Figure 1Expression of Bhlhe40 ( grey ) and Bhlhe41 ( orange ) in the indicated cell populations , data are from the Immgen Database ( microarray data set ) . CMP - common myeloid progenitors . MΦ - macrophages . Flow cytometry detection of the Bhlhe40 ( grey ) and Bhlhe41 ( orange ) transcripts by the PrimeFlow RNA assay in alveolar ( right ) and peritoneal ( left ) macrophages . A probe specific for the Cd8a mRNA ( black line ) was used as a negative control . Results representative of at least four independent experiments . Cell surface phenotype and numbers of alveolar ( C ; digested lung ) and peritoneal ( D ) macrophages in WT and DKO mice . Additional gating for CD11b + cells was applied for peritoneal macrophages as indicated . Levels of CD11b expression on WT and DKO AMs are shown , and median fluorescent intensity for CD11b expression was quantified ( C , bottom left ; 3 mice per genotype ) . Absolute numbers of CD11chiCD11bloSiglecFhi alveolar macrophages from digested lungs ( C ) and CD11b + F4 / 80hi peritoneal macrophages ( D ) from WT and DKO mice are shown ( bottom ; 4 mice per genotype ) .

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

Figure 1Expression of Bhlhe40 (grey) and Bhlhe41 (orange) in the indicated cell populations, data are from the Immgen Database (microarray data set). CMP - common myeloid progenitors. MΦ - macrophages.Flow cytometry detection of the Bhlhe40 (grey) and Bhlhe41 (orange) transcripts by the PrimeFlow RNA assay in alveolar (right) and peritoneal (left) macrophages. A probe specific for the Cd8a mRNA (black line) was used as a negative control. Results representative of at least four independent experiments.Cell surface phenotype and numbers of alveolar (C; digested lung) and peritoneal (D) macrophages in WT and DKO mice. Additional gating for CD11b+ cells was applied for peritoneal macrophages as indicated. Levels of CD11b expression on WT and DKO AMs are shown, and median fluorescent intensity for CD11b expression was quantified (C, bottom left; 3 mice per genotype). Absolute numbers of CD11chiCD11bloSiglecFhi alveolar macrophages from digested lungs (C) and CD11b+F4/80hi peritoneal macrophages (D) from WT and DKO mice are shown (bottom; 4 mice per genotype).


Figure 2Frequency of WT donor ( CD45 . 1 ) , DKO donor ( CD45 . 2 ) and recipient ( CD45 . 1 / 2 ) cells in the indicated BM ( LSK , CMP ) , splenic ( monocytes ) , liver ( Kupffer cells ) , peritoneal and alveolar macrophage populations of lethally irradiated recipients ≥ 6 weeks after transfer of a 1 : 1 mixture of lineage - depleted BM cells from WT and DKO mice . P values < 0 . 05 ( paired Student ' s t - test ) between LSK and other populations are shown for frequencies of WT cells . Three mixed BM chimeras analyzed ; error bars represent s . d . Representative results of three independent experiments . Representative flow - cytometric analysis of lung ( B ) macrophages as in A ( left ) . The expression of CD45 . 1 and CD45 . 2 on donor and recipient cells is shown next to the indicated gates ( right ) . Representative flow - cytometric analysis of peritoneal ( C ) macrophages as in A ( left ) . The expression of CD45 . 1 and CD45 . 2 on donor and recipient cells is shown next to the indicated gates ( right ) . Frequency of WT donor ( CD45 . 1 ) , DKO donor ( CD45 . 2 ) and recipient ( CD45 . 1 / 2 ) cells among the BM LSK cells , splenic monocytes and alveolar macrophages of the recipients that were irradiated with lung shielding , treated with one dose of busulfan as described in Materials and Methods , transferred with a 1 : 1 mixture of lineage - depleted BM cells from WT and DKO mice and analyzed ≥ 8 weeks after transfer . Representative flow cytometry results for alveolar macrophages ( left ) and quantification ( right ) is shown . Surface phenotype of cells from the digested lungs ( right ) and quantification of donor contribution to the AM compartment ( left ) of AM - deficient Csf2rb - / - Csf2rb2 - / - recipients ( CD45 . 2 ) that were subjected to intranasal transfer of a 1 : 1 mixture of sorted fetal liver monocytes from WT ( CD45 . 1 / 2 ) and DKO ( CD45 . 2 ) E18 . 5 embryos at days 1 or 2 after birth and analyzed ≥ 7 weeks after the transfer . Cell surface phenotype of lung cells from a Csf2rb - / - Csf2rb2 - / - mouse that was not subjected to the transfer is shown for comparison . Frequency of donor - and recipient - derived AMs in WT and DKO recipients transferred at days 1 or 2 after birth intranasally with WT CD45 + cells sorted from neonatal lungs and analyzed ≥ 7 weeks after the transfer . Results for quantification ( lower panel ) are pooled from 3 independent experiments with a total of 8 WT and 9 DKO recipients ; various combinations of CD45 . 1 and CD45 . 2 were used to distinguish donor and recipient cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 2Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells in the indicated BM (LSK, CMP), splenic (monocytes), liver (Kupffer cells), peritoneal and alveolar macrophage populations of lethally irradiated recipients ≥ 6 weeks after transfer of a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice. P values < 0.05 (paired Student's t-test) between LSK and other populations are shown for frequencies of WT cells. Three mixed BM chimeras analyzed; error bars represent s.d. Representative results of three independent experiments.Representative flow-cytometric analysis of lung (B) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right).Representative flow-cytometric analysis of peritoneal (C) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right).Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells among the BM LSK cells, splenic monocytes and alveolar macrophages of the recipients that were irradiated with lung shielding, treated with one dose of busulfan as described in Materials and Methods, transferred with a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice and analyzed ≥ 8 weeks after transfer. Representative flow cytometry results for alveolar macrophages (left) and quantification (right) is shown.Surface phenotype of cells from the digested lungs (right) and quantification of donor contribution to the AM compartment (left) of AM-deficient Csf2rb-/-Csf2rb2-/-recipients (CD45.2) that were subjected to intranasal transfer of a 1:1 mixture of sorted fetal liver monocytes from WT (CD45.1/2) and DKO (CD45.2) E18.5 embryos at days 1 or 2 after birth and analyzed ≥ 7 weeks after the transfer. Cell surface phenotype of lung cells from a Csf2rb-/-Csf2rb2-/- mouse that was not subjected to the transfer is shown for comparison.Frequency of donor- and recipient- derived AMs in WT and DKO recipients transferred at days 1 or 2 after birth intranasally with WT CD45+ cells sorted from neonatal lungs and analyzed ≥ 7 weeks after the transfer. Results for quantification (lower panel) are pooled from 3 independent experiments with a total of 8 WT and 9 DKO recipients; various combinations of CD45.1 and CD45.2 were used to distinguish donor and recipient cells.


Figure 3Mixed WT : DKO BM chimeras established as in Figure 2A were injected with EdU on three consecutive days . EdU incorporation was analyzed one day after the last injection . Steady - state WT and DKO mice were injected with EdU and analyzed as in A . Representative flow cytometry plots of EdU incorporation by AMs ( C ) and their quantification ( results pooled from two independent experiments , 7 WT and 6 DKO mice were analyzed in total ) ( B ) is shown . WT ( CD45 . 1 ) and DKO ( CD45 . 2 ) AMs were sorted , mixed in a 1 : 1 ratio , labeled with CellTrace Violet and cultured in the presence of GM - CSF . The ratio of CD45 . 1 and CD45 . 2 cells and CellTrace Violet dilution was analyzed prior to culture ( day 0 ) and after 7 days of culture as described in Materials and Methods . RNA - seq analysis of Maf and Mafb expression in WT and DKO AMs , which were isolated by flow cytometry from mice in steady state ( top ) or from mixed BM chimeras ( bottom ) .

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

Figure 3Mixed WT:DKO BM chimeras established as in Figure 2A were injected with EdU on three consecutive days. EdU incorporation was analyzed one day after the last injection. Steady-state WT and DKO mice were injected with EdU and analyzed as in A. Representative flow cytometry plots of EdU incorporation by AMs (C) and their quantification (results pooled from two independent experiments, 7 WT and 6 DKO mice were analyzed in total) (B) is shown.WT (CD45.1) and DKO (CD45.2) AMs were sorted, mixed in a 1:1 ratio, labeled with CellTrace Violet and cultured in the presence of GM-CSF. The ratio of CD45.1 and CD45.2 cells and CellTrace Violet dilution was analyzed prior to culture (day 0) and after 7 days of culture as described in Materials and Methods.RNA-seq analysis of Maf and Mafb expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom).


Figure 4Staining of bronchoalveolar lavage cytospins from WT and DKO mice with the lipophilic dye Oil Red O . Quantification of Oil Red O - positive cells in WT and DKO mice for individual animals . Results pooled from two independent experiments ; 4 WT and 5 DKO mice were analyzed in total . Flow cytometric analysis of AMs from WT and DKO mice stained with the lipophilic dye BODIPY 493 / 503 . Representative results of two independent experiments with three mice per group . Analysis as in C performed with WT : DKO mixed BM chimeras . Gating on CD45 . 1 and CD45 . 2 was applied to identify cells of WT and DKO donor origin . Representative results of two independent experiments with at least three chimeras . RNA - seq analysis of Acaa1b and Hsd17b4 expression in WT and DKO AMs , which were isolated by flow cytometry from mice in steady state ( top ) or from mixed BM chimeras ( bottom ) .

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

Figure 4Staining of bronchoalveolar lavage cytospins from WT and DKO mice with the lipophilic dye Oil Red O. Quantification of Oil Red O-positive cells in WT and DKO mice for individual animals. Results pooled from two independent experiments; 4 WT and 5 DKO mice were analyzed in total.Flow cytometric analysis of AMs from WT and DKO mice stained with the lipophilic dye BODIPY 493/503. Representative results of two independent experiments with three mice per group. Analysis as in C performed with WT:DKO mixed BM chimeras. Gating on CD45.1 and CD45.2 was applied to identify cells of WT and DKO donor origin. Representative results of two independent experiments with at least three chimeras.RNA-seq analysis of Acaa1b and Hsd17b4 expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom).


Figure 5Comparison of changes in gene expression induced by Bhlhe40 / Bhlhe41 - deficiency in steady - state knockout mice ( horizontal axis ) and in mixed BM chimeras ( vertical axis ) . Log2 transformed DKO / WT fold changes are plotted . Genes with expression of > 5 TPM in at least one of the samples are displayed . Genes highlighted in magenta indicate known and putative regulators of AMs . Closed symbols - Bhlhe40 - bound genes ( Bhlhe40 ChIP - seq peak assigned to the nearest gene within a 50 kb window ) . Open symbols - genes without Bhlhe40 binding . Presence of RNA transcripts ( RNA - seq ; top ) at the Gas7 and Acaa1b genes in WT and DKO AMs ; the ChIP - seq analysis of H3K27 acetylation in WT and DKO AMs ( middle ) and Bhlhe40 binding in WT AMs ( bottom ) . All analyses performed on ex vivo AMs . Gene set enrichment analysis ( GSEA ) on dataset ranked by DKO / WT fold change in steady - state AM RNA - seq using set of genes assigned to top 1 , 000 Bhlhe40 peaks ( ranked by P value ) ( left ) or a random set of 1 , 000 genes ( right ) . Similar results were obtained with all three random sets tested . NES - normalized enrichment score . FDR False Discovery Rate . Pairwise comparison of H3K27 acetylation in WT and DKO AMs performed on 2 - kb regions around the Bhlhe40 peak summits ( right - for all Bhlhe40 peaks called with P value of < 10 − 10 ; center - for top 3 , 000 Bhlhe40 peaks ( ranked by P value ) ) and around WT AM ATAC - seq peaks ( Immgen ) that did not have a Bhlhe40 peak in their close proximity ( filtered as described in Materials and Methods ) ( left ) . Volcano plots show adjusted P values ( vertical axes ) and log2 fold change ( horizontal axes ) for H3K27ac pairwise comparison in WT vs DKO AMs ; genes assigned to some of the Bhlhe40 - binding regions are labeled on the left plot .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis). Log2 transformed DKO/WT fold changes are plotted. Genes with expression of > 5 TPM in at least one of the samples are displayed. Genes highlighted in magenta indicate known and putative regulators of AMs. Closed symbols - Bhlhe40-bound genes (Bhlhe40 ChIP-seq peak assigned to the nearest gene within a 50 kb window). Open symbols - genes without Bhlhe40 binding.Presence of RNA transcripts (RNA-seq; top) at the Gas7 and Acaa1b genes in WT and DKO AMs; the ChIP-seq analysis of H3K27 acetylation in WT and DKO AMs (middle) and Bhlhe40 binding in WT AMs (bottom). All analyses performed on ex vivo AMs.Gene set enrichment analysis (GSEA) on dataset ranked by DKO/WT fold change in steady-state AM RNA-seq using set of genes assigned to top 1,000 Bhlhe40 peaks (ranked by P value) (left) or a random set of 1,000 genes (right). Similar results were obtained with all three random sets tested. NES - normalized enrichment score. FDR False Discovery Rate.Pairwise comparison of H3K27 acetylation in WT and DKO AMs performed on 2-kb regions around the Bhlhe40 peak summits (right - for all Bhlhe40 peaks called with P value of < 10−10; center - for top 3,000 Bhlhe40 peaks (ranked by P value)) and around WT AM ATAC-seq peaks (Immgen) that did not have a Bhlhe40 peak in their close proximity (filtered as described in Materials and Methods) (left). Volcano plots show adjusted P values (vertical axes) and log2 fold change (horizontal axes) for H3K27ac pairwise comparison in WT vs DKO AMs; genes assigned to some of the Bhlhe40-binding regions are labeled on the left plot.


Figure 6Comparison of changes in gene expression induced by Bhlhe40 / Bhlhe41 - deficiency in steady - state knockout mice ( horizontal axis ) and in mixed BM chimeras ( vertical axis ) as shown in Fig . 5 A , but filtered for the indicated signatures ( no TPM filter applied ) . Closed symbols - genes with associated Bhlhe40 binding ( as in Fig . 5 A ) revealed by ChIP - seq . Open symbols - genes without Bhlhe40 binding . Inserts show Bhlhe40 ChIP - seq track for selected dysregulated genes . Flow cytometric comparison of expression of the indicated cell - surface markers ( identified in panel A ) on WT and DKO AMs . A narrow forward / side scatter gate was applied to correct for possible differences in autofluorescence . Representative results of two independent experiments with three and four mice per group .

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 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

Figure 6Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis) as shown in Fig. 5 A, but filtered for the indicated signatures (no TPM filter applied). Closed symbols - genes with associated Bhlhe40 binding (as in Fig. 5 A) revealed by ChIP-seq. Open symbols - genes without Bhlhe40 binding. Inserts show Bhlhe40 ChIP-seq track for selected dysregulated genes.Flow cytometric comparison of expression of the indicated cell-surface markers (identified in panel A) on WT and DKO AMs. A narrow forward/side scatter gate was applied to correct for possible differences in autofluorescence. Representative results of two independent experiments with three and four mice per group.


Figure 2 ( A ) Relative share of the three PIKKs in NCS - responsive phosphorylations . ( C ) The profile of a group of substrates targeted by ATM 20 min after neocarzinostatin ( NCS ) addition and by ATR 1 hr after hydroxyurea ( HU ) addition . Box plots depict 23 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend to the maximum and minimum values which are not farther than 1 . 5 times the interquartile range ( IQR ) . ( D ) Western blotting analysis confirming that the ATM substrate , pS824 / KAP1 is targeted by ATR in response to HU . pS345 / CHK1 - an established ATR substrate - served as a positive control for ATR activation .

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

Figure 2(A) Relative share of the three PIKKs in NCS-responsive phosphorylations.(C) The profile of a group of substrates targeted by ATM 20 min after neocarzinostatin (NCS) addition and by ATR 1 hr after hydroxyurea (HU) addition. Box plots depict 23 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).(D) Western blotting analysis confirming that the ATM substrate, pS824/KAP1 is targeted by ATR in response to HU. pS345/CHK1 - an established ATR substrate - served as a positive control for ATR activation.


Figure 3 ( A ) A cluster of 71 neocarzinostatin ( NCS ) - induced phosphorylations that were enhanced upon continuous inhibition of DNA - PK . Untreated cells are marked UT . This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition . Clusters were obtained using K - means algorithm implemented in Perseus on Z - scored intensities . Box plots depict 71 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1 . 5 times the interquartile range ( IQR ) . ( B ) Depicted are phosphopeptides that responded to NCS treatment in A - T cells . No significant elevation was observed in these phosphorylations in A - T cells following continuous inhibition of DNA - PK . Box plots depict 71 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1 . 5 time the interquartile range ( IQR ) . ( E ) Western blotting analysis of selected phosphorylations in the above cluster showing DNA - PKi - induced elevation . ( F ) Temporal dynamics of DNA - PK - dependent attenuation of pS824 / KAP - 1 .

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

Figure 3(A) A cluster of 71 neocarzinostatin (NCS)-induced phosphorylations that were enhanced upon continuous inhibition of DNA-PK. Untreated cells are marked UT. This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition. Clusters were obtained using K-means algorithm implemented in Perseus on Z-scored intensities. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).(B) Depicted are phosphopeptides that responded to NCS treatment in A-T cells. No significant elevation was observed in these phosphorylations in A-T cells following continuous inhibition of DNA-PK. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 time the interquartile range (IQR).(E) Western blotting analysis of selected phosphorylations in the above cluster showing DNA-PKi-induced elevation.(F) Temporal dynamics of DNA-PK-dependent attenuation of pS824/KAP-1.


Figure 4 ( C ) The first time points at which ' compensated sites ' responded to NCS in WT and A - T cells are presented . ( D ) Temporal kinase dependencies of the ' compensated sites ' in A - T cells . Shown are the numbers of ' compensated sites ' in A - T cells , which depended on each kinase at the 20 and 240 min time points . The early ones depend mainly on DNA - PK , while the later ones - on ATR . ( F ) Western blotting analysis confirming high - throughput results for selected ATM substrates .

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 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

Figure 4(C) The first time points at which 'compensated sites' responded to NCS in WT and A-T cells are presented.(D) Temporal kinase dependencies of the 'compensated sites' in A-T cells. Shown are the numbers of 'compensated sites' in A-T cells, which depended on each kinase at the 20 and 240 min time points. The early ones depend mainly on DNA-PK, while the later ones - on ATR.(F) Western blotting analysis confirming high-throughput results for selected ATM substrates.


Figure 5 ( A ) A group of exclusively ATM - dependent sites that were ATM - dependent one hr after NCS addition , were still phosphorylated or dephosphorylated 4 hr after treatment , but were not affected by ATMi at that time point . Box plots depict 18 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1 . 5 times the interquartile range ( IQR ) . ( B ) This group was enriched for sites that were compensated in an ATR - dependent manner in A - T cells . Box plots depict 9 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1 . 5 times the interquartile range ( IQR ) . ( C ) Western blotting analysis of pS343 / NBS1 , which is compensated upon ATM inhibition , and pS824 / KAP - 1 and pS395 / NUMA , which are not compensated under these conditions .

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 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

Figure 5(A) A group of exclusively ATM-dependent sites that were ATM-dependent one hr after NCS addition, were still phosphorylated or dephosphorylated 4 hr after treatment, but were not affected by ATMi at that time point. Box plots depict 18 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).(B) This group was enriched for sites that were compensated in an ATR-dependent manner in A-T cells. Box plots depict 9 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).(C) Western blotting analysis of pS343/NBS1, which is compensated upon ATM inhibition, and pS824/ KAP-1 and pS395/NUMA, which are not compensated under these conditions.


Figure 6 ( A - F ) Cells were either untreated ( UT ) or treated with neocarzinostatin ( NCS ) in the presence or absence of PIKK inhibitors . Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM .

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Figure 6(A-F) Cells were either untreated (UT) or treated with neocarzinostatin (NCS) in the presence or absence of PIKK inhibitors. Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM.


Figure 1 ( C ) Time - lapse microscopy of cells containing a PsigV - YFP promoter reporter reveals heterogeneous activation of σV in response to lysozyme stress . The stress ( red line ) was added between the 200 mins and 300 min time points ( at 240 min ) . The red arrow highlights a cell with a delayed activation of σV . Scale bar : 5 μm .

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Figure 1(C) Time-lapse microscopy of cells containing a PsigV-YFP promoter reporter reveals heterogeneous activation of σV in response to lysozyme stress. The stress (red line) was added between the 200 mins and 300 min time points (at 240 min). The red arrow highlights a cell with a delayed activation of σV. Scale bar: 5 μm.


Figure 2 ( B ) A priming ( 30 min ) stress of 1 µg / ml lysozyme followed by the high lysozyme stress ( 20 µg / ml ) improves survival . The solid blue lines are the biological repeats for the no priming experiment ( n = 2 ) whereas the solid red lines are the biological repeats for the priming experiment ( n = 4 ) . ( Total number of cells shown for No Priming , N = 2013 and Priming , N = 4937 ) .

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Figure 2(B) A priming (30 min) stress of 1 µg/ml lysozyme followed by the high lysozyme stress (20 µg/ml) improves survival. The solid blue lines are the biological repeats for the no priming experiment (n=2) whereas the solid red lines are the biological repeats for the priming experiment (n=4). (Total number of cells shown for No Priming, N = 2013 and Priming, N = 4937).


Figure 3 ( B , C ) RNA - seq experiment on WT ( JLB130 ) and ΔsigV ( JLB154 ) strains , showing quantification of the absolute expression of individual genes in the presence and absence of lysozyme stress . The shaded grey box represents a ± 5 fold change . DEseq was used to identify genes which were differentially expressed with a 5 % p - value cutoff between the WT and ΔsigV mutant in response to lysozyme treatment ( default DEseq test was used ) . ( B ) Only the sigV operon is strongly activated ( > 5 fold change ) in response to lysozyme stress in WT ( JLB130 ) , as previously reported ( Guariglia - Oropeza and Helmann 2011 ) . ( C ) No genes were strongly upregulated in ΔsigV ( JLB154 ) by lysozyme stress . ( D ) Effect of the overexpression of individual components of the σV pathway ( Biological repeats : WT : n = 8 , sigV + : n = 4 , rsiV + : n = 3 oatA + : n = 4 , yrhK + : n = 3 , sipS + : n = 3 and rasP + : n = 3 , pbpX + : n = 6 ) on the fraction of activated cells . Only overexpression of sigV , rsiV or oatA changed the observed dynamics compared to WT . The histograms of the shown data are shown in Appendix Figure S16 . ( E ) Deleting oatA did not alter the σV activation dynamics . However , deleting sigV or rsiV resulted in no further activation of σV in response to 1 ug / ml lysozyme . n > = 3 biological repeats for all data shown . The histograms of the shown data are shown in Appendix Figure S17 . (

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 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

Figure 3(B, C) RNA-seq experiment on WT (JLB130) and ΔsigV (JLB154) strains, showing quantification of the absolute expression of individual genes in the presence and absence of lysozyme stress. The shaded grey box represents a ± 5 fold change. DEseq was used to identify genes which were differentially expressed with a 5% p-value cutoff between the WT and ΔsigV mutant in response to lysozyme treatment (default DEseq test was used). (B) Only the sigV operon is strongly activated (>5 fold change) in response to lysozyme stress in WT (JLB130), as previously reported (Guariglia-Oropeza and Helmann 2011). (C) No genes were strongly upregulated in ΔsigV (JLB154) by lysozyme stress.(D) Effect of the overexpression of individual components of the σV pathway (Biological repeats: WT: n=8, sigV+: n=4, rsiV+: n=3 oatA+: n=4, yrhK+: n=3, sipS+: n=3 and rasP+: n=3, pbpX+: n=6) on the fraction of activated cells. Only overexpression of sigV, rsiV or oatA changed the observed dynamics compared to WT. The histograms of the shown data are shown in Appendix Figure S16. (E) Deleting oatA did not alter the σV activation dynamics. However, deleting sigV or rsiV resulted in no further activation of σV in response to 1 ug/ml lysozyme. n>=3 biological repeats for all data shown. The histograms of the shown data are shown in Appendix Figure S17. (


Figure 5 ( C ) As predicted , the 2xsigV or 2xsigV - rsiV stains have a homogenous σV response to 1 µg / ml lysozyme , while the 2xrsiV strain has increased heterogeneity . Each bar plot is the average of three biological repeats . The bars correspond to the mean ± s . d . .

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Figure 5(C) As predicted, the 2xsigV or 2xsigV-rsiV stains have a homogenous σV response to 1 µg/ml lysozyme, while the 2xrsiV strain has increased heterogeneity. Each bar plot is the average of three biological repeats. The bars correspond to the mean ± s.d..


Figure 6 ( E , F ) For very long intervals between stress ( 12h ) , the heterogeneity is the same as if the cells had not experienced a prior lysozyme stress ( control ) . N > 50 single - cell traces .

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Figure 6(E, F) For very long intervals between stress (12h), the heterogeneity is the same as if the cells had not experienced a prior lysozyme stress (control). N>50 single-cell traces.


Figure 1 ( A ) Yeast clones harbouring empty bait vector ( pYTH9 , mock ) or those encoding ScAtg8 , LC3B , Atg12 and SUMO1 were transformed with empty prey vector ( pACT2 , mock ) or those encoding fragments of ATG4A , ATG4B , NBR1 , p62 and full - length Nix . Interaction was assessed by yeast growth on the SD ‐ W / ‐ L / ‐ H medium with a β ‐ gal assay . ( B ) GST pulldown assays using cell extracts of Flag ‐ Nix ‐ expressing COS7 cells and immobilized GST or the indicated GST fusions . Coprecipitated Nix was detected with Flag antibodies . ( C ) Purified GST ‐ tagged NixΔTM was cleaved from the GST moiety by thrombin , purified and used for precipitation by GST fusion proteins . Precipitated proteins were analysed by western blotting ( WB ) with Nix antibodies . Ponceau S staining was used to visualize GST ‐ fusion proteins . ( D ) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated ( IP ) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies . β ‐ gal , β ‐ galactosidase ; BafA1 , bafilomycin ‐ A1 ; CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; GST , glutathione ‐ S ‐ transferase ; IgG , immunoglobulin G ; NixΔTM , recombinant Nix lacking a TM ; SUMO1 , small ubiquitin ‐ like modifier 1 ; TCL , total cell lysate ; TM , transmembrane domain .

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

Figure 1(A) Yeast clones harbouring empty bait vector (pYTH9, mock) or those encoding ScAtg8, LC3B, Atg12 and SUMO1 were transformed with empty prey vector (pACT2, mock) or those encoding fragments of ATG4A, ATG4B, NBR1, p62 and full-length Nix. Interaction was assessed by yeast growth on the SD‐W/‐L/‐H medium with a β‐gal assay.(B) GST pulldown assays using cell extracts of Flag‐Nix‐expressing COS7 cells and immobilized GST or the indicated GST fusions. Coprecipitated Nix was detected with Flag antibodies.(C) Purified GST‐tagged NixΔTM was cleaved from the GST moiety by thrombin, purified and used for precipitation by GST fusion proteins. Precipitated proteins were analysed by western blotting (WB) with Nix antibodies. Ponceau S staining was used to visualize GST‐fusion proteins.(D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β‐gal, β‐galactosidase; BafA1, bafilomycin‐A1; CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; GST, glutathione‐S‐transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin‐like modifier 1; TCL, total cell lysate; TM, transmembrane domain.


Figure 2 ( C ) Lysates of COS7 cells transfected with Flag ‐ wt ‐ Nix or the indicated mutants were incubated with immobilized GST ‐ LC3A . The coprecipitated proteins were detected by western blotting ( WB ) with Flag antibodies . BNIP3 , Bcl2 / E1B 19kDa ‐ interacting protein 3 ‐ like protein ; Dr , Danio reio ; GST , glutathione ‐ S ‐ transferase ; Hs , Homo sapiens ; LIR , LC3 ‐ interacting region ; Mm , Mus musculus ; wt , wild type ; Xl , Xenopus laevis .

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Figure 2(C) Lysates of COS7 cells transfected with Flag‐wt‐Nix or the indicated mutants were incubated with immobilized GST‐LC3A. The coprecipitated proteins were detected by western blotting (WB) with Flag antibodies. BNIP3, Bcl2/E1B 19kDa‐interacting protein 3‐like protein; Dr, Danio reio; GST, glutathione‐S‐transferase; Hs, Homo sapiens; LIR, LC3‐interacting region; Mm, Mus musculus; wt, wild type; Xl, Xenopus laevis.


Figure 3Nix recruits EGFP ‐ GABARAP ‐ L1 to stressed mitochondria in reconstituted Nix − / − MEFs . Nix − / − MEFs cotransfected with EGFP ‐ GABARAP ‐ L1 and wt ‐ Nix ( A ) , or Nix ‐ W35A ( B ) , were incubated with and without mitochondrial poison CCCP for 3 h . Cells were then fixed and analysed for colocalization between EGFP ‐ GABARAP ‐ L1 and Nix ( stained with Flag antibodies ) . The final panel in each row shows colocalization between Flag ‐ Nix ‐ Cy3 and GABARAP ‐ L1 using the ' colocalization ' highlighter plug ‐ in for ImageJ software ( NIH , Bethesda , MA , USA ) . ( C ) Quantification was achieved by counting the number of complete colocalizations , EGFP / Cy3 per cell for approximately 30 cells per condition . Results are expressed as a percentage of colocalization ( EGFP : Cy3 ) in control and CCCP ‐ treated cells . Error bars in ( C ) show the standard deviation ( s . d . ) of three independent experiments . Statistical analysis was performed using the Mann - Whitney t ‐ test . CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; DMSO , dimethyl sulfoxide ; EGFP , enhanced green fluorescent protein ; MEF , mouse embryonic fibroblast ; wt , wild type .

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

Figure 3Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs. Nix−/− MEFs cotransfected with EGFP‐GABARAP‐L1 and wt‐Nix (A), or Nix‐W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP‐GABARAP‐L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag‐Nix‐Cy3 and GABARAP‐L1 using the 'colocalization' highlighter plug‐in for ImageJ software (NIH, Bethesda, MA, USA).(C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP‐treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann-Whitney t‐test. CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.


Figure 4 ( A ) Flow cytometry of reticulocyte ‐ enriched blood , cultured in vitro for 3 days , stained with MTR . The blood was taken from mice transplanted with Nix − / − bone marrow and reconstituted with wt ‐ Nix and Nix ‐ W35A viruses . Reticulocytes were induced by phenylhydrazine treatment . The viruses also express GFP . ( B ) Mitochondrial clearance in the GFP ‐ positive fraction of the reticulocyte ‐ enriched blood , as described in panel ( A ) . Mice were reconstituted with wt ‐ Nix , LIR ‐ W35A or empty vector viruses . Data from Nix − / − and Nix + / + mice are shown for comparison . ( C ) Anti ‐ Flag , anti ‐ GFP and anti ‐ β ‐ actin western blots of fetal liver cells transduced with Flag ‐ Nix and Flag ‐ W35A ‐ Nix viruses , normalized to total protein . GFP , green fluorescent protein ; LIR , LC3 ‐ interacting region ; MTR , MitoTracker Red ; wt , wild type .

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Figure 4(A) Flow cytometry of reticulocyte‐enriched blood, cultured in vitro for 3 days, stained with MTR. The blood was taken from mice transplanted with Nix−/− bone marrow and reconstituted with wt‐Nix and Nix‐W35A viruses. Reticulocytes were induced by phenylhydrazine treatment. The viruses also express GFP.(B) Mitochondrial clearance in the GFP‐positive fraction of the reticulocyte‐enriched blood, as described in panel (A). Mice were reconstituted with wt‐Nix, LIR‐W35A or empty vector viruses. Data from Nix−/− and Nix+/+ mice are shown for comparison.(C) Anti‐Flag, anti‐GFP and anti‐β‐actin western blots of fetal liver cells transduced with Flag‐Nix and Flag‐W35A‐Nix viruses, normalized to total protein. GFP, green fluorescent protein; LIR, LC3‐interacting region; MTR, MitoTracker Red; wt, wild type.


Figure 1A , B MARS ( A ) and MARS2 ( B ) mRNA expression levels were measured by real - time PCR in blood samples with 2 and 3 - 4 copies . Relative expression levels were normalized to GAPDH levels , and data were quantified relative to the mRNA expression levels of 2 copy samples . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Unpaired Student ' s t testC , D Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3 - 4 copies . Protein - level intensity shown in ( D ) was quantified using ImageJ and divided by the protein - level intensity of actin . The results were then graphed relative to samples with 2 copies . E , F MARS2 protein expression levels were detected ( E ) and quantified ( F ) . Actin was used as a loading control . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Unpaired Student ' s t test was usedG Cell proliferation was assessed by Ki67 ( red ) immunofluorescent staining in mice neural tube sections at E10 . MARS ( green ) was co - stained . Scale bar : 150 μm . H Double immunofluorescent staining for MARS ( green ) and Ki67 ( red ) in developing heart sections at E10 . Scale bar : 150 μm . I MARS expression was detected in E80 rhesus monkey brain sections ( n = 3 ) by IHC . Local large magnification is shown as framed . Red arrow indicates germinar layer , blue indicates immature neurons . Scale bar : 1mm . MARS protein levels in embryonic chorion ( C ) cells and their corresponding maternal deciduae ( D ) cells were determined by western blot . Each D / C lane pair was quantified relative to D as shown at the bottom . MARS protein levels in embryonic chorion ( C ) cells and their corresponding maternal deciduae ( D ) cells were determined by western blot . The mean MARS levels in all D and C groups were quantified by ImageJ and shown in ( K ) ( n = 6 ) . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs test was usedL MARS2 mRNA expression in embryonic chorion ( C ) and maternal deciduae ( D ) were detected by qPCR ( n = 6 ) . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs test was usedM MARS protein quantification in the brains of foetal rats ( F ) and their corresponding mothers ( M ) from experiment ( O ) ( n = 6 ) . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs test was usedN MARS mRNA levels ( n = 6 ) in the brains of foetal rats ( F ) and their corresponding mothers ( M ) were determined by qPCR ( n = 6 ) . Data are presented as the mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs test was usedO MARS protein levels in foetal rats ( F ) and their corresponding mothers ( M ) were determined by western blot . Each M / F lane pair was quantified relative to M as shown at the bottom . P Representative result of MARS expression was detected by IHC in tumor tissues ( T ) and adjacent noncancer tissues ( N ) of UCC samples . Scale bar : 50 μm . Q Ki67 and MARS expression was analysed by IHC in HCC samples . Scale bar : 20 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 1A, B MARS (A) and MARS2 (B) mRNA expression levels were measured by real-time PCR in blood samples with 2 and 3-4 copies. Relative expression levels were normalized to GAPDH levels, and data were quantified relative to the mRNA expression levels of 2 copy samples. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t testC, D Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3-4 copies. Protein-level intensity shown in (D) was quantified using ImageJ and divided by the protein-level intensity of actin. The results were then graphed relative to samples with 2 copies. E, F MARS2 protein expression levels were detected (E) and quantified (F). Actin was used as a loading control. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test was usedG Cell proliferation was assessed by Ki67 (red) immunofluorescent staining in mice neural tube sections at E10. MARS (green) was co-stained. Scale bar: 150 μm.H Double immunofluorescent staining for MARS (green) and Ki67 (red) in developing heart sections at E10. Scale bar: 150 μm.I MARS expression was detected in E80 rhesus monkey brain sections (n=3) by IHC. Local large magnification is shown as framed. Red arrow indicates germinar layer, blue indicates immature neurons. Scale bar: 1mm.MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. Each D/C lane pair was quantified relative to D as shown at the bottom.MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. The mean MARS levels in all D and C groups were quantified by ImageJ and shown in (K) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was usedL MARS2 mRNA expression in embryonic chorion (C) and maternal deciduae (D) were detected by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was usedM MARS protein quantification in the brains of foetal rats (F) and their corresponding mothers (M) from experiment (O) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was usedN MARS mRNA levels (n=6) in the brains of foetal rats (F) and their corresponding mothers (M) were determined by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was usedO MARS protein levels in foetal rats (F) and their corresponding mothers (M) were determined by western blot. Each M/F lane pair was quantified relative to M as shown at the bottom.P Representative result of MARS expression was detected by IHC in tumor tissues (T) and adjacent noncancer tissues (N) of UCC samples. Scale bar: 50 μm.Q Ki67 and MARS expression was analysed by IHC in HCC samples. Scale bar: 20 μm.


Figure 2B Cell growth was determined in the absence and presence of 0 . 5 mM homocysteine or 0 . 5 mM methionine in the culture media of NE4C cells transfected with pcDNA3 . 1 vector or pcDNA3 . 1 - Flag - Bcl - 2 plasmids ( n = 3 ) . 5μg plasmids were transfected in each 1 × 106 cells . The cell index responds to changes in cell number and cell adhesion . Cleaved caspase 3 ( c - caspase3 ) levels were detected to determine apoptosis levels ( right ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . C , D Apoptotic cells were detected by flow cytometry . The apoptotic rates of untreated , MARS ( C ) - , and MARS2 ( D ) - over - expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . E Cells were treated with different methionine concentrations as indicated . Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS / MARS2 knockdown NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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

Figure 2B Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1-Flag-Bcl-2 plasmids (n=3). 5μg plasmids were transfected in each 1×106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved caspase 3 (c-caspase3) levels were detected to determine apoptosis levels (right). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test.C, D Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)-, and MARS2 (D)-over-expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.E Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


Figure 3HTL and ROS ( superoxide ) levels were compared ( n = 4 ) between NE4C cells transfected with the empty vector , MARS ( A ) plasmids . Data were normalized against that of vector - transfected cells . HTL levels were determined by LC - MS , and ROS levels were determined by DHE staining assay . HTL and ROS ( superoxide ) levels were compared ( n = 4 ) between NE4C cells transfected with the empty vector or MARS2 ( B ) plasmids . Data were normalized against that of vector - transfected cells . HTL levels were determined by LC - MS , and ROS levels were determined by DHE staining assay . C MARS was knocked down by small hairpin RNAs in NE4C cells . MARS knockdown efficiencies were confirmed by western blot . HTL and superoxide levels were quantified ( n = 4 ) relative to vector - transfected NE4C cells . Cells were cultured in 20 μM Hcy - containing media . D Relative superoxide levels were determined by DHE staining assay and compared ( n = 4 ) between NE4C cells with and without MARS knockdown by shRNA . Cells were cultured in 20 μM Hcy - containing media . MARS2 knockdown efficiencies were confirmed by western blot . E NE4C cells with and without MARS knockdown were treated with either Hcy ( 20 μM ) or Met ( 75 μM ) , and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells ( n = 4 ) . F The cellular ROS ( superoxide ) levels were determined ( n = 4 ) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h . The ROS levels were normalized to those of untreated NE4C cells . G Superoxide levels in response to Hcy treatment ( 20 μM , 4 h ) in MARS - expressing and control cells were determined relative to the untreated NE4C cells . H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated . N - Hcy levels in HTL - treated and control cells were detected by western blot . I Protein N - Hcy levels in response to MARS knockdown ( by shRNA ) and over - expression were detected by western blot . J , K Untreated and MARS / MARS2 knockdown NE4C cells were treated with Hcy ( 20 μM ) or HTL ( 10 μM ) , and the N - Hcy ( J ) levels ( n = 4 ) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells . Error bars indicate SEM . Data were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . J , K Untreated and MARS / MARS2 knockdown NE4C cells were treated with Hcy ( 20 μM ) or HTL ( 10 μM ) , and superoxide ( K ) levels ( n = 4 ) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells . Error bars indicate SEM . Data were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

Figure 3HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector, MARS (A) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector or MARS2 (B) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.C MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by western blot. HTL and superoxide levels were quantified (n=4) relative to vector-transfected NE4C cells. Cells were cultured in 20 μM Hcy-containing media.D Relative superoxide levels were determined by DHE staining assay and compared (n=4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy-containing media. MARS2 knockdown efficiencies were confirmed by western blot.E NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n=4).F The cellular ROS (superoxide) levels were determined (n=4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells.G Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS-expressing and control cells were determined relative to the untreated NE4C cells.H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N-Hcy levels in HTL-treated and control cells were detected by western blot.I Protein N-Hcy levels in response to MARS knockdown (by shRNA) and over-expression were detected by western blot.J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and the N-Hcy (J) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and superoxide (K) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Figure 4C SOD activities were down - regulated by in vitro N - homocysteinylation . Flag - tagged SOD1 , SOD1K23W , K122W , K128W ( 3KW ) mutant , SOD2 , and SOD2K44W , K51W , K98W , K106W , K178W ( 5KW ) mutant were expressed in HEK293T cells , affinity purified , and incubated with or without 10 μM HTL in solution ( in vitro ) . After 12 h , the reaction mixture was desalted and concentrated with filter tubes . N - Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2 , respectively ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . D Upon switching N - Hcy substrate lysine ( K ) sites to nonmodifiable tryptophan ( W ) , the N - Hcy unmodifiable mutants displayed lower specific activity than the wide - type ( WT ) SOD . SOD1 ( WT , K23W , K122W , K128W , and 3KW ) and SOD2 ( WT , K44W , K51W , K98W , K106W , K178W , and 5KW ) were expressed in HEK293T cells . The catalytic activity of affinity - purified SOD proteins was determined and normalized to protein levels . Wild - type SOD1 and SOD2 activities were set at 100 % ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . E , F Hcy and HTL treatment in NE4C cells increased N - homocysteinylation of SODs and decreased their activities . Flag - tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells . The cells were transferred to serum - free DMEM with or without 20 μM Hcy ( E ) or 10 μM HTL ( F ) 6 h before harvesting . After affinity purification , the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . G N - Hcy SOD levels were down - regulated in MARS / MARS2 knockdown cells . Flag - tagged SOD1 and SOD2 were each over - expressed in untreated and MARS / MARS2 knockdown NE4C cells . After affinity purification , the N - Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

Figure 4C SOD activities were down-regulated by in vitro N-homocysteinylation. Flag-tagged SOD1, SOD1K23W,K122W,K128W (3KW) mutant, SOD2, and SOD2K44W, K51W,K98W,K106W,K178W (5KW) mutant were expressed in HEK293T cells, affinity purified, and incubated with or without 10 μM HTL in solution (in vitro). After 12 h, the reaction mixture was desalted and concentrated with filter tubes. N-Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2, respectively (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.D Upon switching N-Hcy substrate lysine (K) sites to nonmodifiable tryptophan (W), the N-Hcy unmodifiable mutants displayed lower specific activity than the wide-type (WT) SOD. SOD1 (WT, K23W, K122W, K128W, and 3KW) and SOD2 (WT, K44W, K51W, K98W, K106W, K178W, and 5KW) were expressed in HEK293T cells. The catalytic activity of affinity-purified SOD proteins was determined and normalized to protein levels. Wild-type SOD1 and SOD2 activities were set at 100% (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.E, F Hcy and HTL treatment in NE4C cells increased N-homocysteinylation of SODs and decreased their activities. Flag-tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells. The cells were transferred to serum-free DMEM with or without 20 μM Hcy (E) or 10 μM HTL (F) 6 h before harvesting. After affinity purification, the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.G N-Hcy SOD levels were down-regulated in MARS/MARS2 knockdown cells. Flag-tagged SOD1 and SOD2 were each over-expressed in untreated and MARS/MARS2 knockdown NE4C cells. After affinity purification, the N-Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Figure 5A Levels of β - catenin in response to Hcy treatment in NE4C cells were detected by western blot ( n = 3 ) . Representative western blots are shown , and band intensities were quantified relative to the untreated group . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . B Levels of β - catenin in response to HTL treatment in NE4C cells were detected . Band intensities were normalized to that of untreated cells . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . C TCF activities were measured with a Topflash / Fopflash luciferase reporter system ( n = 4 ) . TCF activities responding to either 20 μM Hcy - or 10 μM HTL - treated were measured by quantifying Topflash / Fopflash relative to the untreated groups . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . D Levels of β - catenin in untreated and MARS knockdown NE4C cells were detected ( n = 3 ) with the presence or absence of 20 μM Hcy in the culture media . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . NE4C cells were co - transfected with NRX - Flag and DVL1 - Myc plasmids and treated with different concentrations of Hcy ( E ) The relative amount of DVL1 that coimmunoprecipitated ( co - IPed ) with NRX - Flag was determined ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . NE4C cells were co - transfected with NRX - Flag and DVL1 - Myc plasmids and treated with different concentrations of HTL ( F ) . The relative amount of DVL1 that coimmunoprecipitated ( co - IPed ) with NRX - Flag was determined ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . G NRX - Flag and DVL1 - Myc were co - transfected in untreated or MARS knockdown NE4C cells . The relative DVL1 levels that co - IPed with NRX - Flag were determined and quantified ( n = 3 ) for treated and control cells cultured with or without 20 μM Hcy . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . Flag - tagged SOD1 and SOD2 were co - over - expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL . ( H ) The levels of β - catenin in cells with and without SOD1 / 2 expression were determined ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . Flag - tagged SOD1 and SOD2 were co - over - expressed in NE4C cells cultured in DMEM and supplemented with HTL . ( I ) Relative levels of DVL1 that co - IPed with NRX - Flag were quantified in SOD - expressing and control cells cultured with or without 10 μM HTL ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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 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 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 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 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 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

Figure 5A Levels of β-catenin in response to Hcy treatment in NE4C cells were detected by western blot (n=3). Representative western blots are shown, and band intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.B Levels of β-catenin in response to HTL treatment in NE4C cells were detected. Band intensities were normalized to that of untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.C TCF activities were measured with a Topflash/Fopflash luciferase reporter system (n=4). TCF activities responding to either 20 μM Hcy- or 10 μM HTL-treated were measured by quantifying Topflash/Fopflash relative to the untreated groups. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.D Levels of β-catenin in untreated and MARS knockdown NE4C cells were detected (n=3) with the presence or absence of 20 μM Hcy in the culture media. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of Hcy (E) The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of HTL (F). The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.G NRX-Flag and DVL1-Myc were co-transfected in untreated or MARS knockdown NE4C cells. The relative DVL1 levels that co-IPed with NRX-Flag were determined and quantified (n=3) for treated and control cells cultured with or without 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL. (H) The levels of β-catenin in cells with and without SOD1/2 expression were determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with HTL. (I) Relative levels of DVL1 that co-IPed with NRX-Flag were quantified in SOD-expressing and control cells cultured with or without 10 μM HTL (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Figure 6B MARS - or MARS2 - catalysed HTL production from Hcy ( 20 μM ) was carried out in vitro in the presence of different AHT concentrations . The amount of HTL produced in each reaction was determined by thin layer chromatography . C MARS and MARS2 activities were determined ( n = 4 ) in the presence of the indicated AHT concentrations in the reactions . AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . D Relative HTL levels were determined ( n = 4 ) for NE4C cells treated with various AHT concentrations for 4 h . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . E AHT ( 71 or 110 μg / kg per day ) was injected into C57BL / 6 mice for 6 days . Plasma HTL levels were detected by LC - MS . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . F Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels ; N - homocysteinylation levels were detected by western blot . NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels ; Band intensities were quantified relative to the untreated group using Image J ( H ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . I Cellular superoxide levels were determined ( n = 4 ) for NE4C cells treated with various AHT concentrations for 4 h . Superoxide levels were normalized to those of untreated NE4C cells . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . J Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT - treated NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . K Levels of β - catenin in untreated , Hcy - , or HTL - treated NE4C cells were detected ( n = 3 ) with the presence or absence of 70 μM AHT in the culture media . Mean β - catenin levels in untreated NE4C cells were set at 100 % . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

Figure 6B MARS- or MARS2-catalysed HTL production from Hcy (20 μM) was carried out in vitro in the presence of different AHT concentrations. The amount of HTL produced in each reaction was determined by thin layer chromatography.C MARS and MARS2 activities were determined (n=4) in the presence of the indicated AHT concentrations in the reactions. AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.D Relative HTL levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.E AHT (71 or 110 μg/kg per day) was injected into C57BL/6 mice for 6 days. Plasma HTL levels were detected by LC-MS. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.F Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; N-homocysteinylation levels were detected by western blot.NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; Band intensities were quantified relative to the untreated group using Image J (H). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.I Cellular superoxide levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Superoxide levels were normalized to those of untreated NE4C cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.J Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT-treated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.K Levels of β-catenin in untreated, Hcy-, or HTL-treated NE4C cells were detected (n=3) with the presence or absence of 70 μM AHT in the culture media. Mean β-catenin levels in untreated NE4C cells were set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.