Figure 3HO - 1 - / - mice have more ECs and less CARs ( p = 0 . 053 ) in BM , while frequency of PaS does not differ . Young HO - 1 - / - mice have lower frequency of Sca - 1high fraction among BM ECs , what resembles phenotype of BM ECs in old mice . Intracellular flow cytometry reveals ( C ) lower levels of SDF - 1α in HO - 1 - / - CARs and ( D ) lower levels of LAP protein in HO - 1 - / - ECs . Analysis of cell surface SCF revealed lower frequency of SCF + ECs in HO - 1 - / - while no differences among CARs . The frequency of ( F ) CD163 + MQs and ( G ) CD163 - MQs in BM of HO - 1 - / - mice is altered .

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

Figure 3HO-1-/- mice have more ECs and less CARs (p = 0.053) in BM, while frequency of PaS does not differ.Young HO-1-/- mice have lower frequency of Sca-1high fraction among BM ECs, what resembles phenotype of BM ECs in old mice.Intracellular flow cytometry reveals (C) lower levels of SDF-1α in HO-1-/- CARs and (D) lower levels of LAP protein in HO-1-/- ECs.Analysis of cell surface SCF revealed lower frequency of SCF+ ECs in HO-1-/- while no differences among CARs.The frequency of (F) CD163+ MQs and (G) CD163- MQs in BM of HO-1-/- mice is altered.


Figure 4Gating strategy of hematopoietic stem and progenitor cells . Young HO - 1 - / - mice possess higher number of ( B ) LT - HSCs , ( C ) ST - HSCs and ( D ) MPPs . Exemplary analysis of cell cycle with Ki67 and nuclear dye . More young HO - 1 - / - LT - HSCs are in G1 and S / G2 / M cell cycle phases . The presented cell cycle analysis is from two independent experiments . Young HO - 1 - / - MPPs do not differ in cell cycling from young HO - 1 + / + MPPs . The presented cell cycle analysis is from two independent experiments . More old HO - 1 - / - LT - HSCs are in G1 phase in comparison to old HO - 1 + / + LT - HSCs , but not in S / G2 / M phase . The presented cell cycle analysis is from two independent experiments . Old MPPs do not differ in cell cycling between genotypes . The presented cell cycle analysis is from two independent experiments . Young HO - 1 - / - LT - HSCs contain more γH2aXhigh cells than young HO - 1 + / + , 378 - 991 cells analyzed from 7 mice / group . Young HO - 1 - / - MPPs contain more γH2aXhigh cells than young HO - 1 + / + , 1693 - 2790 cells analyzed from 7 mice / group . Alkaline comet assay revealed that HO - 1 - / - LT - HSCs possess ( L ) higher oil tail moment and ( M ) more DNA in the comet tail . 1068 - 1189 cells analyzed from 6 mice / group . HSCs from young HO - 1 - / - mice provide worse hematopoietic reconstitution after transplantation than HO - 1 + / + HSCs . Data are shown as mean ± SEM , n = 8 - 9 mice / group .

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

Figure 4Gating strategy of hematopoietic stem and progenitor cells.Young HO-1-/- mice possess higher number of (B) LT-HSCs, (C) ST-HSCs and (D) MPPs.Exemplary analysis of cell cycle with Ki67 and nuclear dye.More young HO-1-/- LT-HSCs are in G1 and S/G2/M cell cycle phases. The presented cell cycle analysis is from two independent experiments. Young HO-1-/- MPPs do not differ in cell cycling from young HO-1+/+ MPPs. The presented cell cycle analysis is from two independent experiments. More old HO-1-/- LT-HSCs are in G1 phase in comparison to old HO-1+/+ LT-HSCs, but not in S/G2/M phase. The presented cell cycle analysis is from two independent experiments. Old MPPs do not differ in cell cycling between genotypes. The presented cell cycle analysis is from two independent experiments.Young HO-1-/- LT-HSCs contain more γH2aXhigh cells than young HO-1+/+, 378-991 cells analyzed from 7 mice/group. Young HO-1-/- MPPs contain more γH2aXhigh cells than young HO-1+/+, 1693-2790 cells analyzed from 7 mice/group.Alkaline comet assay revealed that HO-1-/- LT-HSCs possess (L) higher oil tail moment and (M) more DNA in the comet tail. 1068-1189 cells analyzed from 6 mice/group.HSCs from young HO-1-/- mice provide worse hematopoietic reconstitution after transplantation than HO-1+/+ HSCs. Data are shown as mean ± SEM, n = 8-9 mice/group.


Figure 5RNA - seq revealed 1067 DEGs between young HO - 1 - / - and HO - 1 + / + LT - HSCs . RNA - seq revealed 595 DEGs between old HO - 1 - / - and HO - 1 + / + LT - HSCs . PC1 in PCA analysis on selected 1148 genes separated young HO - 1 + / + LT - HSCs from other groups , clustering the young HO - 1 - / - LT - HSCs together with old LT - HSCs . Hierarchical clustering on the 20 % of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO - 1 + / + LT - HSCs in one and old LT - HSCs with young HO - 1 - / - LT - HSCs in second .

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 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

Figure 5RNA-seq revealed 1067 DEGs between young HO-1-/- and HO-1+/+ LT-HSCs. RNA-seq revealed 595 DEGs between old HO-1-/- and HO-1+/+ LT-HSCs.PC1 in PCA analysis on selected 1148 genes separated young HO-1+/+ LT-HSCs from other groups, clustering the young HO-1-/- LT-HSCs together with old LT-HSCs.Hierarchical clustering on the 20% of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO-1+/+ LT-HSCs in one and old LT-HSCs with young HO-1-/- LT-HSCs in second.


Figure 6Deletion of HO - 1 in myeloid lineage did not cause expansion of LT - HSCs and did not alter their cell cycle status . Long - term PB chimerism derived from HSCs transplanted to HO - 1 - / - recipients is lower than from HSCs transplanted to HO - 1 + / + recipients . Chimerism among BM HSPCs fractions transplanted to HO - 1 - / - recipients are lower than from HSCs transplanted to HO - 1 + / + recipients . Data are shown as mean ± SEM , n = 5 - 12 / grouptotal number of cells derived from HSCs transplanted to HO - 1 - / - recipients are lower than from HSCs transplanted to HO - 1 + / + recipients . Data are shown as mean ± SEM , n = 5 - 12 / groupShort term homing of HSPC to HO - 1 + / + or HO - 1 - / - BM did not differ . Data are shown as mean ± SEM , n = 5 / group , unpaired 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 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 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 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

Figure 6Deletion of HO-1 in myeloid lineage did not cause expansion of LT-HSCs and did not alter their cell cycle status.Long-term PB chimerism derived from HSCs transplanted to HO-1-/- recipients is lower than from HSCs transplanted to HO-1+/+ recipients.Chimerism among BM HSPCs fractions transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/grouptotal number of cells derived from HSCs transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/groupShort term homing of HSPC to HO-1+/+ or HO-1-/- BM did not differ. Data are shown as mean ± SEM, n = 5/group, unpaired t-test.


Figure 7Transplantation of HO - 1 - / - HSCs provides lower chimerism among HSPC fractions in primary recipients . Data are shown as mean ± SEM , n = 7 - 8 mice / group . Transplantation of the same number of donor - derived BM cells from primary recipients provides the same ( C ) PB chimerismTransplantation of the same number of donor - derived BM cells from primary recipients provides the same BM LT - HSC chimerism . Comparison of gene log fold changes in non - transplanted HO - 1 - / - LT - HSCs and HO - 1 - / - LT - HSCs transplanted twice to the wild - type HO - 1 + / + showed that transplantation of HO - 1 - / - LT - HSCs twice to the wild type niche reverses their transcriptional alterations .

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 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 O O O O O O O O O O O O O O O O O O O O O O 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 7Transplantation of HO-1-/- HSCs provides lower chimerism among HSPC fractions in primary recipients. Data are shown as mean ± SEM, n = 7-8 mice/group.Transplantation of the same number of donor-derived BM cells from primary recipients provides the same (C) PB chimerismTransplantation of the same number of donor-derived BM cells from primary recipients provides the same BM LT-HSC chimerism.Comparison of gene log fold changes in non-transplanted HO-1-/- LT-HSCs and HO-1-/- LT-HSCs transplanted twice to the wild-type HO-1+/+ showed that transplantation of HO-1-/- LT-HSCs twice to the wild type niche reverses their transcriptional alterations.


Figure 8Frequency of colonies appearing at day 8 and day 12 from HSCs co - cultured with HO - 1 + / + or HO - 1 - / - MSCs . * p < 0 . 05 , * * * p < 0 . 001 , Fisher exact test . PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed ( C ) 4 distinct clusters of colonies , that differ by frequency of ( D ) CD11b + Gr1 - , ( E ) CD11b + Gr1 + PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed ( F ) cells lacking expression of analyzed lineage markers ( Lin - ) . Analysis of frequency of colonies derived from HO - 1 + / + or from HO - 1 - / - MSC co - cultures among identified clusters . Enrichment in cluster analyzed by Fisher exact test , * * p < 0 . 01 , two - tailed unpaired t - test . Sdf1α mRNA levels in BM of HO - 1 + / + or HO - 1 - / - mice after administration of Noggin . Data are shown as mean ± SEM . * p < 0 . 05 , two - tailed unpaired t - test , n = 10 / groupLT - HSC frequency of LT - HSCs after administration of Noggin to HO - 1 + / + or HO - 1 - / - mice . Data are shown as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , two - tailed unpaired t - test , n = 8 - 10 / groupcell cycling status of LT - HSCs after administration of Noggin to HO - 1 + / + or HO - 1 - / - mice .

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

Figure 8Frequency of colonies appearing at day 8 and day 12 from HSCs co-cultured with HO-1+/+ or HO-1-/- MSCs. *p< 0.05, ***p<0.001, Fisher exact test.PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (C) 4 distinct clusters of colonies, that differ by frequency of (D) CD11b+Gr1-, (E) CD11b+Gr1+PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (F) cells lacking expression of analyzed lineage markers (Lin-).Analysis of frequency of colonies derived from HO-1+/+ or from HO-1-/- MSC co-cultures among identified clusters. Enrichment in cluster analyzed by Fisher exact test, **p<0.01, two-tailed unpaired t-test.Sdf1α mRNA levels in BM of HO-1+/+ or HO-1-/- mice after administration of Noggin. Data are shown as mean ± SEM. *p< 0.05, two-tailed unpaired t-test, n = 10/groupLT-HSC frequency of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice. Data are shown as mean ± SEM. *p< 0.05, **p<0.01, two-tailed unpaired t-test, n = 8-10/groupcell cycling status of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice.


Figure 1A . Assessment of protein levels in different AIFM1 knockout clones . Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells . D . Assessment of protein levels in HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1 - HA ( expression of AIFM1 - HA induced for one day ) . Different amounts of lysates from the indicated cells were analyzed by reducing SDS - PAGE and immunoblotting . While levels of MIA40 were largely unchanged , levels of many MIA40 / CHCHD4 substrates were diminished in AIFM1 knockout cells . N = 3 biological replicates . F . BN - PAGE analyses of HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1 - HA or AIFM1 . Complex I was resolved from isolated mitochondria with blue native electrophoresis ( BN - PAGE ) followed by immunoblotting or in gel determination of the complex I specific activity . Levels of complex I were decreased in AIFM1 knockout cells . N = 3 biological

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 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 O O O O

Figure 1A. Assessment of protein levels in different AIFM1 knockout clones. Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells.D. Assessment of protein levels in HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA (expression of AIFM1-HA induced for one day). Different amounts of lysates from the indicated cells were analyzed by reducing SDS-PAGE and immunoblotting. While levels of MIA40 were largely unchanged, levels of many MIA40/CHCHD4 substrates were diminished in AIFM1 knockout cells. N = 3 biological replicates.F. BN-PAGE analyses of HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA or AIFM1. Complex I was resolved from isolated mitochondria with blue native electrophoresis (BN-PAGE) followed by immunoblotting or in gel determination of the complex I specific activity. Levels of complex I were decreased in AIFM1 knockout cells. N = 3 biological replicates


Figure 3A . In organello import assay with NDUFS5 , NDUFB7 and as control the matrix protein SOD2 . In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells . Non - imported proteins were removed by treatment with Proteinase K . Imported proteins were analyzed by reducing SDS - PAGE and autoradiography . Signals were quantified using ImageQuantTL and the amount of imported protein was plotted . NDUFS5 import is strongly affected by loss of AIFM1 . Import of NDUFB7 is less affected and SOD2 import remained unaffected . This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar . B . Oxidation kinetics of endogenous NDUFB7 and NDUFS5 . HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1 - HA were analyzed by pulse - chase experiments coupled to redox state determination . To this end , cells were incubated with 35S - Met for 5 min ( pulse ) . After removal of 35S - Met , cells were left in " cold " Met for different chase times . Further oxidation was stopped by addition of trichloroacetic acid ( TCA ) . Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40 / CHCHD4 substrates were enriched by immunoprecipitation ( IP ) , and analyzed by non - reducing SDS - PAGE and autoradiography . Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells . NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed . N = 4 biological replicates for NDUFB7 and NDUFS5 . C . Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells . Cells were incubated with 35S - Met for 5 min ( pulse ) , and then MIA40 / CHCHD4 substrates were enriched by immunoprecipitation ( IP ) , and analyzed by non - reducing SDS - PAGE and autoradiography . NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells . D . Interaction between endogenous MIA40 / CHCHD4 and its substrates in wild type and AIFM1 knockout cells . Cells were subjected to native immunoprecipitation ( IP ) against endogenous MIA40 / CHCHD4 . Precipitates were analyzed by SDS - PAGE and immunoblotting against the indicated proteins . In AIFM1 knockout cell interaction between MIA40 / CHCHD4 and its substrates is strongly affected with the interaction between MIA40 / CHCHD4 and NDUFS5 almost completely abolished . This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells , indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40 / CHCHD4 rather than the cause . Quantification using Image lab . Data from 2 experiments were combined and standard deviations are presented . Asterisk indicates background band orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells , respectively .

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 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 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 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

Figure 3A. In organello import assay with NDUFS5, NDUFB7 and as control the matrix protein SOD2. In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells. Non-imported proteins were removed by treatment with Proteinase K. Imported proteins were analyzed by reducing SDS-PAGE and autoradiography. Signals were quantified using ImageQuantTL and the amount of imported protein was plotted. NDUFS5 import is strongly affected by loss of AIFM1. Import of NDUFB7 is less affected and SOD2 import remained unaffected. This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar.B. Oxidation kinetics of endogenous NDUFB7 and NDUFS5. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analyzed by pulse-chase experiments coupled to redox state determination. To this end, cells were incubated with 35S-Met for 5 min (pulse). After removal of 35S-Met, cells were left in "cold" Met for different chase times. Further oxidation was stopped by addition of trichloroacetic acid (TCA). Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells. NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed. N = 4 biological replicates for NDUFB7 and NDUFS5.C. Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells. Cells were incubated with 35S-Met for 5 min (pulse), and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells.D. Interaction between endogenous MIA40/CHCHD4 and its substrates in wild type and AIFM1 knockout cells. Cells were subjected to native immunoprecipitation (IP) against endogenous MIA40/CHCHD4. Precipitates were analyzed by SDS-PAGE and immunoblotting against the indicated proteins. In AIFM1 knockout cell interaction between MIA40/CHCHD4 and its substrates is strongly affected with the interaction between MIA40/CHCHD4 and NDUFS5 almost completely abolished. This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells, indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40/CHCHD4 rather than the cause. Quantification using Image lab. Data from 2 experiments were combined and standard deviations are presented. Asterisk indicates background band orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells, respectively.


Figure 4A . Assessment of MIA40 / CHCHD4 variant - AIFM1 interaction . The indicated MIA40 / CHCHD4 - Strep variants were affinity precipitated ( AP ) under native conditions after stopping thiol - disulfide exchange reactions by NEM incubation . Precipitates were tested for AIFM1 , ALR and MIA40 / CHCHD4 by reducing SDS - PAGE and immunoblotting . 1 % of the total lysate was loaded as input control . M , mock control not expressing MIA40 - Strep . Both , wildtype MIA40 / CHCHD4 and MTS - MIA40 / CHCHD4 but not MIA40 / CHCHD4 ∆ 1 - 40 variants coprecipitated AIFM1 . All mitochondria - localized variants precipitate ALR indicating redox functionality . Arrowhead indicates endogenous MIA40 / CHCHD4 . Emetine - chase experiments to assess the stability of the AIFM1 / CHCHD4 complex . HEK293 cells stably expressing MIA40 / CHCHD4 - Strep were treated for the indicated times with the ribosome inhibitor emetine . Then , cells were lysed under native conditions ( triton X - 100 ) , and MIA40 / CHCHD4 - Strep ( B ) were precipitated . Precipitates were analysed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 interact over a period of at least 6 - 8 hours while the two MIA40 / CHCHD4 substrates , NDUFB7 and AK2 , are quickly released from MIA40 / CHCHD4 ( B , gray box ) . AP , affinity precipitation ; asterisk , background band . C . Emetine - chase experiments to assess the stability of the AIFM1 / CHCHD4 complex . AIFM1 knockout cells stably expressing AIFM1 - HA ( C ) were treated for the indicated times with the ribosome inhibitor emetine . Then , cells were lysed under native conditions ( triton X - 100 ) , and AIFM1 - HA ( C ) were precipitated . Precipitates were analysed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 and AIFM1 interact over a period of at least 6 - 8 hours AP , affinity precipitation ; asterisk , background band . Eluted fractions were subjected to SDS - PAGE and TCE staining ( E ) . AIFM1 and full - length MIA40 / CHCHD4 migrate together indicating formation of a complex . MIA40 / CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40 / CHCHD4 . AIFM1 and ∆ 1 - 40 MIA40 / CHCHD4 do not migrate together emphasizing the importance of the N - terminal amino acids in MIA40 for interaction wth AIFM1 .

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 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 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 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

Figure 4A. Assessment of MIA40/CHCHD4 variant-AIFM1 interaction. The indicated MIA40/CHCHD4-Strep variants were affinity precipitated (AP) under native conditions after stopping thiol-disulfide exchange reactions by NEM incubation. Precipitates were tested for AIFM1, ALR and MIA40/CHCHD4 by reducing SDS-PAGE and immunoblotting. 1% of the total lysate was loaded as input control. M, mock control not expressing MIA40-Strep. Both, wildtype MIA40/CHCHD4 and MTS-MIA40/CHCHD4 but not MIA40/CHCHD4∆1-40 variants coprecipitated AIFM1. All mitochondria-localized variants precipitate ALR indicating redox functionality. Arrowhead indicates endogenous MIA40/CHCHD4.Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. HEK293 cells stably expressing MIA40/CHCHD4-Strep were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and MIA40/CHCHD4-Strep (B) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 interact over a period of at least 6-8 hours while the two MIA40/CHCHD4 substrates, NDUFB7 and AK2, are quickly released from MIA40/CHCHD4 (B, gray box). AP, affinity precipitation; asterisk, background band.C. Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. AIFM1 knockout cells stably expressing AIFM1-HA (C) were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and AIFM1-HA (C) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 and AIFM1 interact over a period of at least 6-8 hours AP, affinity precipitation; asterisk, background band.Eluted fractions were subjected to SDS-PAGE and TCE staining (E). AIFM1 and full-length MIA40/CHCHD4 migrate together indicating formation of a complex. MIA40/CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40/CHCHD4. AIFM1 and ∆1-40 MIA40/CHCHD4 do not migrate together emphasizing the importance of the N-terminal amino acids in MIA40 for interaction wth AIFM1.


Figure 5A . Gel filtration analysis to assess the AIFM1 - MIA40 complex in HEK293 cells . HEK293 cells , cells lacking AIFM1 ( AIFM1 knockout , AIFM1 KO ) or AIFM1 knockout cells complemented with AIFM1 - HA were lysed under native conditions ( triton X - 100 ) , and the cleared lysates subjected to gel filtration analysis . Eluted fractions were subjected to TCA precipitation , resuspension in loading buffer containing SDS and DTT , and subsequent immunoblotting against AIFM1 and MIA40 . Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa ( as judged by comparison to protein markers : apoferritin 443 kDa ; β - amylase 200 kDa ; alcohol dehydrogenase 150 kDa ; bovine serum albumin 66 kDa ; carbonic anhydrase 29 kDa ) . Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40 . This behavior is partially rescued by complementation with AIFM1 - HA . B . Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids ( ∆ 1 - 40 MIA40 / CHCHD4 - Strep ) , which constitute the AIFM1 interaction motif . Experiment was performed as described in ( A ) . AIFM1 and endogenous MIA40 / CHCHD4 migrate in a complex with a size larger than 150 kDa , while N - terminally truncated MIA40 / CHCHD4 migrates as monomer . For number of biological replicates and quantifications , see 5C . C . Quantification of 5A , B and Appendix Figure S9 . Quantification of the areas representing the region of monomeric MIA40 / CHCHD4 and the AIFM1 - MIA40 / CHCHD4 complex , respectively was performed . Quantifications consistently show that AIFM1 knockout , siRNA - mediated depletion of AIFM1 and overexpression of MIA40 / CHCHD4 all resulted in an increase of monomeric MIA40 / CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40 / CHCHD4 are tightly regulated . The numbers below the bars indicate the numbers of biological replicates performed . Averages and standard deviations are presented . D . Levels of AIFM1 and MIA40 / CHCHD4 in mitochondria isolated from the indicated tissues . Ratio of AIFM and MIA40 / CHCHD4 was formed and normalized to ratio in HEK293 cells ( = 1 ) . AIFM1 is in liver and kidney more abundant than MIA40 / CHCHD4 . E . Gel filtration analysis of lysates prepared from mitochondria isolated from the indicated tissues and from HepG2 and HEK293 cells . Experiment was performed as described in ( A ) . AIFM1 and MIA40 / CHCHD4 co - migrate in all samples . Excess AIFM1 migrates at lower molecular mass . Number of biological replicates in quantification plot . Where appropriate , standard deviations are presented . F . Assessing the solubility of MIA40 / CHCHD4 in the IMS . Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling . MIA40 / CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1 - HA . It was released from cells lacking AIFM1 . This indicates that MIA40 / CHCHD4 in wildtype cells is membrane - associated via AIFM1 .

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

Figure 5A. Gel filtration analysis to assess the AIFM1-MIA40 complex in HEK293 cells. HEK293 cells, cells lacking AIFM1 (AIFM1 knockout, AIFM1 KO) or AIFM1 knockout cells complemented with AIFM1-HA were lysed under native conditions (triton X-100), and the cleared lysates subjected to gel filtration analysis. Eluted fractions were subjected to TCA precipitation, resuspension in loading buffer containing SDS and DTT, and subsequent immunoblotting against AIFM1 and MIA40. Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa (as judged by comparison to protein markers: apoferritin 443 kDa; β-amylase 200 kDa; alcohol dehydrogenase 150 kDa; bovine serum albumin 66 kDa; carbonic anhydrase 29 kDa). Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40. This behavior is partially rescued by complementation with AIFM1-HA. B. Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids (∆1-40 MIA40/CHCHD4-Strep), which constitute the AIFM1 interaction motif. Experiment was performed as described in (A). AIFM1 and endogenous MIA40/CHCHD4 migrate in a complex with a size larger than 150 kDa, while N-terminally truncated MIA40/CHCHD4 migrates as monomer. For number of biological replicates and quantifications, see 5C. C. Quantification of 5A,B and Appendix Figure S9. Quantification of the areas representing the region of monomeric MIA40/CHCHD4 and the AIFM1-MIA40/CHCHD4 complex, respectively was performed. Quantifications consistently show that AIFM1 knockout, siRNA-mediated depletion of AIFM1 and overexpression of MIA40/CHCHD4 all resulted in an increase of monomeric MIA40/CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40/CHCHD4 are tightly regulated. The numbers below the bars indicate the numbers of biological replicates performed. Averages and standard deviations are presented.D. Levels of AIFM1 and MIA40/CHCHD4 in mitochondria isolated from the indicated tissues. Ratio of AIFM and MIA40/CHCHD4 was formed and normalized to ratio in HEK293 cells (= 1). AIFM1 is in liver and kidney more abundant than MIA40/CHCHD4.E. Gel filtration analysis of lysates prepared from mitochondria isolated from the indicated tissues and from HepG2 and HEK293 cells. Experiment was performed as described in (A). AIFM1 and MIA40/CHCHD4 co-migrate in all samples. Excess AIFM1 migrates at lower molecular mass. Number of biological replicates in quantification plot. Where appropriate, standard deviations are presented.F. Assessing the solubility of MIA40/CHCHD4 in the IMS. Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling. MIA40/CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1-HA. It was released from cells lacking AIFM1. This indicates that MIA40/CHCHD4 in wildtype cells is membrane-associated via AIFM1.


Figure 6A . Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132 . HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1 - HA were analysed by pulse - chase experiments coupled to redox state determination . Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132 . Conversely , NDUFS5 levels were strongly increased . In AIFM1 knockout cells , large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation - dependent protein import . The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132 . N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5 .

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

Figure 6A. Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analysed by pulse-chase experiments coupled to redox state determination. Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132. Conversely, NDUFS5 levels were strongly increased. In AIFM1 knockout cells, large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation-dependent protein import. The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132. N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5.


f1 ( a - d ) Formation of protein inclusions in SY5Y cells expressing FL ( a ) or the indicated truncated forms of Sph1 ( b - d ) was induced by 12 h lactacystin treatment . After extensive washes , cells were maintained in serum - supplemented ( serum + ) or serum - free ( serum − ) media , and in the absence ( Control ) or presence of 10 mM 3 - MA , an inhibitor of macroautophagy . Graphs show the relative fold difference in the number of cells containing either Sph1 Agm ( left ) or Agg ( right ) observed under different recovery conditions . Values are mean + s . e . ( n = 4 ) . * Significant versus serum + condition . See also Supplementary Figs S1 - S5 .

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

f1(a-d) Formation of protein inclusions in SY5Y cells expressing FL (a) or the indicated truncated forms of Sph1 (b-d) was induced by 12 h lactacystin treatment. After extensive washes, cells were maintained in serum-supplemented (serum+) or serum-free (serum−) media, and in the absence (Control) or presence of 10 mM 3-MA, an inhibitor of macroautophagy. Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (left) or Agg (right) observed under different recovery conditions. Values are mean+s.e. (n=4). *Significant versus serum+ condition. See also Supplementary Figs S1-S5.


f2 ( a - c ) Top : fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL ( a ) , or the indicated truncated Sph1 proteins ( b - c ) treated with lactacystin ( 12 h ) and recovered in basal conditions ( serum + ) . Graphs show the relative fold difference in the number of cells containing either Sph1 Agm ( middle ) or Agg ( bottom ) . Values are mean + s . e . ( n = 3 ) . * Significant versus control . ( d - f ) Relative fold difference in the number of FL ( d ) or truncated ( e - f ) Sph1 Agm ( left ) and Agg ( right ) in WT and Atg5 KO mice MEFs incubated in serum + or serum − conditions . Values are mean + s . e . ( n = 3 ) . * Significant versus serum + condition .

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

f2(a-c) Top: fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL (a), or the indicated truncated Sph1 proteins (b-c) treated with lactacystin (12 h) and recovered in basal conditions (serum+). Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (middle) or Agg (bottom). Values are mean+s.e. (n=3). *Significant versus control.(d-f) Relative fold difference in the number of FL (d) or truncated (e-f) Sph1 Agm (left) and Agg (right) in WT and Atg5 KO mice MEFs incubated in serum+ or serum− conditions. Values are mean+s.e. (n=3). *Significant versus serum+ condition.


f3 ( a - d ) Left : fluorescence images of SY5Y cells co - expressing HA - p38 ( red ) and GFP ( green ) forms of FL ( a ) or the indicated truncated Sph1 ( b - d ) . Cells were treated with lactacystin ( 12 h ) and recovered in the presence or absence of serum . Graphs : percentage of p38 existing alone in Agm or co - aggregating with Sph1 in the forms of Agm or Agg ( left ) , and susceptibility of p38 existing on its own in Agm or co - aggregating with Sph1 proteins in Agm toward serum - stimulated autophagic clearance ( right ) . Values are expressed as folds Agm in the presence of serum and are mean + s . e . ( n = 3 ) . * Significant versus serum + condition calculated using Student ' s t - test . ( e ) Autophagic susceptibility of Agm formed by p38 and ANK1 - p38 fusion proteins . Values are mean + s . e . ( n = 3 ) . * Significant versus control group calculated using Bonferroni test . See also Supplementary Fig . S6 .

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 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

f3(a-d) Left: fluorescence images of SY5Y cells co-expressing HA-p38 (red) and GFP (green) forms of FL (a) or the indicated truncated Sph1 (b-d). Cells were treated with lactacystin (12 h) and recovered in the presence or absence of serum. Graphs: percentage of p38 existing alone in Agm or co-aggregating with Sph1 in the forms of Agm or Agg (left), and susceptibility of p38 existing on its own in Agm or co-aggregating with Sph1 proteins in Agm toward serum-stimulated autophagic clearance (right). Values are expressed as folds Agm in the presence of serum and are mean+s.e. (n=3). *Significant versus serum+ condition calculated using Student's t-test.(e) Autophagic susceptibility of Agm formed by p38 and ANK1-p38 fusion proteins. Values are mean+s.e. (n=3). *Significant versus control group calculated using Bonferroni test. See also Supplementary Fig. S6.


f4 ( a ) Top : colocalization of LAMP1 ( red ) with Agm and Agg of ACA and CACT Sph1 proteins highlighted by anti - GFP ( green ) in SY5Y cells . Arrows indicate colocalization . Bottom : percentage LAMP1 colocalization with Agm and Agg in cells maintained in serum + or serum − media , treated or not with lysosomal inhibitors ( N / L ) . ( b ) Percentage LAMP1 colocalization with Agm in cells co - expressing HA - p38 and the indicated Sph1 proteins maintained in the absence of serum . ( c ) Left : colocalization of LAMP1 ( red ) with GFP - p38 and - ANK1 - p38 Agm in SY5Y cells maintained in the absence of serum . Arrows indicate colocalization . Right : percentage LAMP1 colocalization with p38 and ANK1 - p38 Agm in cells maintained in the presence or absence of serum . ( d , e ) Correlative light and electron microscopy of SY5Y cells expressing the indicated GFP - tagged Sph1 truncations ( d ) , or p38 and ANK1 - p38 ( e ) . Top : phase - contrast fluorescence microscopy ; middle : electron micrographs of the boxed region ; bottom : higher magnification examples of autophagic vacuoles ( yellow arrows ) . # Location of the Agm or Agg structures in both the fluorescence images and the corresponding micrographs . ( f ) Top : colocalization of LC3 ( red ) with GFP - p38 and - ANK1 - p38 Agm in SY5Y cells maintained in the absence of serum . Arrows indicate colocalization . Bottom : percentage colocalization of LC3 with the fluorescent proteins . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition ( a , c , f ) or p38 Agm ( b ) calculated using Bonferroni test . See also Supplementary Fig . S7 .

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

f4(a) Top: colocalization of LAMP1 (red) with Agm and Agg of ACA and CACT Sph1 proteins highlighted by anti-GFP (green) in SY5Y cells. Arrows indicate colocalization. Bottom: percentage LAMP1 colocalization with Agm and Agg in cells maintained in serum+ or serum− media, treated or not with lysosomal inhibitors (N/L).(b) Percentage LAMP1 colocalization with Agm in cells co-expressing HA-p38 and the indicated Sph1 proteins maintained in the absence of serum.(c) Left: colocalization of LAMP1 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Right: percentage LAMP1 colocalization with p38 and ANK1-p38 Agm in cells maintained in the presence or absence of serum.(d,e) Correlative light and electron microscopy of SY5Y cells expressing the indicated GFP-tagged Sph1 truncations (d), or p38 and ANK1-p38 (e). Top: phase-contrast fluorescence microscopy; middle: electron micrographs of the boxed region; bottom: higher magnification examples of autophagic vacuoles (yellow arrows). #Location of the Agm or Agg structures in both the fluorescence images and the corresponding micrographs.(f) Top: colocalization of LC3 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Bottom: percentage colocalization of LC3 with the fluorescent proteins. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,c,f) or p38 Agm (b) calculated using Bonferroni test. See also Supplementary Fig. S7.


f5 ( a ) Fluorescence images showing colocalization patterns of the indicated GFP - p38 variants and Sph1 ( green ) with endogenous p62 ( red ) in SY5Y cells . Arrows indicate colocalization . ( b ) Quantitative FRAP analysis of mCherry - p62 expressed alone ( control ) or co - expressed with the indicated GFP - Sph1 and - p38 variants . Top : time course of changes in relative fluorescence intensity ( RFI ) ; bottom : percentage of p62 in the diffusing fraction and diffusion time ( t1 / 2 ) . Values are mean + s . e . of ten cells analysed over three different experiments . Representative images are shown in Supplementary Fig . S8 . ( c - d ) Top : IBs for p62 and actin in control and p62 knockdown ( p62 KD # 325 or # 788 ) SY5Y cells ( c ) ; and in control and p62 KO MEFs ( d ) . Bottom : autophagic clearance of GFP - ANK1 - p38 Agm in the same cells in response to serum removal . ( e ) Fluorescence images of GFP - p38 , - ANK1 - p38 and - NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1 . Arrows indicate colocalization . ( f , g ) Top : IBs for NBR1 , p62 and actin in control and NBR1 knockdown ( NBR1 KD # 161 ) SY5Y cells ( f ) , and in control and p62 / NBR1 double knockdown ( Double KD # A or # B ) SY5Y cells ( g ) . Bottom : autophagic clearance of GFP - ANK1 - p38 Agm in the same cells . For all densitometric analyses , values are mean + s . e . ( n = 3 ) . * Significant versus serum + condition . See also Supplementary Fig . S8 - S11 .

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 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 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 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

f5(a) Fluorescence images showing colocalization patterns of the indicated GFP-p38 variants and Sph1 (green) with endogenous p62 (red) in SY5Y cells. Arrows indicate colocalization.(b) Quantitative FRAP analysis of mCherry-p62 expressed alone (control) or co-expressed with the indicated GFP-Sph1 and -p38 variants. Top: time course of changes in relative fluorescence intensity (RFI); bottom: percentage of p62 in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Representative images are shown in Supplementary Fig. S8.(c-d) Top: IBs for p62 and actin in control and p62 knockdown (p62 KD #325 or #788) SY5Y cells (c); and in control and p62 KO MEFs (d). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells in response to serum removal.(e) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1. Arrows indicate colocalization.(f,g) Top: IBs for NBR1, p62 and actin in control and NBR1 knockdown (NBR1 KD #161) SY5Y cells (f), and in control and p62/NBR1 double knockdown (Double KD #A or #B) SY5Y cells (g). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells. For all densitometric analyses, values are mean+s.e. (n=3). *Significant versus serum+ condition. See also Supplementary Fig. S8-S11.


f6 ( a - d ) Fluorescence images of GFP - p38 , - ANK1 - p38 and - NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin - 1 ( a ) , Vps34 ( b ) , Atg14L ( c ) and DFCP - 1 ( d ) . Arrows indicate colocalization .

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

f6(a-d) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin-1 (a), Vps34 (b), Atg14L (c) and DFCP-1 (d). Arrows indicate colocalization.


f7 ( a ) Quantitative FRAP analysis of the indicated GFP - p38 variants . Top : representative images . Middle : time course of changes in relative fluorescence intensity ( RFI ) . Bottom : percentage of proteins in the diffusing fraction and diffusion time ( t1 / 2 ) . Values are mean + s . e . of ten cells analysed over three different experiments . Scale bars , 10 μm ( b ) Top : schematic showing all lysine ( K ) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site ( s ) . Bottom : relative fold difference in the number of Agm formed by WT and the different ' K to R ' mutants of ANK1 - p38 fusion protein in SY5Y cells maintained in the presence or absence of serum . ( c ) IBs for HA ( top ) and GFP ( bottom ) of total lysates ( Input ) and GFP immunoprecipitates ( IP ) from the cell lysates of SY5Y cells co - transfected with the indicated HA - ubiquitin constructs and GFP - p38 or - ANK1 - p38 WT or mutant K385R in the presence of lactacystin . ( d ) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination . Values were corrected by levels of GFP immunoprecipitated . ( e ) Top : IF for GFP and K63 - linked ubiquitin ( red ) of SY5Y cells expressing GFP - p38 or - ANK1 - p38 WT and mutant K385R ( green ) . Arrows indicate colocalization . Bottom : percentage of Agm positive for K63 - linked ubiquitin . ( f ) Quantitative FRAP analysis of GFP - ANK1 - p38 K385R and GFP - ANK1 - p38 as in ( a ) . Values are mean + s . e . of ten cells analysed over three different experiments . ( g ) Top : representative images of GFP - ANK1 - p38 K385R Agm in SY5Y cells immunostained for Atg14L , Vps34 and DFCP - 1 . Bottom : percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L , Vps34 and DFCP - 1 under serum − conditions . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition or p38 Agm . # Significant versus ANK1 - p38 Agm . Significance calculated using Bonferroni test . See also Supplementary Fig . S8 .

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 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 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 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

f7(a) Quantitative FRAP analysis of the indicated GFP-p38 variants. Top: representative images. Middle: time course of changes in relative fluorescence intensity (RFI). Bottom: percentage of proteins in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Scale bars, 10 μm(b) Top: schematic showing all lysine (K) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site(s). Bottom: relative fold difference in the number of Agm formed by WT and the different 'K to R' mutants of ANK1-p38 fusion protein in SY5Y cells maintained in the presence or absence of serum.(c) IBs for HA (top) and GFP (bottom) of total lysates (Input) and GFP immunoprecipitates (IP) from the cell lysates of SY5Y cells co-transfected with the indicated HA-ubiquitin constructs and GFP-p38 or -ANK1-p38 WT or mutant K385R in the presence of lactacystin. (d) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination. Values were corrected by levels of GFP immunoprecipitated.(e) Top: IF for GFP and K63-linked ubiquitin (red) of SY5Y cells expressing GFP-p38 or -ANK1-p38 WT and mutant K385R (green). Arrows indicate colocalization. Bottom: percentage of Agm positive for K63-linked ubiquitin.(f) Quantitative FRAP analysis of GFP-ANK1-p38 K385R and GFP-ANK1-p38 as in (a). Values are mean+s.e. of ten cells analysed over three different experiments.(g) Top: representative images of GFP-ANK1-p38 K385R Agm in SY5Y cells immunostained for Atg14L, Vps34 and DFCP-1. Bottom: percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L, Vps34 and DFCP-1 under serum− conditions. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition or p38 Agm. #Significant versus ANK1-p38 Agm. Significance calculated using Bonferroni test. See also Supplementary Fig. S8.


f8 ( a , b ) Autophagic clearance of Agm ( a ) or Agg ( b ) formed by FL WT and mutant K385R or K394R GFP - Sph1 in SY5Y cells , in response to serum removal ( a ) or upon blockage of basal autophagy with 3 - MA ( b ) . ( c , d ) Left : Agm ( c ) or Agg ( d ) of FL GFP - Sph1 ( green ) immunostained for endogenous K63 - linked ubiquitin ( red ) in SY5Y cells . Arrows in ( c ) indicate colocalization . Right : percentage of Agm ( c ) or Agg ( d ) positive for K63 - linked ubiquitin . ( e ) Formation of WT and mutant K385R FL Sph1 ( left ) and ANK1 - p38 ( right ) Agm in response to proteasome inhibition . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition ( a , b ) or WT FL Sph1 or ANK1 - p38 ( c - e ) .

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

f8(a,b) Autophagic clearance of Agm (a) or Agg (b) formed by FL WT and mutant K385R or K394R GFP-Sph1 in SY5Y cells, in response to serum removal (a) or upon blockage of basal autophagy with 3-MA (b).(c,d) Left: Agm (c) or Agg (d) of FL GFP-Sph1 (green) immunostained for endogenous K63-linked ubiquitin (red) in SY5Y cells. Arrows in (c) indicate colocalization. Right: percentage of Agm (c) or Agg (d) positive for K63-linked ubiquitin.(e) Formation of WT and mutant K385R FL Sph1 (left) and ANK1-p38 (right) Agm in response to proteasome inhibition. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,b) or WT FL Sph1 or ANK1-p38 (c-e).


Figure 1Interaction analysis for MIA40 and AK2 . Stable inducible cell lines that express either different variants of MIA40 - Strep ( wildtype and a redox inactive mutant ( MIA40C53S , C55S ) , ( Fischer et al . , 2013 ) or AK2 - HA were treated with N - ethyl maleimide ( NEM ) to stop thiol - disulfide exchange processes and lysed under denaturing conditions . Lysates were analysed by subsequent affinity purification against Strep ( MIA40 ) and immunoblotting . Like classical MIA40 substrates , AK2 interacted with the active site of MIA40 ( lane 6 ) . 0 . 5 % input was loaded as control . Redox state analysis of endogenous AK2 by inverse redox shift assay . HEK293 cells were lysed by TCA precipitation after NEM treatment , lysates were reduced using TCEP and then thiols modified using the alkylating agent mmPEG12 . With this " inverse " method , previously oxidized cysteine thiols are modified with mmPEG12 , which results in a slower migration on SDS - PAGE . Treatment of intact cells with diamide and consequently modification of all cysteines served as control . Likewise , absence of NEM treatment served as ' max ' control . Treatment lysates with NEM instead of mmPEG12 served as minimal shift ( min ) control . A , B , isoforms of AK2 . Both isoforms of AK2 are semi - oxidized in intact cells . Oxidation kinetics experiment to investigate maturation of endogenous AK2 . HEK293 cells were pulse labeled for 10 min with [ 35S ] - methionine and chased with cold methionine for the indicated times . The chase was stopped by TCA precipitation , and then the lysate was treated with mmPEG12 to determine the AK2 redox states , followed by IP against AK2 . Eluates were analyzed by Tris - Tricine - PAGE and autoradiography . With this " direct " redox state determination method reduced AK2 was modified with either three ( isoform B ) or four ( isoform A ) mmPEG12 . Semi - oxidized AK2 was modified with only one ( " B " ) or two ( " A " ) mmPEG12 , respectively . Unmodified and fully reduced cell lysates served as controls . Endogenous AK2 becomes oxidized with a half time of less than 20 min . Oxidation kinetics experiment to investigate the influence of MIA40 on AK2 maturation . Experiment was performed as described in ( D ) , except that 72 h before experiments cells were transfected with control siRNA or siRNA directed against MIA40 . Endogenous AK2 oxidation depends on MIA40 . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 , * * represents p < 0 . 01 and * * * represents 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 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 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 1Interaction analysis for MIA40 and AK2. Stable inducible cell lines that express either different variants of MIA40-Strep (wildtype and a redox inactive mutant (MIA40C53S,C55S), (Fischer et al., 2013) or AK2-HA were treated with N-ethyl maleimide (NEM) to stop thiol-disulfide exchange processes and lysed under denaturing conditions. Lysates were analysed by subsequent affinity purification against Strep (MIA40) and immunoblotting. Like classical MIA40 substrates, AK2 interacted with the active site of MIA40 (lane 6). 0.5% input was loaded as control.Redox state analysis of endogenous AK2 by inverse redox shift assay. HEK293 cells were lysed by TCA precipitation after NEM treatment, lysates were reduced using TCEP and then thiols modified using the alkylating agent mmPEG12. With this "inverse" method, previously oxidized cysteine thiols are modified with mmPEG12, which results in a slower migration on SDS-PAGE. Treatment of intact cells with diamide and consequently modification of all cysteines served as control. Likewise, absence of NEM treatment served as 'max' control. Treatment lysates with NEM instead of mmPEG12 served as minimal shift (min) control. A, B, isoforms of AK2. Both isoforms of AK2 are semi-oxidized in intact cells.Oxidation kinetics experiment to investigate maturation of endogenous AK2. HEK293 cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by TCA precipitation, and then the lysate was treated with mmPEG12 to determine the AK2 redox states, followed by IP against AK2. Eluates were analyzed by Tris-Tricine-PAGE and autoradiography. With this "direct" redox state determination method reduced AK2 was modified with either three (isoform B) or four (isoform A) mmPEG12. Semi-oxidized AK2 was modified with only one ("B") or two ("A") mmPEG12, respectively. Unmodified and fully reduced cell lysates served as controls. Endogenous AK2 becomes oxidized with a half time of less than 20 min.Oxidation kinetics experiment to investigate the influence of MIA40 on AK2 maturation. Experiment was performed as described in (D), except that 72 h before experiments cells were transfected with control siRNA or siRNA directed against MIA40. Endogenous AK2 oxidation depends on MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, ** represents p<0.01 and *** represents p<0.001.


Figure 2Influence of AK2 cysteines on the AK2 redox state . Cells expressing single cysteine to serine mutants of AK2 - HA ( isoform A ) were lysed after NEM treatment , and analyzed by inverse redox shift assay using mmPEG12 , SDS - PAGE and immunoblot against the HA tag . AK2C42S and AK2C92S are present in their completely reduced state indicating presence of a disulfide bond between both cysteines . Influence of AK2 cysteines on cellular localization . Cells expressing single cysteine to serine mutants of AK2 - HA ( isoform A ) were fixed and analyzed by immunofluorescence . The colocalization of AK2 - HA variants with Mitotracker was quantified using Cellprofiler ( Pearson ' s correlation coefficient ) . C42 and C92 are critical for mitochondrial localization of AK2 . The C40S mutant exhibits significantly decreased colocalization with mitochondria . A Kruskall - Wallis rank sum test / Dunn - test was performed . Numbers of analysed cells are indicated in the plot . * * * represents p - value < 0 . 001 compared to wild - type . The black horizontal line represents the median . Data information : Scale bars : 10 μm .

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 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

Figure 2Influence of AK2 cysteines on the AK2 redox state. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were lysed after NEM treatment, and analyzed by inverse redox shift assay using mmPEG12, SDS-PAGE and immunoblot against the HA tag. AK2C42S and AK2C92S are present in their completely reduced state indicating presence of a disulfide bond between both cysteines.Influence of AK2 cysteines on cellular localization. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were fixed and analyzed by immunofluorescence. The colocalization of AK2-HA variants with Mitotracker was quantified using Cellprofiler (Pearson's correlation coefficient). C42 and C92 are critical for mitochondrial localization of AK2. The C40S mutant exhibits significantly decreased colocalization with mitochondria. A Kruskall-Wallis rank sum test/ Dunn-test was performed. Numbers of analysed cells are indicated in the plot. *** represents p-value <0.001 compared to wild-type. The black horizontal line represents the median. Data information: Scale bars: 10 μm.


Figure 3Steady state levels of endogenous AK2 in presence and absence of MIA40 . 72 h before experiments , HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 or ALR . Cell lysates were analysed by immunoblot against the indicated proteins . MIA40 depletion led to a strong decrease in AK2 and ALR levels but not of the control proteins . ALR depletion did not affect AK2 levels . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 , . Stability of endogenous AK2 in the presence and absence of MIA40 . 72 h before experiments HeLa , cells were transfected with control siRNA or siRNAs directed against MIA40 . Cells were pulse labeled for 10 min with [ 35S ] - methionine and chased with cold methionine for the indicated times . The chase was stopped by cell lysis , and AK2 was isolated by denaturing immunoprecipitation . Eluates were analyzed by SDS - PAGE and autoradiography . Stability of endogenous AK2 is significantly decreased upon depletion of MIA40 . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed ; * represents p - value < 0 . 05 compared to wild - type . Stability of cytosolic AK2 - HA cysteine variant . Experiment was performed as in ( B ) except that cells expressing AK2C40S , C42S , C92S - HA were analysed by IP against HA . AK2C40S , C42S , C92S - HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132 . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 , . Steady state levels of AK2 - HA cysteine variants . AK2C40S , C42S , C92S - HA and AK2WT - HA were expressed in the presence or absence of proteasomal inhibitor MG132 . Protein levels at steady state were analysed by immunoblot . All variants are stabilized by proteasomal inhibition . Reported values are the mean of 3 ( AK2WT - HA ) or 5 ( AK2C40S , C42S , C92S - HA ) independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc test was performed . * * * represents p < 0 . 001 . Steady state levels of AK2 - HA cysteine variant . 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5 . AK2C40S , C42S , C92S - HA was stabilized by decreased proteasome levels . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 .

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 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 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 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 3Steady state levels of endogenous AK2 in presence and absence of MIA40. 72 h before experiments, HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 or ALR. Cell lysates were analysed by immunoblot against the indicated proteins. MIA40 depletion led to a strong decrease in AK2 and ALR levels but not of the control proteins. ALR depletion did not affect AK2 levels. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.Stability of endogenous AK2 in the presence and absence of MIA40. 72 h before experiments HeLa, cells were transfected with control siRNA or siRNAs directed against MIA40. Cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by cell lysis, and AK2 was isolated by denaturing immunoprecipitation. Eluates were analyzed by SDS-PAGE and autoradiography. Stability of endogenous AK2 is significantly decreased upon depletion of MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed; * represents p-value <0.05 compared to wild-type.Stability of cytosolic AK2-HA cysteine variant. Experiment was performed as in (B) except that cells expressing AK2C40S,C42S,C92S-HA were analysed by IP against HA. AK2C40S,C42S,C92S-HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.Steady state levels of AK2-HA cysteine variants. AK2C40S,C42S,C92S-HA and AK2WT-HA were expressed in the presence or absence of proteasomal inhibitor MG132. Protein levels at steady state were analysed by immunoblot. All variants are stabilized by proteasomal inhibition. Reported values are the mean of 3 (AK2WT-HA) or 5 (AK2C40S,C42S,C92S-HA) independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. *** represents p<0.001.Steady state levels of AK2-HA cysteine variant. 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5. AK2C40S,C42S,C92S-HA was stabilized by decreased proteasome levels. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.


Figure 4Steady state levels of AK2C40S , C42S , C92S - HA in presence and absence of DPP8 and DPP9 . 72 h before experiments , HEK293 cells stably expressing AK2C40S , C42S , C92S - HA were transfected with control siRNA or siRNAs directed against DPP8 or / and DPP9 . Cell lysates were analysed by immunoblot against the indicated proteins . DPP9 depletion led to an increase in AK2C40S , C42S , C92S - HA levels , while DPP8 depletion did not . Combined depletion of DPP8 and DPP9 increased AK2C40S , C42S , C92S - HA levels even further . This indicates that DPP9 is the main peptidase and DPP8 can serve as its backup . White and black arrow heads indicate the endogenous and HA - tagged AK2 , respectively . The gray arrow head indicates the DPP9 band . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc test was performed . * represents p < 0 . 05 , , # # represent p < 0 . 01 and * * * , # # # represent p < 0 . 001 . Steady state levels of AK2C40S , C42S , C92S - HA upon incubation with the DPP9 inhibitor 1G244 ( Wu et al . , 2009 ) . As ( B ) except that cells were treated for 16 h with inhibitor or DMSO as control . AK2C40S , C42S , C92S - HA levels were strongly increased upon DPP8 / 9 inhibition . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * * * represents p < 0 . 001 . Steady state levels of endogenous AK2 in presence and absence of DPP9 and MIA40 . 72 h before experiments HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 and / or DPP9 . Cell lysates were analysed by immunoblot against the indicated proteins . If import of endogenous AK2 was prevented by depletion of MIA40 , then concomitant depletion of DPPs increased its levels . Strikingly , depletion of DPP9 alone also increased the levels of endogenous AK2 . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc test was performed . * * represents p < 0 . 01 . Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244 . As ( D ) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control . Endogenous AK2 levels were increased upon DPP8 / 9 inhibition . Reported values are the mean of 5 ( 3 d ) and 6 ( 5 d ) independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 , and * * * represents p < 0 . 001 . Steady state levels of AK2 - HA variants with mutated N - termini upon incubation with either 1G244 or vehicle control . HEK293 cells stably expressing AK2 - HA variants were lysed and subjected to immunoblot against the indicated proteins . Levels of DPP8 / 9 - insensitive AK2 variants are strongly increased . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc test was performed . * * represents p < 0 . 01 and # # # , * * * represents p < 0 . 001 . Asterisk denominate statistical comparison with respective control , and hash tag represents comparison to N - WT .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Steady state levels of AK2C40S,C42S,C92S-HA in presence and absence of DPP8 and DPP9. 72 h before experiments, HEK293 cells stably expressing AK2C40S,C42S,C92S-HA were transfected with control siRNA or siRNAs directed against DPP8 or/and DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. DPP9 depletion led to an increase in AK2C40S,C42S,C92S-HA levels, while DPP8 depletion did not. Combined depletion of DPP8 and DPP9 increased AK2C40S,C42S,C92S-HA levels even further. This indicates that DPP9 is the main peptidase and DPP8 can serve as its backup. White and black arrow heads indicate the endogenous and HA-tagged AK2, respectively. The gray arrow head indicates the DPP9 band. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. * represents p<0.05, ,## represent p<0.01 and ***,### represent p<0.001.Steady state levels of AK2C40S,C42S,C92S-HA upon incubation with the DPP9 inhibitor 1G244 (Wu et al., 2009). As (B) except that cells were treated for 16 h with inhibitor or DMSO as control. AK2C40S,C42S,C92S-HA levels were strongly increased upon DPP8/9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. *** represents p<0.001.Steady state levels of endogenous AK2 in presence and absence of DPP9 and MIA40. 72 h before experiments HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 and/or DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. If import of endogenous AK2 was prevented by depletion of MIA40, then concomitant depletion of DPPs increased its levels. Strikingly, depletion of DPP9 alone also increased the levels of endogenous AK2. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01.Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244. As (D) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control. Endogenous AK2 levels were increased upon DPP8/9 inhibition. Reported values are the mean of 5 (3 d) and 6 (5 d) independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, and *** represents p<0.001.Steady state levels of AK2-HA variants with mutated N-termini upon incubation with either 1G244 or vehicle control. HEK293 cells stably expressing AK2-HA variants were lysed and subjected to immunoblot against the indicated proteins. Levels of DPP8/9-insensitive AK2 variants are strongly increased. Reported values are the mean of 4 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01 and ###, *** represents p<0.001. Asterisk denominate statistical comparison with respective control, and hash tag represents comparison to N-WT.


Figure 5Cell fractionation upon inhibition of DPP9 activity . HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO . Cells were subjected to fractionation into ' cytosol ' ( supernatant ) and ' mitochondria ' ( pellet ) by differential digitonin lysis and centrifugation . The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration ( lanes 2 , 5 , 8 , 11 ) irrespective of 1G244 treatment . Likewise , the cytosolic marker protein LDH localized under all conditions to the same fractions . Conversely , endogenous AK2 appeared at this digitonin concentration in the ' cytosol ' fraction but only upon 1G244 treatment ( compare lanes 2 and 5 ) . Thus , inhibition of DPP9 results in appearance of AK2 in the cytosol . Quantification shows loading - control adjusted AK2 - signals of 1G244 - treated ' cytosol ' fraction ( lane 5 ) over DMSO - treated ' cytosol ' fraction ( lane 2 ) . White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . Localization of DPP9 - insensitive AK2 - HA variants . Experiment was performed as described in Figure 2C . Compared to the wild type , DPP9 - insensitive and import - competent AK2 - HA variants co - localized with cytosolic markers indicating partial cytosolic localization . The colocalization of AK2 - HA variants with Mitotracker was quantified using Fiji ( Pearson ' s correlation coefficient ) . A one - way ANOVA with post - hoc Tukey test was performed . Numbers of analysed cells are indicated in the plot . * * represents p < 0 . 01 . Data shown in Figures 2C , 5B , S2 and S5 are derived from the same experiments . The black horizontal line represents the median . Data information : Scale bars : 10 μm . Localization of AK2C40S - HA upon proteasomal inhibition . Experiment was performed as described in Figure 2C . Upon MG132 treatment , colocalization of AK2C40S - HA with the cytosolic marker increased . A one - way ANOVA with post - hoc Tukey test was performed . Numbers of analysed cells are indicated in the plot . * * * represents p < 0 . 001 . The black horizontal line represents the median . Data information : Scale bars : 10 μm .

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 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 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 5Cell fractionation upon inhibition of DPP9 activity. HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO. Cells were subjected to fractionation into 'cytosol' (supernatant) and 'mitochondria' (pellet) by differential digitonin lysis and centrifugation. The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration (lanes 2,5,8,11) irrespective of 1G244 treatment. Likewise, the cytosolic marker protein LDH localized under all conditions to the same fractions. Conversely, endogenous AK2 appeared at this digitonin concentration in the 'cytosol' fraction but only upon 1G244 treatment (compare lanes 2 and 5). Thus, inhibition of DPP9 results in appearance of AK2 in the cytosol. Quantification shows loading-control adjusted AK2-signals of 1G244-treated 'cytosol' fraction (lane 5) over DMSO-treated 'cytosol' fraction (lane 2). White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed.Localization of DPP9-insensitive AK2-HA variants. Experiment was performed as described in Figure 2C. Compared to the wild type, DPP9-insensitive and import-competent AK2-HA variants co-localized with cytosolic markers indicating partial cytosolic localization. The colocalization of AK2-HA variants with Mitotracker was quantified using Fiji (Pearson's correlation coefficient). A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. ** represents p<0.01. Data shown in Figures 2C, 5B, S2 and S5 are derived from the same experiments. The black horizontal line represents the median. Data information: Scale bars: 10 μm.Localization of AK2C40S-HA upon proteasomal inhibition. Experiment was performed as described in Figure 2C. Upon MG132 treatment, colocalization of AK2C40S-HA with the cytosolic marker increased. A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. *** represents p<0.001.The black horizontal line represents the median. Data information: Scale bars: 10 μm.


Figure 6AK2 activity in dependence of its substrate AMP . Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0 . 012 mM AMP . The profiles for both AK2C40S , C232S and AK2C40S , C42S , C92S , C232S are superimposable . Reported values are the mean of at least 3 independent experiments ; error bars represent ± SD . AK2 activity in dependence of its substrate ADP . Maximal activity was observed at around 1 - 2 mM ADP with an apparent KM of 0 . 12 mM and 0 . 1 mM ADP for AK2C40S , C232S and AK2C40S , C42S , C92S , C232S , respectively . The profiles for both AK2C40S , C232S and AK2C40S , C42S , C92S , C232S are thus highly similar . Reported values are the mean of at least 3 independent experiments ; error bars represent ± SD . Activity assay for AK2 - HA variants isolated from HEK293 cells . Indicated AK2 - HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants . Then , the AK2 activity assay described in ( B ) was performed . While import - competent AK2 variants exhibited full activity , a cytosolic AK2 variant with the native N - terminus showed very low activity . This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9 - insensitive cytosolic AK2 variants . The enzymatic inactive K28Q variants served as negative control indicating that only AK2 - HA - dependent activity ways measured by our assay . Reported values are the mean of 2 independent experiments ; error bars represent ± SD .

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 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

Figure 6AK2 activity in dependence of its substrate AMP. Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0.012 mM AMP. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are superimposable. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   AK2 activity in dependence of its substrate ADP. Maximal activity was observed at around 1-2 mM ADP with an apparent KM of 0.12 mM and 0.1 mM ADP for AK2C40S,C232S and AK2C40S,C42S,C92S,C232S, respectively. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are thus highly similar. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   Activity assay for AK2-HA variants isolated from HEK293 cells. Indicated AK2-HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants. Then, the AK2 activity assay described in (B) was performed. While import-competent AK2 variants exhibited full activity, a cytosolic AK2 variant with the native N-terminus showed very low activity. This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9-insensitive cytosolic AK2 variants. The enzymatic inactive K28Q variants served as negative control indicating that only AK2-HA-dependent activity ways measured by our assay. Reported values are the mean of 2 independent experiments; error bars represent ±SD.


Figure 7Steady state levels of NDUFB10 , NDUFS5 , NDUFA8 , COA6 , CHCHD2 , and TIMM9 upon incubation with the DPP9 inhibitor 1G244 . Cells were treated for 24h with inhibitor or DMSO as control , and were then analysed by immunoblot against the indicated proteins . Proteins except TIMM9 are stabilized upon DPP9 inhibition . Reported values are the mean of 3 - 5 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * * represents p < 0 . 01 . Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244 . Cells were treated for 24h with inhibitor or DMSO as control , and were then analysed by immunoblot against the indicated proteins . NDUFB10C107S is stabilized upon DPP9 inhibition . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - test was performed for the respective control and treatment samples . * * represents p < 0 . 01 . Localization of NDUFB10 variants upon 1G244 treatment . Experiment was performed as in ( C ) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins . Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition . Reported values are the mean of 2 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 .

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

Figure 7Steady state levels of NDUFB10, NDUFS5, NDUFA8, COA6, CHCHD2, and TIMM9 upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. Proteins except TIMM9 are stabilized upon DPP9 inhibition. Reported values are the mean of 3-5 independent experiments; error bars represent ±SD. Student's t-test was performed. ** represents p<0.01.Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. NDUFB10C107S is stabilized upon DPP9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed for the respective control and treatment samples. ** represents p<0.01.Localization of NDUFB10 variants upon 1G244 treatment. Experiment was performed as in (C) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins. Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition. Reported values are the mean of 2 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.


Figure 2A Pup survival analysis between P0 - P10 , by sex . Each dot represents the absolute percent of remaining animals per group ( Jag1CTRL n = 211 , male Jag1Ndr / Ndr n = 26 , female Jag1Ndr / Ndr n = 20 , log - rank test , * * * * P < 0 . 0001 ) . B P10 plasma total bilirubin levels ( n = 6 per group , unpaired t test , * * P = 0 . 0091 ) . C P10 plasma thrombin antithrombin levels ( n = 6 per group , unpaired t test , ns , P = 0 . 2788 ) . D P10 serum fibrinogen levels ( n = 6 per group , unpaired t test , ns , P = 0 . 7585 ) . E P10 whole blood prothrombin time ( INR ) ( n = 6 per group , unpaired t test , ns , P > 0 . 9999 ) . F P10 tail bleeding time ( n = 6 per group , unpaired t test , ns , P = 0 . 8916 ) . G - I µCT of P30 skulls . ( G ) Color map depicting wall thickness . Scale bar 3 . 5 mm . ( H ) Skull full thickness volume ( n = 5 - 6 per group , two - way ANOVA . Interaction P = 0 . 7595 ; Sex P = 0 . 1164 , Genotype * * P = 0 . 0012 . Šídák ' s multiple comparisons test : Male Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0227 ; : Female Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 034 ) , ( I ) Compact bone total volume ( n = 5 - 6 per group , two - way ANOVA . Interaction P = 0 . 8631 ; Sex P = 0 . 0901 , Genotype * * P = 0 . 0014 . Šídák ' s multiple comparisons test : Male Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0305 ; Female Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0307 ) . J - K ( J ) Provoked vascular accidents induced by portal vein resin injection , with ( K ) resin leakage quantification outside of blood vessels ( red ) in adult mice ( n = 6 per group , unpaired t test , ns , P = 0 . 1088 ) . L Adult brain Evans blue assay ( Jag1CTRL n = 7 , Jag1Ndr / Ndr n = 8 , unpaired t test , ns , P = 0 . 3807 ) . M Hemorrhagic P3 Jag1Ndr / Ndr brain ( n = 2 / 83 Jag1Ndr / Ndr mice , sex n . d . ) . N Hemorrhagic P15 Jag1Ndr / Ndr retina . Scale bar 20 µm . Sex n . d . O Red blood cells outside P10 Jag1Ndr / Ndr retinal arteriole . Scale bar 10 µm .

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

Figure 2A Pup survival analysis between P0-P10, by sex. Each dot represents the absolute percent of remaining animals per group (Jag1CTRL n=211, male Jag1Ndr/Ndr n=26, female Jag1Ndr/Ndr n= 20, log-rank test, ****P<0.0001).B P10 plasma total bilirubin levels (n=6 per group, unpaired t test, **P=0.0091). C P10 plasma thrombin antithrombin levels (n=6 per group, unpaired t test, ns, P=0.2788).D P10 serum fibrinogen levels (n=6 per group, unpaired t test, ns, P=0.7585).E P10 whole blood prothrombin time (INR) (n=6 per group, unpaired t test, ns, P>0.9999).F P10 tail bleeding time (n=6 per group, unpaired t test, ns, P=0.8916).G-I µCT of P30 skulls. (G) Color map depicting wall thickness. Scale bar 3.5 mm. (H) Skull full thickness volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.7595; Sex P= 0.1164, Genotype **P= 0.0012. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0227; : Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.034), (I) Compact bone total volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.8631; Sex P= 0.0901, Genotype **P= 0.0014. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0305; Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0307).J-K (J) Provoked vascular accidents induced by portal vein resin injection, with (K) resin leakage quantification outside of blood vessels (red) in adult mice (n=6 per group, unpaired t test, ns, P=0.1088).L Adult brain Evans blue assay (Jag1CTRL n=7, Jag1Ndr/Ndr n=8, unpaired t test, ns, P=0.3807).M Hemorrhagic P3 Jag1Ndr/Ndr brain (n= 2/83 Jag1Ndr/Ndr mice, sex n.d.).N Hemorrhagic P15 Jag1Ndr/Ndr retina. Scale bar 20 µm. Sex n.d.O Red blood cells outside P10 Jag1Ndr/Ndr retinal arteriole. Scale bar 10 µm.