (ASO-circPVT1) were subjected immunoblotting analysis E) using antibodies as indicatedMCF7 cells transfected with control ASO (ASO-CTL) or ASO specifically targeting circPVT1 (ASO-circPVT1) were subjected to RT-qPCR analysis to examine the expression of genes as indicated(I, J) MCF7 cells were treated with ASO-CTL, ASO-circPVT1 (50 nM), or fulvestrant (ICI, 1 μM) for duration as indicated followed by cell proliferation assay (I) and colony formation (J) (n = 3 biological replicates, ± s.e.m., **p < 0.01, ***p < 0.001, day 4 by two-tailed Student's t-test).Tumor samples as described in (K) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of genes as indicatedTumor samples as described in (K) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of genes as indicated


Figure 1 ( A , B ) Representative Western blot ( A ) and quantification ( B ) of p62 associated with mitochondria from spinal cords . Protein levels are normalized by subunit ATPβ of mitochondria ( Complex V ) . Mitochondrial p62 levels are increased at 120 days ( symptomatic stage ) in SOD1 - G93A spinal cord relative to Non Tg and wild type SOD1 ( wtSOD1 ) . Results are expressed as mean ± SEM relative to Non Tg at 30 days ; n = 8 mice ( 4 males and 4 females ) . At 120 days , * * p = 0 . 0018 ( Non Tg vs . SOD1 - G93A ) and * * p = 0 . 0011 ( SOD1 - G93A vs . wtSOD1 ) by paired one - way ANOVA with Tukey ' s correction . No other statistically significant differences were found ( paired Friedman ' s test with Dunn ' s correction at 30 days and paired one - way ANOVA with Tukey ' s correction at 60 and 90 days ) . ( C , D ) Representative Western blot ( C ) and quantification ( D ) of Tim23 in homogenates from spinal cords using β - actin as normalizer . Results are expressed as mean ± SEM and relative to Non Tg at 30 days ; n = 6 mice ( 3 males and 3 females ) . * * * p = 0 . 0002 ( Non Tg vs . SOD1 - G93A at 120 days ) by paired one - way ANOVA with Tukey ' s correction . No other statistically significant differences were found ( paired Friedman ' s test with Dunn ' s correction at 30 days and paired one - way ANOVA with Tukey ' s correction at 60 and 90 days ) . Tim23 is decreased in SOD1 - G93A spinal cord relative to Non Tg at 120 days . ( E , F ) Western blot ( E ) and quantification ( F ) of COXI in homogenates from spinal cords . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and relative to Non Tg at 30 days ; n = 6 mice ( 3 males and 3 females ) . * p = 0 . 017 ( Non Tg vs . SOD1 - G93A at 120 days ) by paired Friedman ' s test with Dunn ' s correction . No other statistically significant differences were found ( paired Friedman ' s test with Dunn ' s correction at 30 and 60 days and paired one - way ANOVA with Tukey ' s correction at 90 days ) . COX1 is decreased in SOD1 - G93A spinal cord relative to Non Tg at 120 days . G ) qPCR ( fold change ) of PGC1α mRNA normalized by β - actin mRNA . Results are expressed as mean ± SEM and fold change of Non Tg at 30 days ; n = 6 ( 3 males and 3 females ) for 30 and 120 days , n = 4 ( 2 males and 2 females ) for 60 days and n = 5 ( 3 males and 2 females ) for 90 days . * p = 0 . 035 at 90 days and * * * p = 0 . 0007 at 120 days . Paired Student ' s t test ( for 60 , 90 and 120 days ) and paired Wilcoxon ' s test ( for 30 days ) were used for comparisons .

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

Figure 1(A, B) Representative Western blot (A) and quantification (B) of p62 associated with mitochondria from spinal cords. Protein levels are normalized by subunit ATPβ of mitochondria (Complex V). Mitochondrial p62 levels are increased at 120 days (symptomatic stage) in SOD1-G93A spinal cord relative to Non Tg and wild type SOD1 (wtSOD1). Results are expressed as mean ± SEM relative to Non Tg at 30 days; n=8 mice (4 males and 4 females). At 120 days, **p= 0.0018 (Non Tg vs. SOD1-G93A) and **p= 0.0011 (SOD1-G93A vs. wtSOD1) by paired one-way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one-way ANOVA with Tukey's correction at 60 and 90 days).(C, D) Representative Western blot (C) and quantification (D) of Tim23 in homogenates from spinal cords using β-actin as normalizer. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n=6 mice (3 males and 3 females). ***p= 0.0002 (Non Tg vs. SOD1-G93A at 120 days) by paired one-way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one-way ANOVA with Tukey's correction at 60 and 90 days). Tim23 is decreased in SOD1-G93A spinal cord relative to Non Tg at 120 days.(E, F) Western blot (E) and quantification (F) of COXI in homogenates from spinal cords. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n=6 mice (3 males and 3 females). *p= 0.017 (Non Tg vs. SOD1-G93A at 120 days) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 and 60 days and paired one-way ANOVA with Tukey's correction at 90 days). COX1 is decreased in SOD1-G93A spinal cord relative to Non Tg at 120 days.G) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA. Results are expressed as mean ± SEM and fold change of Non Tg at 30 days; n= 6 (3 males and 3 females) for 30 and 120 days, n= 4 (2 males and 2 females) for 60 days and n= 5 (3 males and 2 females) for 90 days. *p= 0.035 at 90 days and ***p= 0.0007 at 120 days. Paired Student's t test (for 60, 90 and 120 days) and paired Wilcoxon's test (for 30 days) were used for comparisons.


Figure 2A ) Representative images of mt - Keima expressing spinal cord sections of SOD1 - G93A and Non Tg mice at 90 days . Mt - Keima fluorescence with excitation at 458nm is pseudocolored in green and at 543nm is pseudocolored in red . Emissions were recorded sequentially at 600 - 650nm . Scale bar , 150 µm . B ) Representative images of mt - Keima expressing motor neurons in the ventral horn of SOD1 - G93A and Non Tg mice at 90 days , imaged and pseudocolored Scale bar , 20 µm . C ) Quantification of the rate of mitophagy in motor neurons expressed as the percentage of area with high ratio ( 543 / 458nm ) signal normalized by the total mitochondria area . Data were collected from mice at 60 days ( n = 3 , 1 male and 2 females ) , 90 days ( n = 4 , 2 males and 2 females ) , and 120 days ( n = 4 , 2 males and 2 females ) . Number of neurons imaged : 60 days , n = 77 Non Tg and 87 SOD1 - G93A ; 90 days , n = 118 Non Tg and 173 SOD1 - G93A ; 120 days , n = 110 Non Tg and 130 SOD1 - G93A . Comparisons within pairs showed that mitophagy is increased in SOD1 - G93A spinal cords at 60 and 90 days compared to Non Tg . Results are expressed as mean ± SEM . For 60 days , * * p = 0 . 0022 ; for 90 days , * * * p = 0 . 0001 ; for 120 days , p = 0 . 094 , all by unpaired Mann - Whitney test . Comparisons along time showed that mitophagy increases over time in both genotypes . For Non Tg , by unpaired Kruskal - Wallis ' s test with Dunn ' s correction , * * p = 0 . 0046 and * * * p = 0 . 0001 . For SOD1 - G93A , by unpaired Kruskal - Wallis ' s test with Dunn ' s correction , * * p = 0 . 0077 and * * * p = 0 . 0001 .

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

Figure 2A) Representative images of mt-Keima expressing spinal cord sections of SOD1-G93A and Non Tg mice at 90 days. Mt-Keima fluorescence with excitation at 458nm is pseudocolored in green and at 543nm is pseudocolored in red. Emissions were recorded sequentially at 600-650nm. Scale bar, 150 µm.B) Representative images of mt-Keima expressing motor neurons in the ventral horn of SOD1-G93A and Non Tg mice at 90 days, imaged and pseudocolored Scale bar, 20 µm.C) Quantification of the rate of mitophagy in motor neurons expressed as the percentage of area with high ratio (543/458nm) signal normalized by the total mitochondria area. Data were collected from mice at 60 days (n= 3, 1 male and 2 females), 90 days (n= 4, 2 males and 2 females), and 120 days (n= 4, 2 males and 2 females). Number of neurons imaged: 60 days, n= 77 Non Tg and 87 SOD1-G93A; 90 days, n= 118 Non Tg and 173 SOD1-G93A; 120 days, n= 110 Non Tg and 130 SOD1-G93A. Comparisons within pairs showed that mitophagy is increased in SOD1-G93A spinal cords at 60 and 90 days compared to Non Tg. Results are expressed as mean ± SEM. For 60 days, **p= 0.0022; for 90 days, ***p= 0.0001; for 120 days, p= 0.094, all by unpaired Mann-Whitney test. Comparisons along time showed that mitophagy increases over time in both genotypes. For Non Tg, by unpaired Kruskal-Wallis's test with Dunn's correction, **p= 0.0046 and ***p= 0.0001. For SOD1-G93A, by unpaired Kruskal-Wallis's test with Dunn's correction, **p=0.0077 and *** p= 0.0001.


Figure 3Representative Western blot ( A ) of Parkin levels in spinal cord homogenates . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and relative to Non Tg control at 30 days ; n = 8 ( 4 males and 4 females ) . Parkin levels are decreased in SOD1 - G93A at 90 and 120 days compared to Non Tg . quantification ( B ) of Parkin levels in spinal cord homogenates . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and relative to Non Tg control at 30 days ; n = 8 ( 4 males and 4 females ) . Paired Wilcoxon ' s test at 90 days , * * p = 0 . 0078 ; paired Student ' s t test at 120 days , * * p = 0 . 0054 . Paired Wilcoxon ' s test for 30 and 60 days showed no statistically significant differences . Parkin levels are decreased in SOD1 - G93A at 90 and 120 days compared to Non Tg . C ) qPCR ( fold change ) of Parkin mRNA normalized by β - actin mRNA . Results are expressed as mean ± SEM and fold change relative to Non Tg at 30 days of age ; n = 6 ( 3 males and 3 females ) for 30 and 120 days , n = 4 ( 2 males and 2 females ) for 60 days and n = 5 ( 3 males and 2 females ) for 90 days . No statistically significant differences were found by paired Student ' s t test ( 90 days ) and Wilcoxon ' s test ( 30 , 60 and 120 days ) . D , E ) Representative Western blot ( D ) and quantification ( E ) of Parkin levels in spinal cord mitochondria at 120 days . Protein levels are normalized by Complex V . Parkin is reduced in spinal cord mitochondria of SOD1 - G93A mice at 120 days . Results are expressed as mean ± SEM and shown as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * * p = 0 . 0078 , by Wilcoxon ' s t test .

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

Figure 3Representative Western blot (A) of Parkin levels in spinal cord homogenates. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg control at 30 days; n= 8 (4 males and 4 females). Parkin levels are decreased in SOD1-G93A at 90 and 120 days compared to Non Tg.quantification (B) of Parkin levels in spinal cord homogenates. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg control at 30 days; n= 8 (4 males and 4 females). Paired Wilcoxon's test at 90 days, **p= 0.0078; paired Student's t test at 120 days, **p= 0.0054. Paired Wilcoxon's test for 30 and 60 days showed no statistically significant differences. Parkin levels are decreased in SOD1-G93A at 90 and 120 days compared to Non Tg.C) qPCR (fold change) of Parkin mRNA normalized by β-actin mRNA. Results are expressed as mean ± SEM and fold change relative to Non Tg at 30 days of age; n= 6 (3 males and 3 females) for 30 and 120 days, n= 4 (2 males and 2 females) for 60 days and n= 5 (3 males and 2 females) for 90 days. No statistically significant differences were found by paired Student's t test (90 days) and Wilcoxon's test (30, 60 and 120 days).D, E) Representative Western blot (D) and quantification (E) of Parkin levels in spinal cord mitochondria at 120 days. Protein levels are normalized by Complex V. Parkin is reduced in spinal cord mitochondria of SOD1-G93A mice at 120 days. Results are expressed as mean ± SEM and shown as percent of Non Tg; n=8 (4 males and 4 females); **p= 0.0078, by Wilcoxon's t test.


Figure 4A ) Representative Western blot of spinal cord homogenates at 130 days , showing that Parkin is absent in PKO and PKO / G93A mice . B ) Parkin quantification indicates that Parkin is decreased in G93A mice relative to Non Tg at 130 days . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * * p = 0 . 0078 , by Wilcoxon ' s t test . C ) Quantification of SOD1 at 130 days indicates that Parkin knockout does not affect the levels of transgenic mutant human SOD1 expressed in SOD1 - G93A spinal cord . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; no statistically significant differences relative to SOD1 expression were found between G93A and PKO / G93A , p = 0 . 214 , by paired Student ' s t test . Differences between Non Tg and G93A ( by paired Friedman ' s test with Dunn ' s correction ) , * p = 0 . 020 and PKO and PKO / G93A ( by paired one - way ANOVA with Tukey ' s correction ) , * * * p = 0 . 0001 . D , E ) SOD1 filter trap assay ( D ) of spinal cord homogenates at 130 days and quantification of dot intensities ( E ) , showing that PKO / G93A mice have less SOD1 aggregates than G93A . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * * p = 0 . 0078 by paired Wilcoxon ' s test . F ) Representative images of 130 days old lumbar spinal cords sections immunostained for p62 . Scale bar , 200 µm . G ) Quantification of the average number of large p62 - positive inclusions per section at 130 days indicates that PKO PKO / G93A mice have less SOD1 aggregates than G93A . Results are expressed as mean ± SEM ; n = 7 ( 3 males and 4 females ) mice for Non Tg and PKO , n = 10 ( 5 males and 5 females ) mice for G93A and n = 9 ( 4 males and 5 females ) mice for PKO / G93A ; * p = 0 . 010 by unpaired Mann - Whitney test .

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

Figure 4A) Representative Western blot of spinal cord homogenates at 130 days, showing that Parkin is absent in PKO and PKO/G93A mice.B) Parkin quantification indicates that Parkin is decreased in G93A mice relative to Non Tg at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); **p=0.0078, by Wilcoxon's t test.C) Quantification of SOD1 at 130 days indicates that Parkin knockout does not affect the levels of transgenic mutant human SOD1 expressed in SOD1-G93A spinal cord. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); no statistically significant differences relative to SOD1 expression were found between G93A and PKO/G93A, p= 0.214, by paired Student's t test. Differences between Non Tg and G93A (by paired Friedman's test with Dunn's correction), *p= 0.020 and PKO and PKO/G93A (by paired one-way ANOVA with Tukey's correction), ***p= 0.0001.D, E) SOD1 filter trap assay (D) of spinal cord homogenates at 130 days and quantification of dot intensities (E), showing that PKO/G93A mice have less SOD1 aggregates than G93A. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); **p= 0.0078 by paired Wilcoxon's test.F) Representative images of 130 days old lumbar spinal cords sections immunostained for p62. Scale bar, 200 µm.G) Quantification of the average number of large p62-positive inclusions per section at 130 days indicates that PKO PKO/G93A mice have less SOD1 aggregates than G93A. Results are expressed as mean ± SEM; n= 7 (3 males and 4 females) mice for Non Tg and PKO, n= 10 (5 males and 5 females) mice for G93A and n= 9 (4 males and 5 females) mice for PKO/G93A; *p= 0.010 by unpaired Mann-Whitney test.


Figure 5A ) Immunostaining of anterior horn of lumbar spinal cord for ChAT ( in green ) and p62 ( in red ) at 130 days . Scale bar , 10 µm . B ) Quantification of the percentage of ChAT - positive motor neurons ( MN ) containing p62 - positive inclusions at 130 days . PKO PKO / G93A MN have less SOD1 aggregates than G93A , while Non Tg and PKO MN have none . Data were collected from n = 4 ( 2 males and 2 females ) mice for Non Tg and PKO and n = 6 ( 3 males and 3 females ) for G93A and PKO / G93A ( males and females ) . Number of images recorded for each genotype : n = 9 for Non Tg and PKO , n = 16 for G93A and n = 12 for PKO / G93A . Results are expressed as mean ± SEM ; * p = 0 . 032 , by unpaired one - tailed Student ' s t test . C ) Representative Western blots of OPTN , p62 and VCP in spinal cord mitochondrial fractions at 130 days . Protein levels are normalized by Complex V . The quantifications show that mitochondria of PKO / G93A mice have less mitophagy adaptor proteins than G93A relative to Complex V . D ) Quantification of OPTN relative to Complex V at 130 days . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * p = 0 . 039 by paired Student ' s t test . E ) Quantification of p62 , relative to Complex V at 130 days . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * * p = 0 . 0029 by paired Student ' s t test . F ) VCP was quantified using Complex V as loading reference at 130 days . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * p = 0 . 044 by paired Student ' s t test .

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

Figure 5A) Immunostaining of anterior horn of lumbar spinal cord for ChAT (in green) and p62 (in red) at 130 days. Scale bar, 10 µm.B) Quantification of the percentage of ChAT-positive motor neurons (MN) containing p62-positive inclusions at 130 days. PKO PKO/G93A MN have less SOD1 aggregates than G93A, while Non Tg and PKO MN have none. Data were collected from n= 4 (2 males and 2 females) mice for Non Tg and PKO and n= 6 (3 males and 3 females) for G93A and PKO/G93A (males and females). Number of images recorded for each genotype: n= 9 for Non Tg and PKO, n= 16 for G93A and n= 12 for PKO/G93A. Results are expressed as mean ± SEM; *p= 0.032, by unpaired one-tailed Student's t test.C) Representative Western blots of OPTN, p62 and VCP in spinal cord mitochondrial fractions at 130 days. Protein levels are normalized by Complex V. The quantifications show that mitochondria of PKO/G93A mice have less mitophagy adaptor proteins than G93A relative to Complex V.D) Quantification of OPTN relative to Complex V at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); *p= 0.039 by paired Student's t test.E) Quantification of p62, relative to Complex V at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); **p= 0.0029 by paired Student's t test.F) VCP was quantified using Complex V as loading reference at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); *p= 0.044 by paired Student's t test.


Figure 6A ) Kaplan - Meier survival analysis ( Log - Rank test with Holm - Sidak ' s correction for multiple comparisons ) . Mean age of death ( ± SEM ) for G93A was 169 . 3 ± 2 . 5 days and for PKO / G93A was 185 . 6 ± 2 . 2 days , indicating that Parkin knockout prolongs lifespan of SOD1 - G93A mice ; n = 19 for Non Tg ( 9 males and 10 females ) , n = 20 for PKO ( 10 males and 10 females ) , n = 18 for G93A ( 8 males and 10 females ) and n = 19 for PKO / G93A ( 9 males and 10 females ) ; p = 0 . 0000285 . B ) Body weight changes over time , relative to body weight at 45 days that is set at 100 % for each mouse . Parkin knockout delays body weight loss in SOD1 - G93A mice . Results are expressed as mean ± SEM ; n = 19 for Non Tg ( 9 males and 10 females ) , n = 20 for PKO ( 10 males and 10 females ) , n = 18 for G93A ( 8 males and 10 females ) and n = 19 for PKO / G93A ( 9 males and 10 females ) ; * p = 0 . 017 ( at 150 days ) , * p = 0 . 004 ( at 164 days ) , * p = 0 . 002 ( at 171 days ) and * * p = 0 . 001 ( at 178 days ) , by two - way ANOVA with repeated measures and with Holm - Sidak ' s correction for multiple comparisons . C ) Clasping score changes over time . Parkin knockout does not affect clasping onset in SOD1 - G93A mice . An intermediate score of 2 . 5 is indicated by the dotted line . Results are expressed as mean ± SEM ; n = 19 for Non Tg ( 9 males and 10 females ) , n = 20 for PKO ( 10 males and 10 females ) , n = 18 for G93A ( 8 males and 10 females ) and n = 19 for PKO / G93A ( 9 males and 10 females ) . D ) Representative images of NMJs in the soleus muscle at 130 days . Muscles were immunostained for neurofilament H ( NF - H , in red ) and stained with α - bungarotoxin ( α - BTX , in green ) . Scale bar , 20 µm . E ) Quantification of the percentage of innervated NMJs at 130 days indicates that Parkin knockout decreases denervation in SOD1 - G93A mice . Results are expressed as mean ± SEM and were collected from n = 6 mice per group ( 3 males and 3 females ) ; n of images evaluated = 49 for Non Tg ( with 483 NMJs counted ) , 46 for PKO ( 396 NMJs counted ) , 47 for G93A ( 413 NMJs assessed ) and 38 for PKO / G93A ( 325 NMJs counted ) ; * * * p = 0 . 0001 , by unpaired Mann - Whitney test . F ) Representative images of lumbar spinal cord sections at 130 days immunostained with ChAT . Scale bar , 200 µm . G ) Quantification of ChAT - positive MN in the spinal cord ventral horn ( dotted areas ) at 130 days shows that Parkin knockout decreases MN loss in the spinal cord of SOD1 - G93A mice . Results are expressed as mean ± SEM ; n = 10 mice per group in Non Tg , PKO and G93A ( 5 males and 5 females ) and n = 9 for PKO / G93A ( 4 males and 5 females ) ; * p = 0 . 031 , by unpaired Student ' s t test . H H ) Representative images of lumbar spinal cords sections immunostained for GFAP at 130 days . Scale bar , 200 µm . I ) Quantification of the average GFAP staining intensity at 130 days indicates that Parkin knockout decreases spinal cord astrogliosis in SOD1 - G93Amice . Results are expressed as mean ± SEM ; n = 10 mice per group ( 5 males and 5 females ) ; * p = 0 . 033 , by paired Student ' s t test .

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

Figure 6A) Kaplan-Meier survival analysis (Log-Rank test with Holm-Sidak's correction for multiple comparisons). Mean age of death (± SEM) for G93A was 169.3 ± 2.5 days and for PKO/G93A was 185.6 ± 2.2 days, indicating that Parkin knockout prolongs lifespan of SOD1-G93A mice; n=19 for Non Tg (9 males and 10 females), n= 20 for PKO (10 males and 10 females), n= 18 for G93A (8 males and 10 females) and n= 19 for PKO/G93A (9 males and 10 females); p= 0.0000285.B) Body weight changes over time, relative to body weight at 45 days that is set at 100% for each mouse. Parkin knockout delays body weight loss in SOD1-G93A mice. Results are expressed as mean ± SEM; n=19 for Non Tg (9 males and 10 females), n=20 for PKO (10 males and 10 females), n=18 for G93A (8 males and 10 females) and n=19 for PKO/G93A (9 males and 10 females); *p= 0.017 (at 150 days), *p= 0.004 (at 164 days), *p= 0.002 (at 171 days) and **p= 0.001 (at 178 days), by two-way ANOVA with repeated measures and with Holm-Sidak's correction for multiple comparisons.C) Clasping score changes over time. Parkin knockout does not affect clasping onset in SOD1-G93A mice. An intermediate score of 2.5 is indicated by the dotted line. Results are expressed as mean ± SEM; n=19 for Non Tg (9 males and 10 females), n=20 for PKO (10 males and 10 females), n=18 for G93A (8 males and 10 females) and n=19 for PKO/G93A (9 males and 10 females).D) Representative images of NMJs in the soleus muscle at 130 days. Muscles were immunostained for neurofilament H (NF-H, in red) and stained with α-bungarotoxin (α-BTX, in green). Scale bar, 20 µm.E) Quantification of the percentage of innervated NMJs at 130 days indicates that Parkin knockout decreases denervation in SOD1-G93A mice. Results are expressed as mean ± SEM and were collected from n= 6 mice per group (3 males and 3 females); n of images evaluated = 49 for Non Tg (with 483 NMJs counted), 46 for PKO (396 NMJs counted), 47 for G93A (413 NMJs assessed) and 38 for PKO/G93A (325 NMJs counted); ***p= 0.0001, by unpaired Mann-Whitney test.F) Representative images of lumbar spinal cord sections at 130 days immunostained with ChAT. Scale bar, 200 µm. G) Quantification of ChAT-positive MN in the spinal cord ventral horn (dotted areas) at 130 days shows that Parkin knockout decreases MN loss in the spinal cord of SOD1-G93A mice. Results are expressed as mean ± SEM; n = 10 mice per group in Non Tg, PKO and G93A (5 males and 5 females) and n= 9 for PKO/G93A (4 males and 5 females); *p=0.031, by unpaired Student's t test. H H) Representative images of lumbar spinal cords sections immunostained for GFAP at 130 days. Scale bar, 200 µm. I) Quantification of the average GFAP staining intensity at 130 days indicates that Parkin knockout decreases spinal cord astrogliosis in SOD1-G93Amice. Results are expressed as mean ± SEM; n = 10 mice per group (5 males and 5 females); *p= 0.033, by paired Student's t test.


Figure 7A ) Citrate synthase activity in spinal cord homogenates at disease end stage . Results are expressed as mean ± SEM ; n = 8 ( 4 males and 4 females ) mice per group ; * p = 0 . 024 , by paired Student ' s t test . Activities are expressed as nmoles of substrate oxidized per minute per mg of protein . B ) Cytochrome oxidase ( COX ) activity at end stage . Results are expressed as mean ± SEM ; n = 8 ( 4 males and 4 females ) mice per group ; * p = 0 . 032 , by paired Student ' s t test . Activities are expressed as nmoles of substrate oxidized per minute per mg of protein . C ) qPCR ( fold change ) of PGC1α mRNA normalized by β - actin mRNA at 130 days of age . Results are expressed as mean ± SEM and as fold change of the Non Tg ; n = 8 ( 4 males and 4 females ) . No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 250 by paired Wilcoxon ' s test ) ; * p = 0 . 035 paired Friedman ' s test with Dunn ' s correction ( Non Tg vs . G93A ) . No other statistically significant differences were found . D ) qPCR ( fold change ) of PGC1α mRNA normalized by β - actin mRNA at end stage . Results are expressed as mean ± SEM and as fold change of the Non Tg ; n = 8 ( 4 males and 4 females ) . No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 613 by paired Student ' s t test ) . * * p = 0 . 0058 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A comparison ) and * * * p < 0 . 0001 by paired one - way ANOVA with Tukey ' s correction ( for PKO vs . PKO / G93A comparison ) . Parkin knockout improves PGC1α expression at 130 days ( C ) but the effect is lost at end stage ( D ) . E E , F ) Representative Western blots ( E ) and quantification ( F ) of PARIS at disease end stage . Protein levels are normalized by β - actin and results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 99 ) . PARIS levels are unchanged in SOD1 - G93A mice relative to Non Tg at disease end stage .

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

Figure 7A) Citrate synthase activity in spinal cord homogenates at disease end stage. Results are expressed as mean ± SEM; n= 8 (4 males and 4 females) mice per group; *p= 0.024, by paired Student's t test. Activities are expressed as nmoles of substrate oxidized per minute per mg of protein.B) Cytochrome oxidase (COX) activity at end stage. Results are expressed as mean ± SEM; n = 8 (4 males and 4 females) mice per group; *p= 0.032, by paired Student's t test. Activities are expressed as nmoles of substrate oxidized per minute per mg of protein.C) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA at 130 days of age. Results are expressed as mean ± SEM and as fold change of the Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A (p= 0.250 by paired Wilcoxon's test); *p= 0.035 paired Friedman's test with Dunn's correction (Non Tg vs. G93A). No other statistically significant differences were found. D) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA at end stage. Results are expressed as mean ± SEM and as fold change of the Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A (p= 0.613 by paired Student's t test). **p= 0.0058 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A comparison) and ***p<0.0001 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A comparison). Parkin knockout improves PGC1α expression at 130 days (C) but the effect is lost at end stage (D). E E, F) Representative Western blots (E) and quantification (F) of PARIS at disease end stage. Protein levels are normalized by β-actin and results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.99). PARIS levels are unchanged in SOD1-G93A mice relative to Non Tg at disease end stage.


Figure 8A , B ) Western blots ( A ) and quantification ( B ) of Miro1 in spinal cord homogenates at disease end stage . β - actin is used as loading reference . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; * p = 0 . 011 by paired Student ' s t test ( for comparison G93A vs . PKO / G93A ) and * * p = 0 . 003 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) . Parkin knockout increases the levels of Miro1 in SOD1 - G93A mice at disease end stage . C , D ) Western blots ( C ) and quantification ( D ) of Mfn2 in spinal cord homogenates at disease end stage . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 078 by paired Wilcoxon ' s test ) ; * * * p = 0 . 0007 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) and * p = 0 . 037 by paired Friedman ' s test with Dunn ' s correction ( for PKO vs . PKO / G93A ) . Parkin knockout increases the levels of Mfn2 in SOD1 - G93A mice at disease end stage . E ) Western blots of spinal cord mitochondria at disease end stage , probed for lysine 48 ( left panel ) and lysine 63 ( right panel ) ubiquitin chains . Citrate synthase is used as loading control . Asterisks indicate ubiquitinated proteins with different abundance in SOD1 - G93A samples . F , G ) Representative Western blot ( F ) and quantification ( G ) of LC3 II in spinal cord mitochondria at end stage . Protein levels are normalized by Complex V . LC3 II is increased in both G93A and PKO / G93A mitochondria and Parkin knockout does not affect LC3 II levels . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 504 by paired Student ' s t test ) . * p = 0 . 035 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) and * * p = 0 . 0014 by paired one - way ANOVA with Tukey ' s correction ( for PKO vs . PKO / G93A ) H ) Representative Western blot of March5 and Mul1 in spinal cord mitochondria at end stage . I ) Quantification of March5 at disease end stage . Results are expressed as mean ± SEM and percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 742 ) . Protein levels are normalized by Complex V . March5 levels are unaffected by Parkin knockout . J ) Quantification of Mul1 at disease end stage . Results are expressed as mean ± SEM and percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 94 ) . Protein levels are normalized by Complex V . Mul1 levels are unaffected by Parkin knockout .

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

Figure 8A, B) Western blots (A) and quantification (B) of Miro1 in spinal cord homogenates at disease end stage. β-actin is used as loading reference. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; *p= 0.011 by paired Student's t test (for comparison G93A vs. PKO/G93A) and **p= 0.003 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A). Parkin knockout increases the levels of Miro1 in SOD1-G93A mice at disease end stage.C, D) Western blots (C) and quantification (D) of Mfn2 in spinal cord homogenates at disease end stage. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.078 by paired Wilcoxon's test); ***p= 0.0007 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and *p= 0.037 by paired Friedman's test with Dunn's correction (for PKO vs. PKO/G93A). Parkin knockout increases the levels of Mfn2 in SOD1-G93A mice at disease end stage.E) Western blots of spinal cord mitochondria at disease end stage, probed for lysine 48 (left panel) and lysine 63 (right panel) ubiquitin chains. Citrate synthase is used as loading control. Asterisks indicate ubiquitinated proteins with different abundance in SOD1-G93A samples.F, G) Representative Western blot (F) and quantification (G) of LC3 II in spinal cord mitochondria at end stage. Protein levels are normalized by Complex V. LC3 II is increased in both G93A and PKO/G93A mitochondria and Parkin knockout does not affect LC3 II levels. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.504 by paired Student's t test). *p= 0.035 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and **p= 0.0014 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A)H) Representative Western blot of March5 and Mul1 in spinal cord mitochondria at end stage.I) Quantification of March5 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.742). Protein levels are normalized by Complex V. March5 levels are unaffected by Parkin knockout.J) Quantification of Mul1 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.94). Protein levels are normalized by Complex V. Mul1 levels are unaffected by Parkin knockout.


Figure 1A , B NAD and nicotinamide ( NAM ) levels in heart of WT ( n = 5 , n = 6 , respectively ) , mdx ( n = 5 , n = 5 , respectively ) and mdx / CD38 - / - mice ( n = 6 , n = 5 , respectively ) . C , D NAD and NAM levels in diaphragm of WT ( n = 5 , n = 6 , respectively ) , mdx ( n = 4 , n = 5 , respectively ) and mdx / CD38 - / - mice ( n = 6 , n = 5 , respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; B : Kruskal - Wallis followed by Dunn ' s test ; D : Kruskal - Wallis followed by Mann - Whitney tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E , F levels of ADP - ribose ( ADPR ) ( E ) , cyclic ADP - ribose ( cADPR ) ( F ) expressed as nmol / mg protein in heart of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - mice ( n = 5 , n = 5 , n = 6 respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : : ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( G ) qPCR analysis of mRNA levels of Sterile alpha and Toll / interleukin - 1 receptor motif - containing 1 ( SARM1 ) in heart of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - mice ( n = 5 , n = 5 , n = 6 respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . H , I Levels of ADPR ( H ) , cADPR ( I ) expressed as nmol / mg protein in diaphragm of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; ; I : Welch ' s ANOVA followed by Welch ' s t - tests Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( J ) qPCR analysis mRNA levels of SARM1 in diaphragm of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : J : ANOVA Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . K , L Western blot analysis of CD38 protein expression in heart ( K ) and diaphragm ( L ) of WT ( n = 5 , n = 6 , respectively ) and mdx ( n = 5 ) mice . Vinculin is used as housekeeping protein control and the dot plots show the ratio of CD38 / vinculin . Data information : Each dot of the graphs represents a value / mouse . After normality and variance comparison tests , significance was assessed using : unpaired Student ' s t - test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . M , N qPCR analysis of CD38 mRNA levels in heart ( M ) and diaphragm ( N ) of WT ( n = 6 ) and mdx ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : M : unpaired Student ' s t - test ; N : unpaired Welch ' s t - test . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 1A,B NAD and nicotinamide (NAM) levels in heart of WT (n=5, n=6, respectively), mdx (n=5, n=5, respectively) and mdx/CD38-/- mice (n=6, n=5, respectively). C,D NAD and NAM levels in diaphragm of WT (n=5, n=6, respectively), mdx (n=4, n=5, respectively) and mdx/CD38-/- mice (n=6, n=5, respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; B: Kruskal-Wallis followed by Dunn's test; D: Kruskal-Wallis followed by Mann-Whitney tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.E,F levels of ADP-ribose (ADPR) (E), cyclic ADP-ribose (cADPR) (F) expressed as nmol/mg protein in heart of WT (n=6), mdx (n=5) and mdx/CD38-/- mice (n=5, n=5, n=6 respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.(G) qPCR analysis of mRNA levels of Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) in heart of WT (n=6), mdx (n=5) and mdx/CD38-/- mice (n=5, n=5, n=6 respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.H,I Levels of ADPR (H), cADPR (I) expressed as nmol/mg protein in diaphragm of WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; I: Welch's ANOVA followed by Welch's t-tests Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.(J) qPCR analysis mRNA levels of SARM1 in diaphragm of WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: J: ANOVA Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.K, L Western blot analysis of CD38 protein expression in heart (K) and diaphragm (L) of WT (n=5, n=6, respectively) and mdx (n=5) mice. Vinculin is used as housekeeping protein control and the dot plots show the ratio of CD38/vinculin. Data information: Each dot of the graphs represents a value/mouse. After normality and variance comparison tests, significance was assessed using: unpaired Student's t-test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.M, N qPCR analysis of CD38 mRNA levels in heart (M) and diaphragm (N) of WT (n=6) and mdx (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: M: unpaired Student's t-test; N: unpaired Welch's t-test. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


Figure 2A Cardiac function evaluated by echocardiography in mdx / CD38 - / - mice : the dot plots show the left ventricular ( LV ) diastolic ( A1 ) and systolic ( A2 ) inner diameters and LV ejection ( A3 ) fraction in 7 - month - old WT ( n = 5 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 10 ) mice . Data information : : Each dot of the graphs represents a mouse . in duplicate After normality and variance comparison tests , significance was assessed using : A1 ANOVA followed by Fisher ' s LSD test ; A2 , A3 Kruskal - Wallis followed by Dunn ' s test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B Plasma levels of cardiac stress biomarkers : brain natriuretic peptide ( BNP ) ( B1 ) and cardiac troponin I ( cTnI ) ( B2 ) in 7 - month - old WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 11 ) mice . Data information : : Each dot of the graphs represents a mouse . After normality and variance comparison tests , significance was assessed using ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C Cropped images revealed the collagen ( blue ) stained by Masson ' s trichrome staining in the heart of WT , mdx , and mdx / CD38 - / - mice . Dot plot showing the quantification of heart collagen staining area ( % total area ) in 7 - month - old WT , mdx , and mdx / CD38 - / - mice ( n = 4 per group ) . Scale bars : 200 µm . Data information : : Each dot of the graphs represents a mouse . in triplicate After normality and variance comparison tests , significance was assessed using ANOVA followed by Fisher ' s LSD test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . D Isoproterenol - induced heart failure in 3 - month - old mice . The Kaplan - Meier curve shows the survival rate of mdx mice ( NaCl , n = 5 ) ; mdx ( n = 19 ) and mdx / CD38 - / - ( n = 9 ) mice following isoproterenol ( subcutaneous injection ) at 2 . 5 mg / kg / d for 10 days . Data information : one value / mouse . After normality and variance comparison tests , significance was assessed using D : Log - rank Mantel - Cox test and Log - rank test for trend and Gehan - Breslow - Wilcoxon test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E Histogram showing isoproterenol - induced heart hypertrophy in mdx NaCl ( n = 5 ) mice , surviving mdx ( n = 9 ) , mdx / CD38 - / - NaCl ( n = 9 ) and mdx / CD38 - / - ( n = 9 ) mice , expressed as heart weight / body weight ratio ( HW / BW ) . Data information : one value / mouse . After normality and variance comparison tests , significance was assessed using Kruskal - Wallis followed by Mann - Whitney tests Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . F Plasma levels of cardiac stress biomarkers : brain natriuretic peptide ( BNP ) ( F1 ) and cardiac troponin I ( cTnI ) ( F2 ) following isoproterenol treatment in mdx NaCl ( n = 5 ) , surviving mdx ( n = 8 ) , mdx / CD38 - / - NaCl ( n = 5 ) and mdx / CD38 - / - ( n = 8 ) mice . Data information : in duplicate After normality and variance comparison tests , significance was assessed using F2 Kruskal - Wallis followed by Mann - Whitney tests ; F1 : ANOVA followed by Student ' s / Welch ' s t - tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . G , H Time - lapse images recorded by confocal microscopy in " line scanning mode " , showing Ca2 + sparks and waves in cardiomyocytes at rest , extracted from mdx and mdx / CD38 - / - mice ; scale bars : 10 µM ( horizontal ) , 500 ms ( vertical ) . Bar graphs showing the averaged Ca2 + sparks ( G ) and waves ( H ) frequencies in cardiomyocytes isolated from WT ( n = 21 cells ) , mdx ( n = 28 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using Kruskal - Wallis followed by Dunn ' s test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . I Bar graph showing the fractional release following caffeine application in cardiomyocytes from WT ( n = 24 cells ) , mdx ( n = 33 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . J Bar graph showing the post - rest potentiation in cardiomyocytes from WT ( n = 24 cells ) , mdx ( n = 28 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using : Kruskal - Wallis followed by Dunn ' s test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 2A Cardiac function evaluated by echocardiography in mdx/CD38-/- mice: the dot plots show the left ventricular (LV) diastolic (A1) and systolic (A2) inner diameters and LV ejection (A3) fraction in 7-month-old WT (n=5), mdx (n=6) and mdx/CD38-/- (n=10) mice. Data information: : Each dot of the graphs represents a mouse. in duplicate After normality and variance comparison tests, significance was assessed using:A1 ANOVA followed by Fisher's LSD test; A2,A3 Kruskal-Wallis followed by Dunn's test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.B Plasma levels of cardiac stress biomarkers: brain natriuretic peptide (BNP) (B1) and cardiac troponin I (cTnI) (B2) in 7-month-old WT (n=7), mdx (n=6) and mdx/CD38-/- (n=11) mice. Data information: : Each dot of the graphs represents a mouse. After normality and variance comparison tests, significance was assessed using ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.C Cropped images revealed the collagen (blue) stained by Masson's trichrome staining in the heart of WT, mdx, and mdx/CD38-/- mice. Dot plot showing the quantification of heart collagen staining area (% total area) in 7-month-old WT, mdx, and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: : Each dot of the graphs represents a mouse. in triplicate After normality and variance comparison tests, significance was assessed using ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.D Isoproterenol-induced heart failure in 3-month-old mice. The Kaplan-Meier curve shows the survival rate of mdx mice (NaCl, n=5); mdx (n=19) and mdx/CD38-/- (n=9) mice following isoproterenol (subcutaneous injection) at 2.5 mg/kg/d for 10 days. Data information: one value/mouse. After normality and variance comparison tests, significance was assessed using D: Log-rank Mantel-Cox test and Log-rank test for trend and Gehan-Breslow-Wilcoxon test ; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.E Histogram showing isoproterenol-induced heart hypertrophy in mdx NaCl (n=5) mice, surviving mdx (n=9), mdx/CD38-/- NaCl (n=9) and mdx/CD38-/- (n=9) mice, expressed as heart weight/body weight ratio (HW/BW). Data information: one value/mouse. After normality and variance comparison tests, significance was assessed using Kruskal-Wallis followed by Mann-Whitney tests Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.F Plasma levels of cardiac stress biomarkers: brain natriuretic peptide (BNP) (F1) and cardiac troponin I (cTnI) (F2) following isoproterenol treatment in mdx NaCl (n=5), surviving mdx (n=8), mdx/CD38-/- NaCl (n=5) and mdx/CD38-/- (n=8) mice. Data information: in duplicate After normality and variance comparison tests, significance was assessed using F2 Kruskal-Wallis followed by Mann-Whitney tests; F1: ANOVA followed by Student's/ Welch's t-tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.G,H Time-lapse images recorded by confocal microscopy in "line scanning mode", showing Ca2+ sparks and waves in cardiomyocytes at rest, extracted from mdx and mdx/CD38-/- mice; scale bars: 10 µM (horizontal), 500 ms (vertical). Bar graphs showing the averaged Ca2+ sparks (G) and waves (H) frequencies in cardiomyocytes isolated from WT (n=21 cells), mdx (n=28 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using Kruskal-Wallis followed by Dunn's test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.I Bar graph showing the fractional release following caffeine application in cardiomyocytes from WT (n=24 cells), mdx (n=33 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.J Bar graph showing the post-rest potentiation in cardiomyocytes from WT (n=24 cells), mdx (n=28 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using: Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


Figure 3A Measurement of the ventilatory mechanic by barometric plethysmography : dot plots showing inspiratory ( A1 ) and expiratory times ( A2 ) , the relaxation time ( A3 ) and the respiratory frequency ( A4 ) in WT ( n = 8 ) , mdx ( n = 11 ) and mdx / CD38 - / - ( n = 9 ) mice . Data information : Each dot of the graphs represents a mouse . one value / mouse After normality and variance comparison tests , significance was assessed using : A1 , A3 ANOVA followed by Fisher ' s LSD test ; A2 , A4 : Kruskal - Wallis followed by Dunn ' s test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B Images showing muscle fiber typology revealed by immunostaining . Localization of the slow MyHC ( Type I ) fiber and fast MyHCs ( Type IIa and IIb / X ) fibers , along with laminin ( red ) on transverse cross - sections from diaphragm of WT , mdx and mdx / CD38 - / - mice . Scale bars : 100 µm . Histogram showing the percentage of I , IIa and IIb / X fiber type distribution in diaphragm of WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 7 ) mice . Data information : one value / mouse After normality and variance comparison tests , significance was assessed using : ; B : Chi - square test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C Fiber size distribution in the diaphragm of WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 7 ) mice . Data information : one value / mouse After normality and variance comparison tests , significance was assessed using : C : Kolmogorov - Smirnov test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . D Images revealing the collagen ( blue ) by Masson ' s trichrome staining in diaphragm of WT , mdx , and mdx / CD38 - / - mice . Quantification of collagen staining area ( % total area ) in the diaphragm of WT , mdx and mdx / CD38 - / - mice ( n = 4 per group ) . Scale bars : 200 µm . Data information : Each dot of the graphs represents a mouse . D in duplicate ; After normality and variance comparison tests , significance was assessed using : : ANOVA followed by Fisher ' s LSD test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E Embryonic myosin expression : immunostaining showing its localization along with laminin ( green ) on transverse cross - sections from diaphragm of WT , mdx and mdx / CD38 - / - mice . Scale bars : 50 µm . Histogram showing the relative proportion of embryonic myosin area ( % total area ) in diaphragm of WT ( n = 7 ) , mdx ( n = 8 ) and mdx / CD38 - / - ( n = 8 ) mice . Data information : Each dot of the graphs represents a mouse . E one value / mouse . After normality and variance comparison tests , significance was assessed using : E : Welch ' s ANOVA followed by Welch ' s t - tests . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 3A Measurement of the ventilatory mechanic by barometric plethysmography: dot plots showing inspiratory (A1) and expiratory times (A2), the relaxation time (A3) and the respiratory frequency (A4) in WT (n=8), mdx (n=11) and mdx/CD38-/- (n=9) mice. Data information: Each dot of the graphs represents a mouse. one value/mouse After normality and variance comparison tests, significance was assessed using: A1,A3 ANOVA followed by Fisher's LSD test; A2, A4 : Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.B Images showing muscle fiber typology revealed by immunostaining. Localization of the slow MyHC (Type I) fiber and fast MyHCs (Type IIa and IIb/X) fibers, along with laminin (red) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 100 µm. Histogram showing the percentage of I, IIa and IIb/X fiber type distribution in diaphragm of WT (n=7), mdx (n=6) and mdx/CD38-/- (n=7) mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: ; B: Chi-square test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.C Fiber size distribution in the diaphragm of WT (n=7), mdx (n=6) and mdx/CD38-/- (n=7) mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: C: Kolmogorov-Smirnov test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.D Images revealing the collagen (blue) by Masson's trichrome staining in diaphragm of WT, mdx, and mdx/CD38-/- mice. Quantification of collagen staining area (% total area) in the diaphragm of WT, mdx and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. D in duplicate; After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.E Embryonic myosin expression: immunostaining showing its localization along with laminin (green) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 50 µm. Histogram showing the relative proportion of embryonic myosin area (% total area) in diaphragm of WT (n=7), mdx (n=8) and mdx/CD38-/- (n=8) mice. Data information: Each dot of the graphs represents a mouse. E one value/mouse. After normality and variance comparison tests, significance was assessed using: E: Welch's ANOVA followed by Welch's t-tests. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


Figure 4A Images showing immunostaining of myeloid cells showing F4 / 80 ( macrophage marker ) , Ly - 6G / 6C ( monocyte , granulocyte and neutrophil marker ) , CD8 ( cytotoxic T lymphocyte marker ) and IL - 6 positive cells in diaphragm of 7 - month - old WT ( n = 8 ) , mdx ( n = 8 ) and mdx / CD38 - / - ( n = 7 or 8 ) mice . Lower panel : histograms quantifying the percentage of F4 / 80 ( A1 ) mdx / CD38 - / - ( n = 7 ) , LY6 ( A2 ) mdx / CD38 - / - ( n = 8 ) , CD8 ( A3 ) mdx / CD38 - / - ( n = 7 ) , IL - 6 ( A4 ) mdx / CD38 - / - ( n = 8 ) positive cells . Scale bars : 50 µm . Data information : Each dot of the graphs represents a mouse . A one value / mouse After normality and variance comparison tests , significance was assessed using : A1 - 4 : Welch ' s ANOVA followed by Welch ' s t - tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B qPCR analysis of mRNA levels of interleukin - 1β ( IL - 1ß ) ( B1 ) and - 6 ( IL - 6 ) ( B2 ) , cyclin - dependent kinase inhibitor 1 ( p21 ) ( B3 ) transforming growth factor - β ( TGF - β ) ( B4 ) , and senescence markers ( cell - cycle inhibitor p16 , INK4a ) ( B5 ) , Collagen Type I Alpha 1 Chain ( Col1a1 ) ( B6 ) in diaphragm of 20 - month - old WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse . B in duplicate . After normality and variance comparison tests , significance was assessed using : B1 - 6 : ANOVA followed by Fisher ' s LSD test . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 4A Images showing immunostaining of myeloid cells showing F4/80 (macrophage marker), Ly-6G/6C (monocyte, granulocyte and neutrophil marker), CD8 (cytotoxic T lymphocyte marker) and IL-6 positive cells in diaphragm of 7-month-old WT (n=8), mdx (n=8) and mdx/CD38-/- (n=7 or 8 ) mice. Lower panel: histograms quantifying the percentage of F4/80 (A1) mdx/CD38-/- (n=7), LY6 (A2) mdx/CD38-/- (n=8), CD8 (A3) mdx/CD38-/- (n=7), IL-6 (A4) mdx/CD38-/- (n=8) positive cells. Scale bars: 50 µm. Data information: Each dot of the graphs represents a mouse. A one value/mouse After normality and variance comparison tests, significance was assessed using: A1-4: Welch's ANOVA followed by Welch's t-tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.B qPCR analysis of mRNA levels of interleukin-1β (IL-1ß) (B1) and -6 (IL-6) (B2), cyclin-dependent kinase inhibitor 1 (p21) (B3) transforming growth factor-β (TGF-β) (B4), and senescence markers (cell-cycle inhibitor p16, INK4a) (B5), Collagen Type I Alpha 1 Chain (Col1a1) (B6) in diaphragm of 20-month-old WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse. B in duplicate. After normality and variance comparison tests, significance was assessed using: B1-6: ANOVA followed by Fisher's LSD test. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.