Figure 2Clustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of IBM ( A ) Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 ; CDCA , chenodeoxycholic acid ; OH - Kyn , 3 - hydroxy - DL - kynurenine SDMA , symmetric dimethylarginine . Clustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of NMD patients ( B ) Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . C3 , component 3 ; CDCA , chenodeoxycholic acidClustering of metabolome data in patients and controls ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites in blood of MIRAS carriers ( C ) . Data information Colours in VIP score and volcano plots indicate the same most relevant and / or significantly changed metabolites among all patients groups . cAMP , cyclic AMP ; C3 , component 3 ; GABA , γ - aminobutyric acid HIAA , 5 - Hydroxyindole - 3 - acetic acid ; IMP , inosine monophosphate ; OH - Kyn , 3 - hydroxy - DL - kynurenine ; OH - Trp , hydroxytryptophan ; SDMA , symmetric dimethylarginine .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IBM (A) Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid; OH-Kyn, 3-hydroxy-DL-kynurenine SDMA, symmetric dimethylarginine.Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of NMD patients (B) Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acidClustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MIRAS carriers (C). Data information Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. cAMP, cyclic AMP; C3, component 3; GABA, γ-aminobutyric acid HIAA, 5-Hydroxyindole-3-acetic acid; IMP, inosine monophosphate; OH-Kyn, 3-hydroxy-DL-kynurenine; OH-Trp, hydroxytryptophan; SDMA, symmetric dimethylarginine.


Figure 3A Relative values of single metabolites and creatine / creatinine ratios in blood of primary MD , IBM and NMD patients , and MIRAS carriers compared to controls . Data information : All data represent mean ± SD . For individual metabolites : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two sample T - test ) . For creatine / creatinine ( cr / crn ) ratio : * P = 0 . 022 , * * P = 0 . 005 , * * * P = 0 . 0001 ( Kruskal - Wallis test with Dunn ´ s multiple comparisons test or Mann - Whitney test ) . B Relative values of single metabolites in blood of adult IOSCA ( marked " IOSCA " ) patients and one IOSCA child patient compared to controls . Data information : All data represent mean ± 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 B-PANEL_START O O O O O O O O O O O O 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 Relative values of single metabolites and creatine/creatinine ratios in blood of primary MD, IBM and NMD patients, and MIRAS carriers compared to controls. Data information: All data represent mean ± SD. For individual metabolites: *P < 0.05, **P < 0.01, ***P < 0.001 (two sample T-test). For creatine/creatinine (cr/crn) ratio: *P = 0.022, **P = 0.005, ***P = 0.0001 (Kruskal-Wallis test with Dunn´s multiple comparisons test or Mann-Whitney test).B Relative values of single metabolites in blood of adult IOSCA (marked "IOSCA") patients and one IOSCA child patient compared to controls. Data information: All data represent mean ± SD.


Figure 4Metabolomes of muscle of MIRAS ( A ) patients ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites . Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . AMP , adenosine monophosphate NAD , nicotinamide adenine dinucleotide TCA , taurocholic acidMetabolomes of muscle of PEO ( B ) patients ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites . Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . car . , carnitine NAD , nicotinamide adenine dinucleotideD Relative values of metabolites in muscle of MIRAS , PEO and MELAS / MIDD patients compared to controls . All data represent mean ± SD . * P = 0 . 031 , * * P = 0 . 008 ( two sample T - test ) . UDP , uridine diphosphate .

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

Figure 4Metabolomes of muscle of MIRAS (A) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. AMP, adenosine monophosphate NAD, nicotinamide adenine dinucleotide TCA, taurocholic acidMetabolomes of muscle of PEO (B) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. car., carnitine NAD, nicotinamide adenine dinucleotideD Relative values of metabolites in muscle of MIRAS, PEO and MELAS/MIDD patients compared to controls. All data represent mean ± SD. *P = 0.031, **P = 0.008 (two sample T-test). UDP, uridine diphosphate.


Figure 6A Separate specificity and sensitivity ( ROC ) of sorbitol , alanine , myoinositol and cystathionine ( left ) and conventional blood biomarkers lactate and pyruvate , and cytokine FGF21 ( right ) in blood of MIRAS , PEO and MELAS / MIDD patients ( n = 20 ) compared to controls ( n = 30 ) . Data information : ROC analysis : AUC of sorbitol 0 . 81 ( 95 % CI 0 . 68 - 0 . 94 , P = 0 . 0003 ) , alanine 0 . 81 ( 95 % CI 0 . 66 - 0 . 94 , P = 0 . 0003 ) , myoinositol 0 . 79 ( 95 % CI 0 . 66 - 0 . 91 , P = 0 . 0007 ) and cystathionine 0 . 78 ( 95 % CI 0 . 65 - 0 . 91 , P = 0 . 001 ) . AUC of conventional biomarkers : FGF21 0 . 87 ( 95 % CI 0 . 74 - 0 . 99 , P = 0 . 0001 ) , lactate 0 . 86 ( 95 % CI 0 . 76 - 0 . 97 , P = 0 . 0001 ) and pyruvate 0 . 78 ( 95 % CI 0 . 64 - 0 . 93 , P = 0 . 0017 ) . B Combined ' multi - biomarker ' value of sorbitol / alanine / myoinositol / cystathionine ; ROC - analysis ( left ) ; mean centroids MD , IBM and NMD patients , and MIRAS carriers compared to controls ( right ) . Data information : ROC analysis AUC of ' multi - biomarker ' 0 . 94 ( 95 % CI 0 . 89 - 0 . 995 , P = 0 . 0001 ) . Mean centroid data represent mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( 1 - way ANOVA with Dunnett ´ s multiple comparison test ) .

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

Figure 6A Separate specificity and sensitivity (ROC) of sorbitol, alanine, myoinositol and cystathionine (left) and conventional blood biomarkers lactate and pyruvate, and cytokine FGF21 (right) in blood of MIRAS, PEO and MELAS/MIDD patients (n = 20) compared to controls (n = 30). Data information: ROC analysis: AUC of sorbitol 0.81 (95% CI 0.68 - 0.94, P = 0.0003), alanine 0.81 (95% CI 0.66 - 0.94, P = 0.0003), myoinositol 0.79 (95% CI 0.66 - 0.91, P = 0.0007) and cystathionine 0.78 (95% CI 0.65 - 0.91, P = 0.001). AUC of conventional biomarkers: FGF21 0.87 (95% CI 0.74 - 0.99, P = 0.0001), lactate 0.86 (95% CI 0.76 - 0.97, P = 0.0001) and pyruvate 0.78 (95% CI 0.64 - 0.93, P = 0.0017).B Combined 'multi-biomarker' value of sorbitol/alanine/myoinositol/cystathionine; ROC-analysis (left); mean centroids MD, IBM and NMD patients, and MIRAS carriers compared to controls (right). Data information: ROC analysis AUC of 'multi-biomarker' 0.94 (95% CI 0.89 - 0.995, P = 0.0001). Mean centroid data represent mean ± SD. **P < 0.01, ***P < 0.001 (1-way ANOVA with Dunnett´s multiple comparison test).


Figure 1B OsPRR73 confers salinity stress tolerance in rice . The seedlings of Dongjin ( DJ , the wild type control ) , osprr73 , and the complementation line of osprr73 ( abbreviated as Com - L1 ) grown under 12 h light / 12 h dark conditions for 28 days ( left panel ) , transferred to 180 mM NaCl for 21 days ( middle panel ) and recovered for 12 and 24 days ( the two right panels ) respectively . Scale bar , 5 cm . C Statistical analysis of the survival rate of DJ , osprr73 and Com - L1 plants in ( B ) after recovery 12 days . Data are presented as mean ± SD . ( n from 4 biological replicates , and 24 plants were tested in each of biological replicates ) . ( * * * ) P ≤ 0 . 001 were generated by student ' s t - test . E Null mutant of osprr73 in Nipponbare ( NIP ) background by genome editing displayed hypersensitivity to NaCl treatment . Scale bar , 5 cm . F Survival rate of NIP ( wild type of CRISPR lines ) and osprr73 - C ( C stands for CRISPR ) plants in ( E ) after recovery 9 days . Data are presented as mean ± SD . n = 4 biological replicates , 24 plants for each biological replicate . ( * ) P ≤ 0 . 05 and ( * * * ) P ≤ 0 . 001 were generated by student ' s t - test . Quantiﬁcation of chlorophyll content of WT and osprr73 mutants Data are presented as mean ± SD . n = 3 , biological replicates , and asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 , ( * * * ) P ≤ 0 . 001 . Quantiﬁcation of electrolyte leakage of WT and osprr73 mutants with or without 180 mM NaCl treatment for 7 days . Data are presented as mean ± SD . n = 3 , biological replicates , and asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 , ( * * * ) P ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B OsPRR73 confers salinity stress tolerance in rice. The seedlings of Dongjin (DJ, the wild type control), osprr73, and the complementation line of osprr73 (abbreviated as Com-L1) grown under 12 h light/12 h dark conditions for 28 days (left panel), transferred to 180 mM NaCl for 21 days (middle panel) and recovered for 12 and 24 days (the two right panels) respectively. Scale bar, 5 cm. C Statistical analysis of the survival rate of DJ, osprr73 and Com-L1 plants in (B) after recovery 12 days. Data are presented as mean ± SD. (n from 4 biological replicates, and 24 plants were tested in each of biological replicates). (***) P ≤ 0.001 were generated by student's t-test.E Null mutant of osprr73 in Nipponbare (NIP) background by genome editing displayed hypersensitivity to NaCl treatment. Scale bar, 5 cm. F Survival rate of NIP (wild type of CRISPR lines) and osprr73-C (C stands for CRISPR) plants in (E) after recovery 9 days. Data are presented as mean ± SD. n = 4 biological replicates, 24 plants for each biological replicate. (*) P ≤0.05 and (***) P ≤ 0.001 were generated by student's t-test.Quantiﬁcation of chlorophyll content of WT and osprr73 mutants Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.Quantiﬁcation of electrolyte leakage of WT and osprr73 mutants with or without 180 mM NaCl treatment for 7 days. Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.


Figure 3A , B The Na + and K + contents in the shoots and roots of DJ and osprr73 mutant plants with or without 180 mM NaCl treatment for 7 days . Na + ( A ) and K + ( B ) contents were measured with ICP method . Data represent means ± SD , ( n = 3 , biological replicates ) . ( * * ) P ≤ 0 . 001 and ( * ) P ≤ 0 . 001 indicate significant difference by student t - test . The abbreviation of n . s . stands for no significant . C Ratio of Na + to K + was calculated with their respective content in ( A ) and ( B ) . Data represent mean ± SD ( n = 3 , biological replicates ) . For each of biological replicates , 6 individual plants were measured . The asterisk represents significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , and n . s . indicates no significant . D Four - weeks old seedlings of DJ and osprr73 were treated with 90 mM Na2SO4 for 14 d ( middle panel ) and recovered for additional 7 days ( right panel ) . Scale bar , 5 cm . E Statistical analysis of survival rates with Na2SO4 treatment in ( D ) . Data represent means ± SD . n = 3 biological replicates , 24 plants for each biological replicate . ( * * * ) P ≤ 0 . 001 indicates significant difference by student t - test . F The seedlings of DJ and osprr73 mutant were treated with 90 mM MgCl2 for 14 days ( middle panel ) and recovered for 7 days ( right panel ) . Scale bar , 5 cm . G Statistical analysis of survival rates with MgCl2 treatment in ( F ) . The survival rate of DJ and osprr73 plants in MgCl2 stress were calculated after recovery 7 days . Data represent means ± SD . n = 2 biological replicates , 24 plants of each replicate . H The seedlings of DJ and osprr73 mutant were treated with 180 mM mannitol for 36 days ( middle panel ) and recovered for 7 days ( right panel ) . Scale bar , 5 cm . I The survival rate of DJ and osprr73 plants in mannitol stress . Data represent means ± SD . n = 3 biological replicates , 24 plants of each replicate . The abbreviation of n . s . stands for no significant generated by student t - test .

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

Figure 3A, B The Na+ and K+ contents in the shoots and roots of DJ and osprr73 mutant plants with or without 180 mM NaCl treatment for 7 days. Na+ (A) and K+ (B) contents were measured with ICP method. Data represent means ± SD, (n = 3, biological replicates). (**) P ≤ 0.001 and (*) P ≤ 0.001 indicate significant difference by student t-test. The abbreviation of n.s. stands for no significant. C Ratio of Na+ to K+ was calculated with their respective content in (A) and (B). Data represent mean ± SD (n = 3, biological replicates). For each of biological replicates, 6 individual plants were measured. The asterisk represents significant difference among means by student's t-test with (*) P ≤0.05, and n.s. indicates no significant.D Four-weeks old seedlings of DJ and osprr73 were treated with 90 mM Na2SO4 for 14 d (middle panel) and recovered for additional 7 days (right panel). Scale bar, 5 cm. E Statistical analysis of survival rates with Na2SO4 treatment in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants for each biological replicate. (***) P ≤ 0.001 indicates significant difference by student t-test. F The seedlings of DJ and osprr73 mutant were treated with 90 mM MgCl2 for 14 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm. G Statistical analysis of survival rates with MgCl2 treatment in (F). The survival rate of DJ and osprr73 plants in MgCl2 stress were calculated after recovery 7 days. Data represent means ± SD. n = 2 biological replicates, 24 plants of each replicate. H The seedlings of DJ and osprr73 mutant were treated with 180 mM mannitol for 36 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm. I The survival rate of DJ and osprr73 plants in mannitol stress. Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. The abbreviation of n.s. stands for no significant generated by student t-test.


Figure 4A , B The expression level of OsHKT2 ; 1 and OsHKT1 ; 5 in osprr73 mutant and the wild control Dongjin ( DJ ) plants with or without salinity stress in multiple time points . DJ Data represent means ± SD . n = 3 , technical replicates . The representative data is from at least two individual biological replicates . The asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 and ( * * * ) P ≤ 0 . 001 . C Transient expression assay in the infiltrated leaves of N . benthamiana showing OsPRR73 can repress OsHKT2 ; 1 transcription . Left panel showing the bioluminescence signal of OsHKT2 ; 1 and OsPRR73 . Bioluminescence of co - transformation of either GFP or 35S : GFP - OsPRR73 ( GFP - OsPRR73 ) with OsHKT2 ; 1pro : LUC in N . benthamiana . D Quantiﬁcation of bioluminescence intensity of OsHKT2 ; 1pro : LUC shown in ( C ) . Data represent means ± SD . n = 10 . The asterisks indicate the significant difference by student ' s t - test with ( * * * ) P ≤ 0 . 001 . At least two biological replicates were conducted with similar results . E ChIP - qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2 ; 1 promoter by OsPRR73 , compared to wild type DJ controls . The amplicon of Ubiquitin was taken as a negative control . Two - week - old seedlings were harvested at ZT12 . Data represent means ± SD ( n = 3 , technical repeats ) . The experiments were performed at least two biological replicates with similar result . Top scheme indicates the locations of amplicons for ChIP analysis . The asterisks represent significant difference among means by student ' s t - test with ( * * ) P ≤ 0 . 01 . F EMSA assay with the OsPRR73 incubated with the probes designed for the S4 region of OsHKT2 ; 1 promoter . Unlabelled probes were used as competitor with indicated folds . MBP alone was used as negative control . Arrowhead marks the shifted positive bands .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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, B The expression level of OsHKT2;1 and OsHKT1;5 in osprr73 mutant and the wild control Dongjin (DJ) plants with or without salinity stress in multiple time points. DJ Data represent means ± SD. n = 3, technical replicates. The representative data is from at least two individual biological replicates. The asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01 and (***) P ≤ 0.001.C Transient expression assay in the infiltrated leaves of N. benthamiana showing OsPRR73 can repress OsHKT2;1 transcription. Left panel showing the bioluminescence signal of OsHKT2;1 and OsPRR73. Bioluminescence of co-transformation of either GFP or 35S: GFP-OsPRR73 (GFP-OsPRR73) with OsHKT2;1pro: LUC in N. benthamiana. D Quantiﬁcation of bioluminescence intensity of OsHKT2;1pro: LUC shown in (C). Data represent means ± SD. n = 10. The asterisks indicate the significant difference by student's t-test with (***) P ≤ 0.001. At least two biological replicates were conducted with similar results.E ChIP-qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2;1 promoter by OsPRR73, compared to wild type DJ controls. The amplicon of Ubiquitin was taken as a negative control. Two-week-old seedlings were harvested at ZT12. Data represent means ± SD (n =3, technical repeats). The experiments were performed at least two biological replicates with similar result. Top scheme indicates the locations of amplicons for ChIP analysis. The asterisks represent significant difference among means by student's t-test with (**) P ≤ 0.01.F EMSA assay with the OsPRR73 incubated with the probes designed for the S4 region of OsHKT2;1 promoter. Unlabelled probes were used as competitor with indicated folds. MBP alone was used as negative control. Arrowhead marks the shifted positive bands.


Figure 5A Coimmunoprecipitation assay showing the physical interaction between OsPRR73 and HDAC10 in vivo . CoIP was conducted with GFP - Trap beads , and GFP and FLAG antibodies were used to detect OsPRR73 and HDAC10 in immunoprecipitants as indicated . B Split luciferase complementation ( SLC ) assay showing OsPRR73 interacts with HDAC10 in the co - infiltrated N . benthamiana leaves . C Bimolecular - fluorescence complementation ( BiFC ) assay shows the interaction between OsPRR73 and HDAC10 occurs in nucleus . BF represents bright field . Scale bar , 40 µm . D N - terminus of OsPRR73 containing PR domain is required and sufficient for interacting with HDAC10 . Upper panel shows the schematic illustration of full - length and the truncated OsPRR73 proteins . CoIP was performed with GFP - Trap beads , and immunoprecipitants were detected by FLAG for HADC10 and GFP for truncated proteins of OsPRR73 respectively . E , F Chromatin Immunoprecipitation - qPCR ( ChIP - qPCR ) assay to detect the enriched promoter regions of OsHKT2 ; 1 by using the H3K9ac ( E ) and H3 ( F ) antibodies . Two - week - old seedlings were harvested at ZT12 . The locations of amplicons were described in Figure 4E . Data represent means ± SD . n = 3 technical replicates . The asterisk indicates the significant difference by student ' s t - test with ( * ) P ≤ 0 . 05 . The experiments were repeated at least twice with similar result .

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

Figure 5A Coimmunoprecipitation assay showing the physical interaction between OsPRR73 and HDAC10 in vivo. CoIP was conducted with GFP-Trap beads, and GFP and FLAG antibodies were used to detect OsPRR73 and HDAC10 in immunoprecipitants as indicated.B Split luciferase complementation (SLC) assay showing OsPRR73 interacts with HDAC10 in the co-infiltrated N.benthamiana leaves.C Bimolecular-fluorescence complementation (BiFC) assay shows the interaction between OsPRR73 and HDAC10 occurs in nucleus. BF represents bright field. Scale bar, 40 µm.D N-terminus of OsPRR73 containing PR domain is required and sufficient for interacting with HDAC10. Upper panel shows the schematic illustration of full-length and the truncated OsPRR73 proteins. CoIP was performed with GFP-Trap beads, and immunoprecipitants were detected by FLAG for HADC10 and GFP for truncated proteins of OsPRR73 respectively.E, F Chromatin Immunoprecipitation-qPCR (ChIP-qPCR) assay to detect the enriched promoter regions of OsHKT2;1 by using the H3K9ac (E) and H3 (F) antibodies. Two-week-old seedlings were harvested at ZT12. The locations of amplicons were described in Figure 4E. Data represent means ± SD. n = 3 technical replicates. The asterisk indicates the significant difference by student's t-test with (*) P ≤ 0.05. The experiments were repeated at least twice with similar result.


Figure 6D The oshkt2 ; 1 seedling is more tolerant to the treatment of NaCl . The oshkt2 ; 1 and NIP , its wild type , four - week - old seedlings grown under NaCl conditions for 0 days ( left panel ) , then were transferred to 200 mM NaCl for 28 days ( middle panel ) , and recovered for 7 days ( right panel ) . Scale bar , 5 cm . E Statistical analysis of survival rate in ( D ) . Data represent means ± SD . n = 3 biological replicates , 24 plants of each replicate . ( * ) P ≤ 0 . 05 was generated by student ' s t - test .

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Figure 6D The oshkt2;1 seedling is more tolerant to the treatment of NaCl. The oshkt2;1 and NIP, its wild type, four-week-old seedlings grown under NaCl conditions for 0 days (left panel), then were transferred to 200 mM NaCl for 28 days (middle panel), and recovered for 7 days (right panel). Scale bar, 5 cm. E Statistical analysis of survival rate in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. (*) P ≤ 0.05 was generated by student's t-test.


Figure 7A Malondialdehyde ( MDA ) content in leaves of 14 - d - old DJ and osprr73 plants treated by 180 mM NaCl for 3 d and normal condition . Data represent means ± SD . n = 6 , biological replicates . The asterisks indicate the significant difference ( * * * ) P ≤ 0 . 001 by student ' s t - test . B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM NaCl treatment for 3 days . Scale bar , 1 cm . C H2O2 concentration was detected by using H2DCFDA in the root tip of DJ and osprr73 mutant plants . Scale bar , 5 mm . - NaCl and + NaCl represented the untreated or treated plants by NaCl respectively . D Quantitative analysis of H2O2 concentration in the root of DJ and osprr73 mutant . Data represent means ± SD . n ≥ 6 . The asterisks indicate the significant difference ( * * * ) P ≤ 0 . 001 by student ' s t - test . E , F Measurement of SOD ( Superoxide dismutase ) ( E ) and CAT ( Catalase ) ( F ) enzymes activity in shoots of DJ and osprr73 seedlings with or without NaCl stress . Data represent means ± SD . n = 3 technical replicates . Data are representative from two independent biological replicates with similar result . The asterisks indicate the significant difference ( * ) P ≤ 0 . 05 by student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O 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 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 7A Malondialdehyde (MDA) content in leaves of 14-d-old DJ and osprr73 plants treated by 180 mM NaCl for 3 d and normal condition. Data represent means ± SD. n = 6, biological replicates. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM NaCl treatment for 3 days. Scale bar, 1 cm.C H2O2 concentration was detected by using H2DCFDA in the root tip of DJ and osprr73 mutant plants. Scale bar, 5 mm. -NaCl and +NaCl represented the untreated or treated plants by NaCl respectively.D Quantitative analysis of H2O2 concentration in the root of DJ and osprr73 mutant. Data represent means ± SD. n ≥ 6. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.E, F Measurement of SOD (Superoxide dismutase) (E) and CAT (Catalase) (F) enzymes activity in shoots of DJ and osprr73 seedlings with or without NaCl stress. Data represent means ± SD. n = 3 technical replicates. Data are representative from two independent biological replicates with similar result. The asterisks indicate the significant difference (*) P ≤ 0.05 by student's t-test.


Figure 1 ( e ) Gene Set Enrichment Analysis ( GSEA ) of the genes differentially expressed between YAP / TAZ - deficient ( siY / T ) and control siRNA ( siCtrl ) transfected HLE cells showed an enrichment for genes involved in the regulation of lipid peroxidation . ( g ) Basal lipid peroxidation levels increased with the loss of function of YAP / TAZ . HLE - shLuc and HLE - shY / T cells were stained with C11 - BODIPY 581 / 591 . Reduced - Bodipy was measured by flow cytometry using a 488 nm laser , and oxidized - Bodipy was measured with a 561 nm laser . A significant shift of oxidized - Bodipy occurred upon depletion of YAP / TAZ . Results represent three independent experiments .

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Figure 1(e) Gene Set Enrichment Analysis (GSEA) of the genes differentially expressed between YAP/TAZ-deficient (siY/T) and control siRNA (siCtrl) transfected HLE cells showed an enrichment for genes involved in the regulation of lipid peroxidation.(g) Basal lipid peroxidation levels increased with the loss of function of YAP/TAZ. HLE-shLuc and HLE-shY/T cells were stained with C11-BODIPY 581/591. Reduced-Bodipy was measured by flow cytometry using a 488 nm laser, and oxidized-Bodipy was measured with a 561 nm laser. A significant shift of oxidized-Bodipy occurred upon depletion of YAP/TAZ. Results represent three independent experiments.


Figure 2 ( b ) Quantitative RT - PCR analysis confirmed the dependency of SLC7A11 gene expression on YAP / TAZ . HLE cells were transfected with control siRNA ( siCtrl ) or siRNA against YAP / TAZ ( siY / T ) and cultured with DMSO or 6μM Sorafenib for 18 hours . RNA was extracted and analyzed by quantitative RT - PCR . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments . ( f ) Binding of YAP and TAZ to a DNA fragment containing the TEAD binding motif in the SLC7A11 promoter . ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control . DNA fragments were amplified using the primers specific for TEAD binding motif in the SLC7A11 promoter region shown in ( e ) . The non - coding region NC10 served as negative control , and the bona fide TEAD target gene CYR61 served as positive control . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments . ( i ) Representative images of immuno - histochemical staining of SLC7A11 and YAP proteins in HCC samples from patients . Scale bars , 50μm . ( j ) Quantification of the immunohistochemical stainings shown in ( i ) revealed a positive correlation of YAP and SLC7A11 expression ( N = 10 ) . Statistical significance was calculated using Pearson correlation analysis .

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Figure 2(b) Quantitative RT-PCR analysis confirmed the dependency of SLC7A11 gene expression on YAP/TAZ. HLE cells were transfected with control siRNA (siCtrl) or siRNA against YAP/TAZ (siY/T) and cultured with DMSO or 6μM Sorafenib for 18 hours. RNA was extracted and analyzed by quantitative RT-PCR. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.(f) Binding of YAP and TAZ to a DNA fragment containing the TEAD binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for TEAD binding motif in the SLC7A11 promoter region shown in (e). The non-coding region NC10 served as negative control, and the bona fide TEAD target gene CYR61 served as positive control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.(i) Representative images of immuno-histochemical staining of SLC7A11 and YAP proteins in HCC samples from patients. Scale bars, 50μm. (j) Quantification of the immunohistochemical stainings shown in (i) revealed a positive correlation of YAP and SLC7A11 expression (N=10). Statistical significance was calculated using Pearson correlation analysis.


Figure 3 ( a ) SLC7A11 protein levels decreased upon siRNA - mediated depletion of ATF4 but not of NRF2 either with or without Sorafenib treatment , and Sorafenib promoted the expression of ATF4 . HLE cells were transfected with siCtrl , siATF4 or siNRF2 and treated with or without 6μM Sorafenib for 18 hours . The expression of SLC7A11 , ATF4 and NRF2 was determined by immunoblotting . GAPDH served as loading control . Results represent three independent experiments . ( f ) Colony formation assay demonstrating that the ferroptosis inhibitor Ferrostatin - 1 ( Fer ) reversed Sorafenib - induced cell death in ATF4 - deficient HCC cells . HLE cells transfected with siCtrl or siATF4 were treated with Sorafenib ( 8μM ) or DMSO plus either DMSO or Ferrostatin - 1 ( Fer ; 5μM ) for 2 weeks . Results represent three independent experiments . ( g ) The forced expression of SLC7A11 rescued cell death induced by ATF4 ablation . HLE cells were transfected with a construct coding for SLC7A11 ( SLC7A11 OE ) or empty vector ( EV ) and with siCtrl or siATF4 every other day . Cells were then treated with either DMSO or 6μM Sorafenib for 2 weeks . Colony formation was visualized by crystal violet staining . Results represent three independent experiments . ( h ) Immunohistochemical staining of ATF4 in HCC and adjacent non - neoplastic areas from patients . Tumor tissues showed a higher expression of ATF4 . Scale bar , 100μm . ( i ) Quantification of ATF4 - positive cells in tumor and non - tumor samples showed that HCC tumors present higher ATF4 levels . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using unpaired t - test . ( j ) Representative images of immuno - histochemical staining of ATF4 and SLC7A11 proteins in HCC samples from patients . Scale bars , 50μm .

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Figure 3(a) SLC7A11 protein levels decreased upon siRNA-mediated depletion of ATF4 but not of NRF2 either with or without Sorafenib treatment, and Sorafenib promoted the expression of ATF4. HLE cells were transfected with siCtrl, siATF4 or siNRF2 and treated with or without 6μM Sorafenib for 18 hours. The expression of SLC7A11, ATF4 and NRF2 was determined by immunoblotting. GAPDH served as loading control. Results represent three independent experiments.(f) Colony formation assay demonstrating that the ferroptosis inhibitor Ferrostatin-1 (Fer) reversed Sorafenib-induced cell death in ATF4-deficient HCC cells. HLE cells transfected with siCtrl or siATF4 were treated with Sorafenib (8μM) or DMSO plus either DMSO or Ferrostatin-1 (Fer; 5μM) for 2 weeks. Results represent three independent experiments.(g) The forced expression of SLC7A11 rescued cell death induced by ATF4 ablation. HLE cells were transfected with a construct coding for SLC7A11 (SLC7A11 OE) or empty vector (EV) and with siCtrl or siATF4 every other day. Cells were then treated with either DMSO or 6μM Sorafenib for 2 weeks. Colony formation was visualized by crystal violet staining. Results represent three independent experiments.(h) Immunohistochemical staining of ATF4 in HCC and adjacent non-neoplastic areas from patients. Tumor tissues showed a higher expression of ATF4. Scale bar, 100μm. (i) Quantification of ATF4-positive cells in tumor and non-tumor samples showed that HCC tumors present higher ATF4 levels. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using unpaired t-test. (j) Representative images of immuno-histochemical staining of ATF4 and SLC7A11 proteins in HCC samples from patients. Scale bars, 50μm.


Figure 4 ( a ) YAP / TAZ deficiency repressed Sorafenib - induced expression of ATF4 , whereas ATF4 depletion had no effect on YAP / TAZ protein levels . HLE cells were transfected with siCtrl or siATF4 or siY / T and treated with 6μM Sorafenib or not for 18 hours . ATF4 and YAP / TAZ protein levels were analyzed by immunoblotting . GAPDH served as loading control . Results represent three independent experiments . ( d ) Cellular fractionation revealed that high cell density , and with it , lower YAP / TAZ activity reduced the levels of nuclear ATF4 . HLE cells were seeded at different cell numbers , and cell lysates of high cell density cells ( HD ) and low cell density ( LD ) were fractionated into cytoplasmic and nuclear fractions and analyzed for ATF4 and YAP / TAZ by immunoblotting . GAPDH served as loading control for total lysate and cytoplasmic proteins , Lamin A / C served as loading control for nuclear proteins . Results represent three independent experiments . ( h ) Representative images of immuno - histochemical staining of ATF4 and YAP proteins in HCC samples from patients . Scale bars , 50μm . ( i ) Quantification of the immunohistochemical staining in ( j ) showed a positive correlation of ATF4 and YAP expression ( N = 18 ) . Statistical significance was calculated using Pearson correlation analysis .

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Figure 4(a) YAP/TAZ deficiency repressed Sorafenib-induced expression of ATF4, whereas ATF4 depletion had no effect on YAP/TAZ protein levels. HLE cells were transfected with siCtrl or siATF4 or siY/T and treated with 6μM Sorafenib or not for 18 hours. ATF4 and YAP/TAZ protein levels were analyzed by immunoblotting. GAPDH served as loading control. Results represent three independent experiments.(d) Cellular fractionation revealed that high cell density, and with it, lower YAP/TAZ activity reduced the levels of nuclear ATF4. HLE cells were seeded at different cell numbers, and cell lysates of high cell density cells (HD) and low cell density (LD) were fractionated into cytoplasmic and nuclear fractions and analyzed for ATF4 and YAP/TAZ by immunoblotting. GAPDH served as loading control for total lysate and cytoplasmic proteins, Lamin A/C served as loading control for nuclear proteins. Results represent three independent experiments.(h) Representative images of immuno-histochemical staining of ATF4 and YAP proteins in HCC samples from patients. Scale bars, 50μm. (i) Quantification of the immunohistochemical staining in (j) showed a positive correlation of ATF4 and YAP expression (N=18). Statistical significance was calculated using Pearson correlation analysis.


Figure 5 ( b ) Quantitative RT - PCR analysis verified that depletion of YAP / TAZ or ATF4 declined the expression levels of SLC7A11 and CHAC1 . HLE cells were transfected with siCtrl , siY / T or siATF4 and cultured with 6μM Sorafenib for 18 hours . Quantitative RT - PCR was conducted to determine SLC7A11 and CHAC1 mRNA levels . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using Two - way ANOVA . Results represent 3 independent experiments . ( e ) Binding of YAP and TAZ to DNA fragments containing the AARE binding motif in the SLC7A11 promoter . ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control . DNA fragments were amplified using the primers specific for AARE binding motif in the SLC7A11 promoter region . The non - coding region NC10 served as negative control , and the bona fide TEAD target genes CYR61 and ANKRD1 served as positive controls . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments . ( g ) YAP / TAZ bind to DNA fragments containing the AARE binding motif within the SLC7A11 promoter via ATF4 . HLE cells were cultured with 6μM Sorafenib for 18 hours before harvest . In a 1st round ChIP ATF4 was immunoprecipitated with antibody against ATF4 , rabbit IgG was used as control . DNA - protein immunocomplexes were eluted and in a 2nd round ChIP antibody against YAP / TAZ was used to precipitate DNA fragments which were then amplified and analyzed by quantitative PCR for the AARE motif in the SLC7A11 promoter . NC10 served as negative PCR control . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments .

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Figure 5(b) Quantitative RT-PCR analysis verified that depletion of YAP/TAZ or ATF4 declined the expression levels of SLC7A11 and CHAC1. HLE cells were transfected with siCtrl, siY/T or siATF4 and cultured with 6μM Sorafenib for 18 hours. Quantitative RT-PCR was conducted to determine SLC7A11 and CHAC1 mRNA levels. Data are shown as mean ± standard deviation (SD).Statistical significance was calculated using Two-way ANOVA. Results represent 3 independent experiments.(e) Binding of YAP and TAZ to DNA fragments containing the AARE binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for AARE binding motif in the SLC7A11 promoter region. The non-coding region NC10 served as negative control, and the bona fide TEAD target genes CYR61 and ANKRD1 served as positive controls. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.(g) YAP/TAZ bind to DNA fragments containing the AARE binding motif within the SLC7A11 promoter via ATF4. HLE cells were cultured with 6μM Sorafenib for 18 hours before harvest. In a 1st round ChIP ATF4 was immunoprecipitated with antibody against ATF4, rabbit IgG was used as control. DNA-protein immunocomplexes were eluted and in a 2nd round ChIP antibody against YAP/TAZ was used to precipitate DNA fragments which were then amplified and analyzed by quantitative PCR for the AARE motif in the SLC7A11 promoter. NC10 served as negative PCR control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.


Figure 6Combination of YAP / TAZ deficiency and Sorafenib treatment suppressed tumor growth in a HCC xenograft model . SNU398 - shLuc or SNU398 - shYAP / TAZ ( shY / T ) cells were transplanted into the flanks of immunodeficient NSG mice . Once the tumors were palpable , mice were treated with 20mg / kg Sorafenib or vehicle control , and tumor sizes were measured twice a week ( b ) . Tumor weights were also recorded after sacrifice of the mice Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using two - way ANOVA analysis . Mouse numbers of shLuc + Vehicle , shY / T + Vechicle and shY / T + Srf were 4 per cohort , mouse numbers of shLuc + Srf were 3 per cohort . ( e ) Pharmacological inhibition of glutathione synthesis and function sensitizes HCC tumors to Sorafenib therapy . SNU398 cells were transplanted into the flanks of immunodeficient NSG mice . Once the tumors were palpable , mice were treated with vehicle control or with 20mg / kg Sorafenib alone , or with 20mg / kg Sorafenib and 20mM BSO in the drinking water or 120mg / kg SSA per os . Tumor sizes were measured twice a week . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using two - way ANOVA analysis . Mouse numbers were 8 for the experimental cohorts Vehicle , Srf , and Srf + SSA and 7 for Srf + BSO . ( g ) Quantification of 4 - HNE - positive cells of the immunohistochemical stainings described in ( f ) . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Each data point represents the average of 3 technical replicates of one histological section , the numbers of tumor samples are given in ( e ) .

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Figure 6Combination of YAP/TAZ deficiency and Sorafenib treatment suppressed tumor growth in a HCC xenograft model. SNU398-shLuc or SNU398-shYAP/TAZ (shY/T) cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with 20mg/kg Sorafenib or vehicle control, and tumor sizes were measured twice a week (b). Tumor weights were also recorded after sacrifice of the mice Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers of shLuc + Vehicle, shY/T + Vechicle and shY/T + Srf were 4 per cohort, mouse numbers of shLuc + Srf were 3 per cohort.(e) Pharmacological inhibition of glutathione synthesis and function sensitizes HCC tumors to Sorafenib therapy. SNU398 cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with vehicle control or with 20mg/kg Sorafenib alone, or with 20mg/kg Sorafenib and 20mM BSO in the drinking water or 120mg/kg SSA per os. Tumor sizes were measured twice a week. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers were 8 for the experimental cohorts Vehicle, Srf, and Srf + SSA and 7 for Srf + BSO.(g) Quantification of 4-HNE-positive cells of the immunohistochemical stainings described in (f). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Each data point represents the average of 3 technical replicates of one histological section, the numbers of tumor samples are given in (e).


Figure 1B ) Electrophoretic mobility shift assay ( EMSA ) to examine binding affinities of the GST - KNL2518 - 560 fragment for the CENP - A or H3 nucleosome . The molar ratio of KNL2518 - 560 against nucleosomes are indicated . D ) - F ) EMSA to examine binding affinities of the GST - KNL2 fragment for the CENP - A nucleosome : D , GST - KNL2347 - 560 ; E , GST - KNL2456 - 560 ; F , GST - KNL2456 - 516 ( CC - upstream ) .

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Figure 1B) Electrophoretic mobility shift assay (EMSA) to examine binding affinities of the GST-KNL2518-560 fragment for the CENP-A or H3 nucleosome. The molar ratio of KNL2518-560 against nucleosomes are indicated.D)-F) EMSA to examine binding affinities of the GST-KNL2 fragment for the CENP-A nucleosome: D, GST-KNL2347-560; E, GST-KNL2456-560; F, GST-KNL2456-516 (CC-upstream).


Figure 3B ) EMSA to examine contribution of the ggKNL2 R527 residue to the CENP - A nucleosome binding . D ) Expression of the WT or R527A mutant GFP - KNL2 in KNL2 conditional knock out chicken DT40 cells ( cKO - KNL2 ) . Expression of full - length KNL2 WT was conditionally turned off by tetracycline ( tet ) addition in cKO - KNL2 cells ( None ) . GFP - fused KNL2 WT or R527A mutant was stably expressed in the cKO - KNL2 cells . These cells were cultured in the presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control . E ) Localization analyses of GFP - fused KNL2 WT or R527A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , and DNA was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm . F ) Growth of the cKO - KNL2 cells expressing GFP - fused KNL2 WT or R527A mutant . The upper panel shows examined cell numbers at the indicated time after tet addition ( + tet : KNL2 OFF ) . The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number . The lower panel shows examined untreated cells ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 cells ( None ) were also examined .

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Figure 3B) EMSA to examine contribution of the ggKNL2 R527 residue to the CENP-A nucleosome binding.D) Expression of the WT or R527A mutant GFP-KNL2 in KNL2 conditional knock out chicken DT40 cells (cKO-KNL2). Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition in cKO-KNL2 cells (None). GFP-fused KNL2 WT or R527A mutant was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.E) Localization analyses of GFP-fused KNL2 WT or R527A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or R527A mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


Figure 4B ) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP - A nucleosome binding . D ) Expression of GFP - KNL2 WT or CENP - A C - terminal tail binding mutants in cKO - KNL2 cells . Expression of full - length KNL2 WT was conditionally turned off by tetracycline ( tet ) addition to the cKO - KNL2 cells ( None ) . GFP - fused KNL2 WT , F535A , W536A or F535A / W536A was stably expressed in the cKO - KNL2 cells . These cells were cultured in the presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control . E ) Localization analyses of GFP - fused KNL2 WT and F535A , W536A or F535A / W536A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , and DNA was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm . F ) Growth of the cKO - KNL2 cells expressing GFP - fused KNL2 WT or each CENP - A C - terminal tail binding mutant . The upper panel shows examined cell numbers at the indicated time after tet addition ( + tet : KNL2 OFF ) . The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number . The lower panel shows examined untreated cells ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 cells ( None ) were also examined .

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Figure 4B) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP-A nucleosome binding.D) Expression of GFP-KNL2 WT or CENP-A C-terminal tail binding mutants in cKO-KNL2 cells. Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition to the cKO-KNL2 cells (None). GFP-fused KNL2 WT, F535A, W536A or F535A/W536A was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.E) Localization analyses of GFP-fused KNL2 WT and F535A, W536A or F535A/W536A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or each CENP-A C-terminal tail binding mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


Figure 6A ) Immunostaining with anti - Mis18α antibody in AID - based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h . Clear colocalization of KNL2 signals with Mis18α was observed ( Insets ) . DNA was stained with DAPI . Scale bar indicates 10 µm . B ) AID - based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP - tagged CENP - A were used . After addition of IAA , SNAP - CENP - A signals were visualized by TMR - Star staining . CENP - T was stained as a kinetochore marker . Representative images were presented . Mitotic centromere localization of each KNL2 mutant was shown as a merged image ( Insets ) : SNAP - CENP - A ( red ) and each KNL2 mutant ( green ) . DNA was stained with DAPI . Scale bar indicates 10 µm .

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Figure 6A) Immunostaining with anti-Mis18α antibody in AID-based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h. Clear colocalization of KNL2 signals with Mis18α was observed (Insets). DNA was stained with DAPI. Scale bar indicates 10 µm.B) AID-based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP-tagged CENP-A were used. After addition of IAA, SNAP-CENP-A signals were visualized by TMR-Star staining. CENP-T was stained as a kinetochore marker. Representative images were presented. Mitotic centromere localization of each KNL2 mutant was shown as a merged image (Insets): SNAP-CENP-A (red) and each KNL2 mutant (green). DNA was stained with DAPI. Scale bar indicates 10 µm.


Figure 7A ) KNL2 ( red ) localization in CENP - C conditional knockout chicken DT40 cells ( cKO - CENP - C ) with or without CENP - C expression . DNA was stained with DAPI ( blue ) . CENP - T was stained as a kinetochore marker ( light blue ) . Scale bar indicates 10 μm . B ) KNL2 intensities were evaluated in six cells before and after CENP - C knock out . KNL2 signal intensities on the mitotic centromeres ( 14 - 90 centromeres ) in each cell were plotted with the average and the standard deviation . The ratio of KNL2 intensities between cells before and after CENP - C knock out was estimated . KNL2 intensities were reduced by ~ 55 % , when CENP - C was knocked out . C ) Competitive pull - down assays for the CENP - A nucleosome binding of ggKNL2 and ggCENP - C . A complex of GST - KNL2456 - 560 with the CENP - A nucleosome ( CA - nuc ) was incubated in the absence or presence of MBP - CENP - C619 - 690 ( right panel ) or phosphorylated MBP - CENP - C619 - 690 ( middle panel ) and pulled down by GST affinity . The concentration of CENP - C619 - 690 was increased from 0 μM to 1 μM ( lanes 3 - 7 ) . The ratio of band intensities of pull - downed histones ( * ) to GST - KNL2456 - 560 in each lane ( lanes 3 - 7 ) was estimated and presented in the bar graph ( left panel ) . Data are mean with standard deviations calculated from the technical replicates , n = 3 .

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Figure 7A) KNL2 (red) localization in CENP-C conditional knockout chicken DT40 cells (cKO-CENP-C) with or without CENP-C expression. DNA was stained with DAPI (blue). CENP-T was stained as a kinetochore marker (light blue). Scale bar indicates 10 μm.B) KNL2 intensities were evaluated in six cells before and after CENP-C knock out. KNL2 signal intensities on the mitotic centromeres (14-90 centromeres) in each cell were plotted with the average and the standard deviation. The ratio of KNL2 intensities between cells before and after CENP-C knock out was estimated. KNL2 intensities were reduced by ~55%, when CENP-C was knocked out.C) Competitive pull-down assays for the CENP-A nucleosome binding of ggKNL2 and ggCENP-C. A complex of GST-KNL2456-560 with the CENP-A nucleosome (CA-nuc) was incubated in the absence or presence of MBP-CENP-C619-690 (right panel) or phosphorylated MBP-CENP-C619-690 (middle panel) and pulled down by GST affinity. The concentration of CENP-C619-690 was increased from 0 μM to 1 μM (lanes 3-7). The ratio of band intensities of pull-downed histones (*) to GST-KNL2456-560 in each lane (lanes 3-7) was estimated and presented in the bar graph (left panel). Data are mean with standard deviations calculated from the technical replicates, n=3.


Figure 1Immunohistochemistry of NSMCE2 on mouse testis . The inset illustrates the accumulation of NSMCE2 on the XY body . Scale bar , 50 μm . Immunofluorescence ( IF ) of NSMCE2 and SCP3 on isolated mouse spermatocytes . Threads of NSMCE2 can be detected on regions of weak SCP3 signal , indicative of asynapsis ( arrowheads ) . Scale bar , 2 . 5 μm . Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes . Representative image of NSMCE2 and BRCA1 foci in control and MMS ‐ treated MEFs . Scale bar , 2 . 5 μm . Quantification of NSMCE2 and BRCA1 foci in wild ‐ type MEFs exposed to IR ( 10 Gy ; 1 ‐ h recovery ) , MMS ( 1 mM for 1 h ; 24 ‐ h recovery ) , and PARP inhibitor ( 1 μM for 1 h ; 24 ‐ h recovery ) . Data indicate mean values and are representative of three independent experiments . Error bars indicate SD . Representative image of IR ‐ ( 10 Gy ; 1 ‐ h recovery ) and MMS ‐ ( 1 mM for 1 ‐ h ; 24 ‐ h recovery ) induced NSMCE2 and γH2AX foci in MEFs . Scale bar , 2 . 5 μm .

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Figure 1Immunohistochemistry of NSMCE2 on mouse testis. The inset illustrates the accumulation of NSMCE2 on the XY body. Scale bar, 50 μm.Immunofluorescence (IF) of NSMCE2 and SCP3 on isolated mouse spermatocytes. Threads of NSMCE2 can be detected on regions of weak SCP3 signal, indicative of asynapsis (arrowheads). Scale bar, 2.5 μm.Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes.Representative image of NSMCE2 and BRCA1 foci in control and MMS‐treated MEFs. Scale bar, 2.5 μm.Quantification of NSMCE2 and BRCA1 foci in wild‐type MEFs exposed to IR (10 Gy; 1‐h recovery), MMS (1 mM for 1 h; 24‐h recovery), and PARP inhibitor (1 μM for 1 h; 24‐h recovery). Data indicate mean values and are representative of three independent experiments. Error bars indicate SD.Representative image of IR‐ (10 Gy; 1‐h recovery) and MMS‐ (1 mM for 1‐h; 24‐h recovery) induced NSMCE2 and γH2AX foci in MEFs. Scale bar, 2.5 μm.


Figure 2Brightfield microscopy of 2 . 5 dpc embryos obtained from crosses of Nsmce2GT / + mice . The right panel is of the same 2 embryos shown on the left panel , 36 h after being cultured in vitro . The arrow indicates embryos that showed asymmetric cell sizes when isolated , which degenerated upon culture . These embryos were identified as Nsmce2GT / GT through genotyping . Examples of the three genotypes found at Mendelian frequencies ( see Appendix Table S1 ) on 2 . 5 dpc embryos . Examples of the type of nuclei found on Nsmce2GT / GT and Nsmce2 + ( + / + or + / GT ) 2 . 5 dpc embryos , 12 h after in vitro culture . Mutant embryos were identified due to the absence of NSMCE2 signal ( green ) . DAPI was used to stain DNA ( blue ) . White arrows indicate examples of cells with condensed chromosomes or irregular shapes , which are hardly seen in wild type embryos .

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Figure 2Brightfield microscopy of 2.5 dpc embryos obtained from crosses of Nsmce2GT/+ mice. The right panel is of the same 2 embryos shown on the left panel, 36 h after being cultured in vitro. The arrow indicates embryos that showed asymmetric cell sizes when isolated, which degenerated upon culture. These embryos were identified as Nsmce2GT/GT through genotyping.Examples of the three genotypes found at Mendelian frequencies (see Appendix Table S1) on 2.5 dpc embryos.Examples of the type of nuclei found on Nsmce2GT/GT and Nsmce2+ (+/+ or +/GT) 2.5 dpc embryos, 12 h after in vitro culture. Mutant embryos were identified due to the absence of NSMCE2 signal (green). DAPI was used to stain DNA (blue). White arrows indicate examples of cells with condensed chromosomes or irregular shapes, which are hardly seen in wild type embryos.


Figure 3Immunoprecipitation of SMC5 ( and IgG , as a control ) from a 48 ‐ h culture of B lymphocytes of wt and Nsmce2SD / SD animals . NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB . The image of the Ponceau staining is shown as a loading control . IF of NSMCE2 foci in MMS ‐ treated Nsmce2 + / + and Nsmce2SD / SD MEFs . Scale bar , 2 . 5 μm . Representative picture of 6 ‐ month ‐ old Nsmce2 + / + and Nsmce2SD / SD littermates . Kaplan - Meyer survival curves of Nsmce2 + / + and Nsmce2SD / SD mice . The P ‐ value was calculated with the Mantel - Cox long ‐ rank test .

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Figure 3Immunoprecipitation of SMC5 (and IgG, as a control) from a 48‐h culture of B lymphocytes of wt and Nsmce2SD/SD animals. NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB. The image of the Ponceau staining is shown as a loading control.IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm.Representative picture of 6‐month‐old Nsmce2+/+ and Nsmce2SD/SD littermates.Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2SD/SD mice. The P‐value was calculated with the Mantel-Cox long‐rank test.


Figure 4A Kaplan - Meyer survival curves of Nsmce2 + / + and Nsmce2 + / GT mice . The P ‐ value was calculated with the Mantel - Cox long ‐ rank test . * * * P 0 . 001 . B Tumor incidence found on Nsmce2 + / + and Nsmce2 + / GT mice at the time of death ( i . e . moribund mice ) . C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2 + / + and Nsmce2 + / GT MEFs . β ‐ actin was used as a loading control . Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample . D Sister chromatid exchange ( SCE ) events found per metaphase on Nsmce2 + / + and Nsmce2 + / GT MEFs . * * * P 0 . 001 . E , F Relative percentages of micronuclei ( E ) and polynucleated cells ( F ) on cultures of Nsmce2 + / + and Nsmce2 + / GT MEFs . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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Figure 4A Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2+/GT mice. The P‐value was calculated with the Mantel-Cox long‐rank test. ***P 0.001.B Tumor incidence found on Nsmce2+/+ and Nsmce2+/GT mice at the time of death (i.e. moribund mice).C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2+/+ and Nsmce2+/GT MEFs. β‐actin was used as a loading control. Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample.D Sister chromatid exchange (SCE) events found per metaphase on Nsmce2+/+ and Nsmce2+/GT MEFs. ***P 0.001.E, F Relative percentages of micronuclei (E) and polynucleated cells (F) on cultures of Nsmce2+/+ and Nsmce2+/GT MEFs. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


Figure 5NSMCE2 levels assessed by WB in UQ . CreERT2 transgenic Nsmce2 + / + and Nsmce2lox / lox littermate MEFs , 24 h after exposure to 4 ‐ OHT . Tubulin levels are shown as a loading control . Clonogenic assay on immortalized UQ . CreERT2 / Nsmce2lox / lox MEFs . The picture shows the colonies detected after 10 days of growth in the presence or absence of 4 ‐ OHT , upon staining with crystal violet . NSMCE2 and BRCA1 foci in MMS ‐ treated ( 1 mM for 1 h ; 24 ‐ h recovery ) UQ . CreERT2 / Nsmce2lox / lox MEFs , 48 h after being treated ( or not ) with 4 ‐ OHT . Scale bar , 5 μm . SCE events per metaphase on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in the presence of 4 ‐ OHT for 72 h . * * * P 0 . 001 . Example of SCE events ( arrows ) found on Nsmce2 ‐ deleted MEFs . SCE events at telomeres on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in the presence of 4 ‐ OHT for 72 h , as detected by CO ‐ FISH ( see ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 . Telomere lengths of UQ . CreERT2 Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h . Example of the intensity of telomere signals ( yellow ) used for ( H ) . Percentage of cells with micronuclei found on cultures of UQ . CreERT2 / Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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Figure 5NSMCE2 levels assessed by WB in UQ.CreERT2 transgenic Nsmce2+/+ and Nsmce2lox/lox littermate MEFs, 24 h after exposure to 4‐OHT. Tubulin levels are shown as a loading control.Clonogenic assay on immortalized UQ.CreERT2/Nsmce2lox/lox MEFs. The picture shows the colonies detected after 10 days of growth in the presence or absence of 4‐OHT, upon staining with crystal violet.NSMCE2 and BRCA1 foci in MMS‐treated (1 mM for 1 h; 24‐h recovery) UQ.CreERT2/Nsmce2lox/lox MEFs, 48 h after being treated (or not) with 4‐OHT. Scale bar, 5 μm.SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001.Example of SCE events (arrows) found on Nsmce2‐deleted MEFs.SCE events at telomeres on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h, as detected by CO‐FISH (see ). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.Telomere lengths of UQ.CreERT2 Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h.Example of the intensity of telomere signals (yellow) used for (H).Percentage of cells with micronuclei found on cultures of UQ.CreERT2/Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


Figure 6Intrauterine dwarfism observed upon NSMCE2 deletion on 17 . 5 dpc embryos . Pregnant females were treated at 14 . 5 dpc for 3 days with intraperitoneal injections of 4 ‐ OHT . The image represents the difference in size observed between Nsmce2 + / + and Nsmce2lox / lox embryos carrying the UQ . CreERT2 transgene ( Nsmce2 + / + and Nsmce2lox / lox , from now on ) . Representative picture of Nsmce2 + / + and Nsmce2lox / lox animals fed for 40 weeks with a 4 ‐ OHT ‐ containing diet ( see also Movies EV1 and EV2 ) . Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox / lox mice . Percentage of fat against total body mass on Nsmce2 + / + and Nsmce2lox / lox animals . * * * P 0 . 001 . White bloodcell counts ( WBC ) from Nsmce2 + / + and Nsmce2lox / lox animals . * * * P 0 . 001 . Percentage of γH2AX ‐ positive micronuclei found on the intestinal epithelia of Nsmce2 + / + and Nsmce2lox / lox animals ( see Appendix Fig S11 ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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Figure 6Intrauterine dwarfism observed upon NSMCE2 deletion on 17.5 dpc embryos. Pregnant females were treated at 14.5 dpc for 3 days with intraperitoneal injections of 4‐OHT. The image represents the difference in size observed between Nsmce2+/+ and Nsmce2lox/lox embryos carrying the UQ.CreERT2 transgene (Nsmce2+/+ and Nsmce2lox/lox, from now on).Representative picture of Nsmce2+/+ and Nsmce2lox/lox animals fed for 40 weeks with a 4‐OHT‐containing diet (see also Movies EV1 and EV2).Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox/lox mice.Percentage of fat against total body mass on Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.White bloodcell counts (WBC) from Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.Percentage of γH2AX‐positive micronuclei found on the intestinal epithelia of Nsmce2+/+ and Nsmce2lox/lox animals (see Appendix Fig S11). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


Figure 7A WB illustrating the depletion of NSMCE2 in B cells isolated from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes , 48 h after being stimulated in vitro with lipopolysaccharide ( LPS ) . Tubulin was used as a loading control . B Representative FACS analysis from the B ‐ cell cultures mentioned in ( A ) . DNA content was measured with propidium iodide ( PI ) . Note the presence of cells with > 4n DNA content in NSMCE2 ‐ deleted cells . C , D Frequency of origin firing ( C ) and fork rate ( D ) were analyzed by DNA fiber analyses ( see ) from fibers prepared from B ‐ cell cultures explained in ( A ) . No significant differences were observed between Nsmce2 + / + and Nsmce2lox / loxB cells . Data are representative of two independent experiments . Error bars indicate SD . Between 200 and 300 tracks were measured to estimate fork rate and 300 - 500 for the calculation of origin firing frequencies . E Representative images of the DNA fibers used for the analysis explained in ( C , D ) . CldU ( red ) and IdU ( green ) channels are shown .

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Figure 7A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS). Tubulin was used as a loading control.B Representative FACS analysis from the B‐cell cultures mentioned in (A). DNA content was measured with propidium iodide (PI). Note the presence of cells with > 4n DNA content in NSMCE2‐deleted cells.C, D Frequency of origin firing (C) and fork rate (D) were analyzed by DNA fiber analyses (see ) from fibers prepared from B‐cell cultures explained in (A). No significant differences were observed between Nsmce2+/+ and Nsmce2lox/loxB cells. Data are representative of two independent experiments. Error bars indicate SD. Between 200 and 300 tracks were measured to estimate fork rate and 300-500 for the calculation of origin firing frequencies.E Representative images of the DNA fibers used for the analysis explained in (C, D). CldU (red) and IdU (green) channels are shown.


Figure 8Representative pictures of the spleens from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes . Numbers of B and T cells from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes evaluated by flow cytometry ( B220 : B ‐ cell marker ; CD3 : T ‐ cell marker ) . SCE events per metaphase on B cells obtained from CD19 + / Cre animals of the indicated genotypes , 72 h after being stimulated in vitro with LPS . * * P 0 . 01 , * * * P 0 . 001 . Analysis of forward ( FSC ) and side ( SSC ) scatter parameters of B cells obtained from CD19 + / Cre animals of the indicated genotypes , 48 h after being stimulated in vitro with LPS . The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2 . Representative images of B ‐ cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS . Insets provide a magnified view of an individual cell ( arrowhead ) for better comparison of the observed differences in cell size . Scale bar , 100 μm . Examples of the types of nuclei found on Blm and Nsmce2 double ‐ mutant B cells ( compared with wild ‐ type B cells ) , in cells obtained from ( E ) . DAPI was used to stain DNA . Whereas wt nuclei are regular in size , double ‐ mutant cells invariably presented enlarged , multilobulated , and irregular nuclei . Scale bar , 2 . 5 μm .

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Figure 8Representative pictures of the spleens from 2‐month‐old CD19+/Cre animals of the indicated genotypes.Numbers of B and T cells from 2‐month‐old CD19+/Cre animals of the indicated genotypes evaluated by flow cytometry (B220: B‐cell marker; CD3: T‐cell marker).SCE events per metaphase on B cells obtained from CD19+/Cre animals of the indicated genotypes, 72 h after being stimulated in vitro with LPS. **P 0.01, ***P 0.001.Analysis of forward (FSC) and side (SSC) scatter parameters of B cells obtained from CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with LPS. The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2.Representative images of B‐cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS. Insets provide a magnified view of an individual cell (arrowhead) for better comparison of the observed differences in cell size. Scale bar, 100 μm.Examples of the types of nuclei found on Blm and Nsmce2 double‐mutant B cells (compared with wild‐type B cells), in cells obtained from (E). DAPI was used to stain DNA. Whereas wt nuclei are regular in size, double‐mutant cells invariably presented enlarged, multilobulated, and irregular nuclei. Scale bar, 2.5 μm.


Figure 1B Western blot analyses after induced expression of FLAG - tagged UPF3A in WT HEK293 and HeLa cells ( n = 1 ) . Tubulin serves as control . The asterisk indicates unspecific bands . C Fraction of expressed genes ( genes with non - zero counts in DESeq2 ) were calculated which exhibit individual or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) and / or alternative splicing ( AS ) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis ( cutoffs are indicated ) . AS and DTU events were collapsed on the gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR ( ISAR ) using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution and corrected for multiple testing using the Benjamini - Hochberg method . D Volcano plot showing the differential gene expression analyses from the RNA - Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A . The log2 fold change is plotted against the - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . OE = overexpression . E Read coverage of SMG5 from WT HEK293 and HeLa RNA - seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer ( IGV ) snapshot . Differential gene expression ( from DESeq2 ) is indicated as log2 fold change ( log2FC ) on the right . Schematic representation of the protein coding transcript below .

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Figure 1B Western blot analyses after induced expression of FLAG-tagged UPF3A in WT HEK293 and HeLa cells (n=1). Tubulin serves as control. The asterisk indicates unspecific bands.C Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR (ISAR) using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.D Volcano plot showing the differential gene expression analyses from the RNA-Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A. The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. OE = overexpression.E Read coverage of SMG5 from WT HEK293 and HeLa RNA-seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer (IGV) snapshot. Differential gene expression (from DESeq2) is indicated as log2 fold change (log2FC) on the right. Schematic representation of the protein coding transcript below.


Figure 2A Western blot analysis of WT and UPF3A KO cells ( clones 4 , 14 and 20 ) with the indicated siRNA treatments ( n = 1 ) . UPF3A and UPF3B protein levels were detected , Tubulin serves as control . The asterisk indicates unspecific bands . B Quantitative RT - PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC ) ( n = 3 for RSRC2 and SRSF2 , n = 4 for ZFAS1 ) . C Fraction of expressed genes ( genes with non - zero counts in DESeq2 ) were calculated which exhibit individual or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) and / or alternative splicing ( AS ) events in the indicated conditions using the respective computational analysis ( cutoffs are indicated ) . AS and DTU events were collapsed on the gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution and corrected for multiple testing using the Benjamini - Hochberg method . D , E Volcano plots showing the differential gene expression analyses from the indicated RNA - Seq datasets ( D UPF3A KO clone 14 , E UPF3A KO clone 20 ) . The log2 fold change is plotted against the - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg

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Figure 2A Western blot analysis of WT and UPF3A KO cells (clones 4, 14 and 20) with the indicated siRNA treatments (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.B Quantitative RT-PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC) (n=3 for RSRC2 and SRSF2, n=4 for ZFAS1).C Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.D,E Volcano plots showing the differential gene expression analyses from the indicated RNA-Seq datasets (D UPF3A KO clone 14, E UPF3A KO clone 20). The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method