B . HEK293 cells were transfected with a pool of siRNA against p53 ( sip53 ) or with control siRNA ( siScr ) for 24 h and treated for additional 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM and CHK2 , as well as p53 and GSNOR were assessed by Western blot . Phospho : basal level ratios of ATM and CHK2 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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. HEK293 cells were transfected with a pool of siRNA against p53 (sip53) or with control siRNA (siScr) for 24 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, as well as p53 and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.


C . HCT116 cells expressing the wild type form of p53 ( p53wt ) , or an empty vector ( Empty , selected as negative control ) , were treated for 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM and CHK2 , p53 and GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . Phospho : basal level ratios of ATM and CHK2 , along with densitometry of p53 and GSNOR immunoreactive bands are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HCT116 cells expressing the wild type form of p53 (p53wt), or an empty vector (Empty, selected as negative control), were treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, p53 and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2, along with densitometry of p53 and GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.


A , B . HEK293 cells treated for 2 , 4 and 8 h with 100 μM H2O2 . After treatment , cells were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or MitoSox ( B ) to evaluate the production of H2O2 or mitochondrial superoxide , respectively . Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells ( arbitrarily set as 1 ) and represent the means ± SD of n ≥ 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

O O O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. HEK293 cells treated for 2, 4 and 8 h with 100 μM H2O2. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.


C . HEK293 were transfected for 48 h with siRNA against ATM ( siATM ) , GSNOR ( siGSNOR ) , or control siRNA ( scramble , siScr ) . Afterwards , they were treated for 8 h with 100 μM H2O2 and mitophagy was assessed by Western blot of different mitochondrial complex subunits [ i . e . , NDUFB8 ( complex I ) , SDHB ( complex II ) , MTCO2 ( complex IV ) ] . Basal and phospho ATM , as well as GSNOR were used to check the efficiency of siRNA - mediated knock down . Vinculin was used as loading control .

O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O B-GENEPROD 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-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O

C. HEK293 were transfected for 48 h with siRNA against ATM (siATM), GSNOR (siGSNOR), or control siRNA (scramble, siScr). Afterwards, they were treated for 8 h with 100 μM H2O2 and mitophagy was assessed by Western blot of different mitochondrial complex subunits [i.e., NDUFB8 (complex I), SDHB (complex II), MTCO2 (complex IV)]. Basal and phospho ATM, as well as GSNOR were used to check the efficiency of siRNA-mediated knock down. Vinculin was used as loading control.


D . Densitometry of mitochondrial protein immunoreactive bands of panel C ( normalized to Vinculin ) is indicated as H2O2 - treated versus untreated cells ( CTR ) and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant .

O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Densitometry of mitochondrial protein immunoreactive bands of panel C (normalized to Vinculin) is indicated as H2O2-treated versus untreated cells (CTR) and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant.


E . In the same experimental settings , mitophagy was also assessed by RT - qPCR relative quantitation of D - loop ( selected as measure of mtDNA ) normalized to genomic actin ( gActin ) . Results shown are the means ± SD of n = 8 experiments * * * p < 0 . 001 ; n . s . , not significant .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. In the same experimental settings, mitophagy was also assessed by RT-qPCR relative quantitation of D-loop (selected as measure of mtDNA) normalized to genomic actin (gActin). Results shown are the means ± SD of n = 8 experiments ***p<0.001; n.s., not significant.


F . U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 4 h with 100 μM H2O2 . Mitophagy was assessed by RT - qPCR relative quantitation of D - loop normalized to genomic actin ( gActin ) . Results shown are the means ± SD of n = 6 experiments . * p < 0 . 05 ; * * * p < 0 . 001 .

O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 4 h with 100 μM H2O2. Mitophagy was assessed by RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 6 experiments. *p<0.05; ***p<0.001.


In the same experimental settings , mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . Percentage of mitochondria merging with LC3 - positive puncta calculated by Fiji analysis software using the open - source plugin ComDet v . 0 . 3 . 7 . Values are expressed as % of mitochondria ( TOM20 + particles ) co - localizing with LC3 / cell and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n ≥ 7 different cells analyzed . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O 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-SUBCELLULAR O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 7 different cells analyzed. *p<0.05; **p<0.01; ***p<0.001.


H . In the same experimental settings , mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . Anti - TOM20 ( red ) was used to visualize mitochondria ; anti - LC3 ( green ) was used to identify autophagosomes .

O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O

H. In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes.


A , B . ATMWT U2OS cells were treated with 10 μM CCCP for 8 h . After treatment , cells were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or MitoSox ( B ) to evaluate the production of H2O2 or mitochondrial superoxide , respectively . Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells ( arbitrarily set as 1 ) and represent the means ± SD of n = 3 independent experiments . * * * p < 0 . 001 .

O O O O B-GENEPROD B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. ATMWT U2OS cells were treated with 10 μM CCCP for 8 h. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. ***p< 0.001.


C . In the same experimental settings , basal and phospho - active forms of ATM , and GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls .

O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O

C. In the same experimental settings, basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls.


U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . Mitophagy was evaluated by : ( D ) RT - qPCR relative quantitation of D - loop normalized to genomic actin ( gActin ) . Results shown are the means ± SD of n = 4 experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. Mitophagy was evaluated by: (D) RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 4 experiments. *p<0.05; **p<0.01; ***p<0.001.


U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . ( E fluorescence microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . Anti - TOM20 ( red ) was used to visualize mitochondria ; anti - LC3 ( green ) was used to identify autophagosomes .

B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O

U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. (E fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes.


U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . F ) fluorescence microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . Percentage of mitochondria merging with LC3 - positive puncta calculated by Fiji analysis software using the open - source plugin ComDet v . 0 . 3 . 7 . Values are expressed as % of mitochondria ( TOM20 + particles ) co - localizing with LC3 / cell and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n ≥ 9 different cells . * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . , not significant .

B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O 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-SUBCELLULAR O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. F) fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 9 different cells. **p<0.01; ***p<0.001; n.s., not significant.


U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . ( G Western blot of different subunits of mitochondrial proteins , i . e . , NDUFB8 ( complex I ) , SDHA and SDHB ( complex II ) , MTCO2 ( complex IV ) and voltage - dependent anion channel ( VDAC ) . Tubulin , LDH and Vinculin were used as loading controls .

B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. (G Western blot of different subunits of mitochondrial proteins, i.e., NDUFB8 (complex I), SDHA and SDHB (complex II), MTCO2 (complex IV) and voltage-dependent anion channel (VDAC). Tubulin, LDH and Vinculin were used as loading controls.


U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . H ) Densitometry of mitochondrial protein immunoreactive bands and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant .

B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. H) Densitometry of mitochondrial protein immunoreactive bands and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant.


B . HEK293 cells overexpressing the wild type form of GSNOR ( GSNORwt ) or an empty vector ( Empty ) were subjected to combined treatment ( 200 μM H2O2 + 400 μM DPTA ) in the presence or absence of the ATM inhibitor KU55933 ( KU ) . Analysis of dead cells were performed with Trypan blue exclusion assay . Western blot analysis of GSNOR is shown as inset in the graph to substantiate transfection efficiency . Data , shown as fold change of dead cells relative to untreated cells ( arbitrarily set to 1 ) , represent the mean count ± SEM of n = 4 experiments done in duplicate * p < 0 . 05 ; with respect to Empty cells .

O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) in the presence or absence of the ATM inhibitor KU55933 (KU). Analysis of dead cells were performed with Trypan blue exclusion assay. Western blot analysis of GSNOR is shown as inset in the graph to substantiate transfection efficiency. Data, shown as fold change of dead cells relative to untreated cells (arbitrarily set to 1), represent the mean count ± SEM of n = 4 experiments done in duplicate *p<0.05; with respect to Empty cells.


C . Representative optic microscopy image of GSNORwt and Empty cells upon 24 h treatment with H2O2 + DPTA in the presence KU .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

C. Representative optic microscopy image of GSNORwt and Empty cells upon 24 h treatment with H2O2 + DPTA in the presence KU.


D . Western blot analysis of Caspase3 and PARP1 in GSNORwt and Empty cells treated as in panel C . Tubulin was used as loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O

D. Western blot analysis of Caspase3 and PARP1 in GSNORwt and Empty cells treated as in panel C. Tubulin was used as loading control.


E . HEK293 cells overexpressing the wild type form of GSNOR ( GSNORwt ) or an empty vector ( Empty ) were transfected with siRNA against Pakin ( siParkin ) or control siRNA ( scramble , siScr ) . Afterwards , they were subjected to combined treatment ( 200 μM H2O2 + 400 μM DPTA ) and viability assessed by Alamar blue ( AB ) fluorescent assay . Data , shown as fold change of viable cells , refer to AB fluorescence ( relative to untreated cells , arbitrarily set to 1 ) and represent the means ± SD of n = 6 independent experiments . * * , p < 0 . 01 ; n . s . , not significant .

O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were transfected with siRNA against Pakin (siParkin) or control siRNA (scramble, siScr). Afterwards, they were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) and viability assessed by Alamar blue (AB) fluorescent assay. Data, shown as fold change of viable cells, refer to AB fluorescence (relative to untreated cells, arbitrarily set to 1) and represent the means ± SD of n = 6 independent experiments. **, p<0.01; n.s., not significant.


F . Western blot of Gsnor was assessed in mouse adult fibroblasts ( MAFs ) , obtained from wild type ( WT ) and Gsnor - null ( KO ) mice treated with 200 or 400 μM H2O2 for 24 h .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O

F. Western blot of Gsnor was assessed in mouse adult fibroblasts (MAFs), obtained from wild type (WT) and Gsnor-null (KO) mice treated with 200 or 400 μM H2O2 for 24 h.


WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2 , or 400 μM DPTA , or a combination of both . Cell viability was evaluated by LIVE / DEAD assay .

O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay.


H . WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2 , or 400 μM DPTA , or a combination of both . Cell viability was evaluated by LIVE / DEAD assay . Scale bar = 50 µm . Data , shown as % of dead ( red ) cells , represent the mean count ± SD of n = 3 different fields of three independent experiments . * p < 0 . 05 ; n . s . , not significant with respect to WT MAFs .

O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O

H. WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. Scale bar = 50 µm. Data, shown as % of dead (red) cells, represent the mean count ± SD of n = 3 different fields of three independent experiments. *p<0.05; n.s., not significant with respect to WT MAFs.


A , B . Jurkat cells were treated for 10 and 30 min with PMA ( 200 ng / ml ) and ionomycin ( iono ) ( 300 ng / ml ) . After treatment , cells were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or DAF - FM - DA ( B ) to evaluate the production of H2O2 or NO , respectively . Values are expressed as units of DCF or DAF - FM fluorescence relative to untreated cells ( arbitrarily set as 1 ) and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n = 4 ( A ) and n = 6 ( B ) independent experiments . * p < 0 . 01 ; * * p < 0 . 001 ; * * * p < 0 . 001 .

O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Jurkat cells were treated for 10 and 30 min with PMA (200 ng/ml) and ionomycin (iono) (300 ng/ml). After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or DAF-FM-DA (B) to evaluate the production of H2O2 or NO, respectively. Values are expressed as units of DCF or DAF-FM fluorescence relative to untreated cells (arbitrarily set as 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 (A) and n = 6 (B) independent experiments. *p<0.01; **p<0.001; ***p<0.001.


C . ( left ) Jurkat cells were treated for 24 h with H2O2 ( 50 μM ) or PMA / ionomycin ( 200 + 300 ng / ml ) . Basal and phospho - active form of ATM , and GSNOR were assessed by Western blot . Vinculin was used as loading control . ( right ) Phospho : basal level ratios of ATM along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. (left) Jurkat cells were treated for 24 h with H2O2 (50 μM) or PMA/ionomycin (200 + 300 ng/ml). Basal and phospho-active form of ATM, and GSNOR were assessed by Western blot. Vinculin was used as loading control. (right) Phospho:basal level ratios of ATM along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.


D . Jurkat cells were treated for 24 h with H2O2 ( 50 μM ) or PMA / ionomycin ( 200 + 300 ng / ml ) , in the presence or absence of ATM inhibitor ( KU55933 ) ( 5 μM ) or GSNOR inhibitor ( N6022 ) ( 25 μM ) . Cell death was assessed cytofluorimetrically upon staining with propidium iodide ( PI ) . Values are expressed as % of sub - G1 population of PI - stained cells and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n = 5 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 .

O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Jurkat cells were treated for 24 h with H2O2 (50 μM) or PMA/ionomycin (200 + 300 ng/ml), in the presence or absence of ATM inhibitor (KU55933) (5 μM) or GSNOR inhibitor (N6022) (25 μM). Cell death was assessed cytofluorimetrically upon staining with propidium iodide (PI). Values are expressed as % of sub-G1 population of PI-stained cells and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 5 independent experiments. *p<0.05; **p<0.01.


E , F . CD4 + T cells were incubated for 30 min with anti - CD3 , anti - CD28 and anti - CD49d ( stimulation ) . After stimulation , cells were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( E ) or DAF - FM - DA ( F ) to fluorometrically evaluate the production of H2O2 or NO , respectively . Values are shown as units of DCF or DAF - FM fluorescence relative to non - stimulated cells ( arbitrarily set as 1 ) . Values are shown as fold change and represent the median plus range with all the experimental points of n = 3 independent experiments . * * p < 0 . 01 .

O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F. CD4+ T cells were incubated for 30 min with anti-CD3, anti-CD28 and anti-CD49d (stimulation). After stimulation, cells were incubated with 5 μM 2',7'-H2DCF-DA (E) or DAF-FM-DA (F) to fluorometrically evaluate the production of H2O2 or NO, respectively. Values are shown as units of DCF or DAF-FM fluorescence relative to non-stimulated cells (arbitrarily set as 1). Values are shown as fold change and represent the median plus range with all the experimental points of n = 3 independent experiments. **p < 0.01.


G . CD4 + T cells were stimulated for 96 h with anti - CD3 , anti - CD28 and anti - CD49d in the presence or absence of ATM inhibitor ( KU , upper panel ) or GSNOR inhibitor ( N6022 , bottom panel ) . Cell death was assessed cytofluorometrically upon staining with Annexin V ( AnV ) and PI . Values are expressed as fold change of AnV + / PI + cells relative to control ( CD4 + without inhibitor , vehicle , arbitrarily set to 1 ) and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n = 4 independent experiments . * p < 0 . 05 ; * * * p < 0 . 001 .

O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. CD4+ T cells were stimulated for 96 h with anti-CD3, anti-CD28 and anti-CD49d in the presence or absence of ATM inhibitor (KU, upper panel) or GSNOR inhibitor (N6022, bottom panel). Cell death was assessed cytofluorometrically upon staining with Annexin V (AnV) and PI. Values are expressed as fold change of AnV+/PI+ cells relative to control (CD4+ without inhibitor, vehicle, arbitrarily set to 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 independent experiments. *p<0.05; ***p<0.001.


H . In the same experimental settings , populations of stimulated ( proliferating ) CD4 + T cells were identified cytofluorometrically and included in rectangles . Values ( as % of total population ) are shown in red in each representative plot identify and summed up in a graph as the means ± SD of n = 3 independent experiments . * p < 0 . 05 ; * * * p < 0 . 001 .

O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. In the same experimental settings, populations of stimulated (proliferating) CD4+ T cells were identified cytofluorometrically and included in rectangles. Values (as % of total population) are shown in red in each representative plot identify and summed up in a graph as the means ± SD of n = 3 independent experiments. *p<0.05; ***p<0.001.


[ A ] The same MVs from GB03 caused opposed impacts on plants grown in different medium . The petri dishes contain plastic partitions ( red dotted lines ) that separate different medium . 0 . 5 MS and 0 . 05 MS indicate 1 / 2 - strength ( nutrient - sufficient ) and 1 / 20th - strength ( nutrient - deficient ) Murashige & Skoog medium . Images were taken at 11 days after treatments ( DAT ) .

O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O

[A] The same MVs from GB03 caused opposed impacts on plants grown in different medium. The petri dishes contain plastic partitions (red dotted lines) that separate different medium. 0.5 MS and 0.05 MS indicate 1/2-strength (nutrient-sufficient) and 1/20th-strength (nutrient-deficient) Murashige & Skoog medium. Images were taken at 11 days after treatments (DAT).


[ B ] Quantification of total leaf area per plant ( square centimeter , cm2 ) . Values correspond to the means ± SE of three biological replicates . Different letters denote significant differences at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O O O O O B-TISSUE B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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] Quantification of total leaf area per plant (square centimeter, cm2). Values correspond to the means ± SE of three biological replicates. Different letters denote significant differences at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ C ] Effective quantum yield of photosystem II in plants . n = 3 biological replicates , mean ± SE . Asterisks denote significant differences at * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 , Dunnett ' s multiple comparison test ( comparing with the control ) within each group of the same DAT

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[C] Effective quantum yield of photosystem II in plants. n=3 biological replicates, mean± SE. Asterisks denote significant differences at * p < 0.05, ** p < 0.01 and *** p < 0.001, Dunnett's multiple comparison test (comparing with the control) within each group of the same DAT


[ D ] Cell death visualization by trypan blue staining of 11 DAT leaves . Bar = 1mm

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O B-TISSUE O O O O

[D] Cell death visualization by trypan blue staining of 11 DAT leaves. Bar= 1mm


[ A ] Plants grown with 0 . 05 MS and GMVs ( G 0 . 05 ) showed hyper - induction of IPS1 and PS2 gene expression in both shoots and roots , compared to plants grown with 0 . 05 MS alone ( C 0 . 05 ) . Values are means ± SE of three biological replicates .

O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

[A] Plants grown with 0.05 MS and GMVs (G 0.05) showed hyper-induction of IPS1 and PS2 gene expression in both shoots and roots, compared to plants grown with 0.05 MS alone (C 0.05). Values are means ± SE of three biological replicates.


[ B ] Root acid phosphatase activity as detected by the blue color from the 5 - bromo - 4 - chloro - 3 - indolyl phosphate p - toluidine salt ( BCIP ) treatment . Image contrast enhanced for improved visualization of the blue color in roots

[B] Root acid phosphatase activity as detected by the blue color from the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) treatment. Image contrast enhanced for improved visualization of the blue color in roots


Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - accumulation of anthocyanin ( C ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-accumulation of anthocyanin (C) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - induction of IPS1 gene expression ( D ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-induction of IPS1 gene expression (D) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ E ] Pi supplementation to the 0 . 05 MS medium partially restored GMV - induced plant vigor , as indicated by increases in total leaf area per plant . [ F ] A low Pi level ( LP ) , which was equal to 1 / 20 of that in the 0 . 5 MS medium , in plant growth medium blocked GMV - induced plant vigor . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE B-EXP_ASSAY O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[E] Pi supplementation to the 0.05 MS medium partially restored GMV-induced plant vigor, as indicated by increases in total leaf area per plant. [F] A low Pi level (LP), which was equal to 1/20 of that in the 0.5 MS medium, in plant growth medium blocked GMV-induced plant vigor. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ G ] The Arabidopsis phr1phl1 mutant showed substantially decreased growth - promotion , compared to the wild type plants . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O O O B-ORGANISM B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[G] The Arabidopsis phr1phl1 mutant showed substantially decreased growth-promotion, compared to the wild type plants. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ A ] Images of Arabidopsis plants exposed to four individual GMV components , including diacetyl ( DA ) , 2 , 3 - butanediol ( BTDL ) , 2 - methyl - 1 - propanol ( MP ) and acetoin ( ATN ) . The compounds were applied at dosages that , when the compounds totally evaporate from the agar - containing solid droplets , would yield in volatile concentrations of 9 . 7 µg ( DA ) , 32 . 5 µg ( BTDL ) , 7 . 9 µg ( MP ) and 28 . 5 µg ( ATN ) per mL free space in the petri dish , which resembled the 1 : 3 . 3 : 0 . 8 : 2 . 9 ratio among the four GMV components in natural GMVs22 .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[A] Images of Arabidopsis plants exposed to four individual GMV components, including diacetyl (DA), 2,3-butanediol (BTDL), 2-methyl-1-propanol (MP) and acetoin (ATN). The compounds were applied at dosages that, when the compounds totally evaporate from the agar-containing solid droplets, would yield in volatile concentrations of 9.7 µg (DA), 32.5 µg (BTDL), 7.9 µg (MP) and 28.5 µg (ATN) per mL free space in the petri dish, which resembled the 1 : 3.3 : 0.8 : 2.9 ratio among the four GMV components in natural GMVs22.


anthocyanin hyper accumulation ( B ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

anthocyanin hyper accumulation (B) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


PSR gene hyper induction ( C ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PSR gene hyper induction (C) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Compared to the wild type plants , phr1phl1 showed substantially decreased anthocyanin hyper accumulation ( D ) triggered by DA . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O O O B-ORGANISM O B-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Compared to the wild type plants, phr1phl1 showed substantially decreased anthocyanin hyper accumulation (D) triggered by DA. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Compared to the wild type plants , phr1phl1 showed substantially decreased PSR gene hyper induction ( E ) triggered by DA . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Compared to the wild type plants, phr1phl1 showed substantially decreased PSR gene hyper induction (E) triggered by DA. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ A ] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[A] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ B ] DA elevates SA accumulation levels in Pi - deficient Arabidopsis . Means ± SE of three biological replicates . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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] DA elevates SA accumulation levels in Pi-deficient Arabidopsis. Means ± SE of three biological replicates. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ C ] Images of plants grown with different treatments using DA ( 9 . 7 µg per mL free space ) or SA ( 100 µM in plant growth medium ) .

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[C] Images of plants grown with different treatments using DA (9.7 µg per mL free space) or SA (100 µM in plant growth medium).


Exogenous application of SA mimics DA - induced anthocyanin accumulation ( D ) patterns in Pi - deficient plants . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Exogenous application of SA mimics DA-induced anthocyanin accumulation (D) patterns in Pi-deficient plants. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Exogenous application of SA mimics DA - induced IPS1 gene induction ( E ) patterns in Pi - deficient plants . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Exogenous application of SA mimics DA-induced IPS1 gene induction (E) patterns in Pi-deficient plants. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Compared to the wild type plants , the NahG transgenic plants showed altered responses to DA under Pi - deficiency condition , as shown by plant images ( F )

O O O O O B-ORGANISM O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-ORGANISM B-EXP_ASSAY O O O

Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition, as shown by plant images (F)


Compared to the wild type plants , the NahG transgenic plants showed altered responses to DA under Pi - deficiency condition and quantification of anthocyanin accumulation levels ( G ) . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O O O B-ORGANISM O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition and quantification of anthocyanin accumulation levels (G). Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ A ] Clustering of DA - regulated immunity - related genes in Arabidopsis with sufficient Pi , as revealed by RNAseq analysis .

O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O

[A] Clustering of DA-regulated immunity-related genes in Arabidopsis with sufficient Pi, as revealed by RNAseq analysis.


[ B ] DA suppresses plant ROS burst induced by flg22 or elf18 . ROS accumulation was represented in relative luminescence units ( RLU ) . Data bar indicate mean ± SE ( n = 24 ) readings for 60 min . Three independent experiments were performed and similar results were observed . * * p < 0 . 01 , student t - test .

O O O B-SMALL_MOLECULE O B-ORGANISM B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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] DA suppresses plant ROS burst induced by flg22 or elf18. ROS accumulation was represented in relative luminescence units (RLU). Data bar indicate mean ± SE (n=24) readings for 60 min. Three independent experiments were performed and similar results were observed. **p < 0.01, student t-test.


[ C ] DA induces gene expression of AZI1 in Arabidopsis . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group . Bar indicates mean ± SE ( n = 3 ) , two biological replicates were analyzed with similar results .

O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[C] DA induces gene expression of AZI1 in Arabidopsis. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group. Bar indicates mean ± SE (n=3), two biological replicates were analyzed with similar results.


[ D ] flg22 - induced MPK3 / MPK6 phosphorylation in Arabidopsis is not decreased by DA . Kinase assays were performed at 5 min after flg22 treatment . Three independent experiments were performed and similar results were observed .

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[D] flg22-induced MPK3/MPK6 phosphorylation in Arabidopsis is not decreased by DA. Kinase assays were performed at 5 min after flg22 treatment. Three independent experiments were performed and similar results were observed.


[ E ] Plant resistance to the pathogen Pst DC3000 is not reduced by DA . The severity of pathogen infection was measured as colony forming units ( CFUs ) per mg FW plants at 0 and 3 days after inoculation . Data bar indicate mean ± SE ( n = 8 ) , two independent experiments were performed . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group .

O O O O O O O O B-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[E] Plant resistance to the pathogen Pst DC3000 is not reduced by DA. The severity of pathogen infection was measured as colony forming units (CFUs) per mg FW plants at 0 and 3 days after inoculation. Data bar indicate mean ± SE (n=8), two independent experiments were performed. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group.


[ F ] Root colonization of GB03 in wild type Arabidopsis ( Col - 0 ) and the mutants / transgenic lines npr1 , NahG , and rbohd . * * * p < 0 . 001compared with Col - 0 without DA treatment , n = 9 biological replicates , student t - test .

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[F] Root colonization of GB03 in wild type Arabidopsis (Col-0) and the mutants/transgenic lines npr1, NahG, and rbohd. *** p < 0.001compared with Col-0 without DA treatment, n= 9 biological replicates, student t-test.


[ G ] Bacteria chemotaxis with different concentrations of DA . Data point indicates mean ± SE ( n = 6 ) . Two independent experiments were performed and similar results were observed . * * * p < 0 . 001 , student t - test .

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[G] Bacteria chemotaxis with different concentrations of DA. Data point indicates mean± SE (n=6). Two independent experiments were performed and similar results were observed. *** p < 0.001, student t-test.


( A ) Antibacterial activity of NGJ1 , T6SS - 1 - and T6SS - 2 - strains against target bacteria .

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(A) Antibacterial activity of NGJ1, T6SS-1- and T6SS-2- strains against target bacteria.


( A ) Growth curve of recombinant BL21 ( DE3 ) cells upon IPTG mediated induction of proteins , represented as colored line graphs .

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(A) Growth curve of recombinant BL21 (DE3) cells upon IPTG mediated induction of proteins, represented as colored line graphs.


( B ) Bacterial two - hybrid assay reflecting interaction between proteins of T6SS apparatus and effector operons . Interaction between T25 - zip and T18 - zip was used as positive control while pKNT25 and pUT18C ( empty vectors ) was used as negative control . Appearance of blue color suggests positive interaction while absence of color suggests lack of interaction .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O 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-EXP_ASSAY O O O O O O O B-EXP_ASSAY O

(B) Bacterial two-hybrid assay reflecting interaction between proteins of T6SS apparatus and effector operons. Interaction between T25-zip and T18-zip was used as positive control while pKNT25 and pUT18C (empty vectors) was used as negative control. Appearance of blue color suggests positive interaction while absence of color suggests lack of interaction.


( C ) Yeast two - hybrid assay reflecting interaction of TseTBg1 and TseTBg2 effector proteins with the corresponding PAAR ( PAARTseTBg ) proteins encoded in their operon . Interaction between P53 and SV40 large T - antigen ( T ) proteins was used as positive control . The pGBKT7 and pGADT7 ( empty vectors ) were used as negative control .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Yeast two-hybrid assay reflecting interaction of TseTBg1 and TseTBg2 effector proteins with the corresponding PAAR (PAARTseTBg) proteins encoded in their operon. Interaction between P53 and SV40 large T-antigen (T) proteins was used as positive control. The pGBKT7 and pGADT7 (empty vectors) were used as negative control.


( A , B ) Effect of purified TseTBg ( TseTBg1 / TseTBg2 ) and one of the variants ( TseTBg2D116A , K129A ) proteins on degradation of bacterial genomic DNA .

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(A, B) Effect of purified TseTBg (TseTBg1/TseTBg2) and one of the variants (TseTBg2D116A,K129A) proteins on degradation of bacterial genomic DNA.


( C , D ) RNase activity of TseTBg effector proteins , in presence or absence of cognate TsiTBg immunity proteins .

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(C, D) RNase activity of TseTBg effector proteins, in presence or absence of cognate TsiTBg immunity proteins.


( E ) Effect of TseTBg1 , TseTBg2 , TseTBg2D116A , K129A proteins on degradation of pHM1 , a broad host - range plasmid isolated from E . coli and NGJ1 . ( F ) Effect of TseTBg1 and TseTBg2 proteins on degradation of pHM1 , reintroduced into E . coli from NGJ1 .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O

(E) Effect of TseTBg1, TseTBg2, TseTBg2D116A,K129A proteins on degradation of pHM1, a broad host-range plasmid isolated from E. coli and NGJ1. (F) Effect of TseTBg1 and TseTBg2 proteins on degradation of pHM1, reintroduced into E. coli from NGJ1.


( G ) Effect of ectopic expression of DNA adenine methylase ( Dam ) protein on TseTBg proteins mediated degradation of plasmid DNA .

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(G) Effect of ectopic expression of DNA adenine methylase (Dam) protein on TseTBg proteins mediated degradation of plasmid DNA.


( E , F ) Graph showing the effect of native TsiTBg and HTH or Imm52 domain deleted variant of TsiTBg proteins on Gus reporter gene expression ( quantified using 4 - methylumbelliferyl ß - D - glucuronide , MUG , as substrate ) under the external ( pExtTseTBg1 / pExtTseTBg2 ) and internal ( pIntTseTBg1 / pIntTseTBg2 ) promoters in E . coli .

O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O

(E, F) Graph showing the effect of native TsiTBg and HTH or Imm52 domain deleted variant of TsiTBg proteins on Gus reporter gene expression (quantified using 4-methylumbelliferyl ß-D-glucuronide, MUG, as substrate) under the external (pExtTseTBg1/pExtTseTBg2) and internal (pIntTseTBg1/pIntTseTBg2) promoters in E. coli.


( A ) Interaction of external ( pExtTseTBg1 ) and internal ( pIntTseTBg1 ) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein . ( B ) Interaction of pExtTseTBg1 and pIntTseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain deleted variant of TseTBg1 protein ( TsiTBg1ΔHTH ) . ( C ) Interaction of pExtTseTBg2 and pIntTseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein . ( D ) Interaction of control ( random ) DNA with the TsiTBg1 protein .

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(A) Interaction of external (pExtTseTBg1) and internal (pIntTseTBg1) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein. (B) Interaction of pExtTseTBg1 and pIntTseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain deleted variant of TseTBg1 protein (TsiTBg1ΔHTH). (C) Interaction of pExtTseTBg2 and pIntTseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein. (D) Interaction of control (random) DNA with the TsiTBg1 protein.


( A ) MCF10A - eGFP - LC3 cells were left untreated ( Control ) or treated with 5 μM rapamycin ( Rapa ) , 500 ng / ml TRAIL or 10 ng / ml TNF for 24 h . When indicated , 10 mM 3 ‐ MA or 5 μg / ml TRAIL ‐ R2 antagonist antibody was added 1 h before the drugs . Representative confocal images ( 20 μm scale bars ) and the percentages of cells with LC3 translocation are shown .

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(A) MCF10A-eGFP-LC3 cells were left untreated (Control) or treated with 5 μM rapamycin (Rapa), 500 ng/ml TRAIL or 10 ng/ml TNF for 24 h. When indicated, 10 mM 3‐MA or 5 μg/ml TRAIL‐R2 antagonist antibody was added 1 h before the drugs. Representative confocal images (20 μm scale bars) and the percentages of cells with LC3 translocation are shown.


( B ) MCF10A - eGFP - LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting ( left ) , or stimulated with 500 ng / ml TRAIL for 24 h and analysed for LC3 translocation ( right ) .

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(B) MCF10A-eGFP-LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting (left), or stimulated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (right).


( C ) MCF10A cells left untreated ( Control ) or treated with 500 ng / ml TRAIL or 5 μM rapamycin ( Rapa ) for 24 h were analysed for LC3 ‐ I , LC3 ‐ II and tubulin by immunoblotting . Pepstatin A and E64d were added to the cells 4 h before harvesting . Similar results were obtained in two independent experiments .

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(C) MCF10A cells left untreated (Control) or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa) for 24 h were analysed for LC3‐I, LC3‐II and tubulin by immunoblotting. Pepstatin A and E64d were added to the cells 4 h before harvesting. Similar results were obtained in two independent experiments.


( D ) MCF10A cells were treated with 5 μM rapamycin ( Rapa ) or indicated concentrations of TRAIL for 24 h , and the increase in the degradation of long ‐ lived proteins as compared with the untreated cultures ( Control ) was measured .

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(D) MCF10A cells were treated with 5 μM rapamycin (Rapa) or indicated concentrations of TRAIL for 24 h, and the increase in the degradation of long‐lived proteins as compared with the untreated cultures (Control) was measured.


( E ) MCF10A - eGFP - LC3 cells transfected with the indicated siRNAs were left untreated 48 h later ( Control ) or treated with 500 ng / ml TRAIL for 24 h and analysed for the DNA content by flow cytometry . The percentage of apoptotic cells with sub ‐ G1 DNA content is shown . The values represent mean ± s . d . for three ( A , D ) or four ( B , E ) independent experiments . * P ‐ value 0 . 05 , * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with control sample ( A - left panel and D ) , with TRAIL ‐ treated cells without anti ‐ TRAIL ‐ R2 ( A - right panel ) or with TRAIL ‐ treated cells without siRNA ( B , E ) .

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(E) MCF10A-eGFP-LC3 cells transfected with the indicated siRNAs were left untreated 48 h later (Control) or treated with 500 ng/ml TRAIL for 24 h and analysed for the DNA content by flow cytometry. The percentage of apoptotic cells with sub‐G1 DNA content is shown. The values represent mean±s.d. for three (A, D) or four (B, E) independent experiments. *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with control sample (A-left panel and D), with TRAIL‐treated cells without anti‐TRAIL‐R2 (A-right panel) or with TRAIL‐treated cells without siRNA (B, E).


( A ) Protein lysates from MCF10A cells left untreated or treated with 500 ng / ml TRAIL or 5 μM rapamycin ( Rapa , positive control ) for 24 h were analysed by immunoblotting for phosphorylated p70S6K ( P ‐ p70S6K ) , total p70S6K and GAPDH ( loading control ) .

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(A) Protein lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa, positive control) for 24 h were analysed by immunoblotting for phosphorylated p70S6K (P‐p70S6K), total p70S6K and GAPDH (loading control).


( B ) Immunoblot analysis of the levels of P ‐ ACC and tubulin ( loading control ) in lysates from MCF10A cells left untreated or treated with 500 ng / ml TRAIL for 24 h , 1 mM AICAR for 4 h ( positive control ) or 5 μM rapamycin for 24 h ( negative control ) .

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(B) Immunoblot analysis of the levels of P‐ACC and tubulin (loading control) in lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL for 24 h, 1 mM AICAR for 4 h (positive control) or 5 μM rapamycin for 24 h (negative control).


( C ) Immunoblot analysis of the level of P ‐ ACC and GAPDH ( loading control ) in lysates from MCF10A cells treated with 500 ng / ml TRAIL for the indicated times .

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(C) Immunoblot analysis of the level of P‐ACC and GAPDH (loading control) in lysates from MCF10A cells treated with 500 ng/ml TRAIL for the indicated times.


( D ) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately ( upper panel ) or after additional 24 h ( middle panel ) or 2 h ( lower panel ) incubation with ( + ) or without ( − ) 500 ng / ml TRAIL .

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(D) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately (upper panel) or after additional 24 h (middle panel) or 2 h (lower panel) incubation with (+) or without (−) 500 ng/ml TRAIL.


( E ) MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng / ml TRAIL were analysed for LC3 translocation ( top ) and the DNA content ( bottom ) . The values represent mean ± s . d . for four independent experiments . * P ‐ value 0 . 05 and * * * P ‐ value 0 . 001 as compared with TRAIL ‐ treated cells without siRNA . Similar results were obtained in two ( A , B and D ) or three ( C , D ) independent experiments .

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(E) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng/ml TRAIL were analysed for LC3 translocation (top) and the DNA content (bottom). The values represent mean±s.d. for four independent experiments. *P‐value 0.05 and ***P‐value 0.001 as compared with TRAIL‐treated cells without siRNA. Similar results were obtained in two (A, B and D) or three (C, D) independent experiments.


( A ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs ( top ) and proteins ( bottom by RT - PCR and immunoblotting , respectively . Similar results were obtained in three independent experiments .

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(A) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top) and proteins (bottom by RT-PCR and immunoblotting, respectively. Similar results were obtained in three independent experiments.


( B ) MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h , and left untreated or treated with 500 ng / ml TRAIL for 2 h , 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h ( starvation ) were analysed for P ‐ ACC and tubulin ( loading control ) expression by immunoblotting . Similar results were obtained in two independent experiments .

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(B) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h, and left untreated or treated with 500 ng/ml TRAIL for 2 h, 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h (starvation) were analysed for P‐ACC and tubulin (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.


( C ) Cells transfected as in ( B ) and left untreated or treated with 500 ng / ml TRAIL , 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation . The values represent mean ± s . d . of three independent experiments . * * * P ‐ value 0 . 001 as compared with cells treated in a same way , but transfected without siRNA .

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(C) Cells transfected as in (B) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation. The values represent mean±s.d. of three independent experiments. ***P‐value 0.001 as compared with cells treated in a same way, but transfected without siRNA.


( A ) The lysates of MCF10A cells treated with 500 ng / ml TRAIL for the indicated times were analysed by immunoblotting . The control lane ( 0 min TRAIL ) originates from the same blot and has been treated and exposed identically . Similar results were obtained in three independent experiments .

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(A) The lysates of MCF10A cells treated with 500 ng/ml TRAIL for the indicated times were analysed by immunoblotting. The control lane (0 min TRAIL) originates from the same blot and has been treated and exposed identically. Similar results were obtained in three independent experiments.


( B , C ) The lysates of MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h and left untreated ( − ) or treated with 500 ng / ml TRAIL for 2 h were analysed by immunoblotting . ( C , lower panel ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT - PCR . Similar results were obtained in two independent experiments .

O O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(B, C) The lysates of MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and left untreated (−) or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting. (C, lower panel) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT-PCR. Similar results were obtained in two independent experiments.


( D , E ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h , treated as in Figure 3C and analysed for LC3 translocation ( left ) and sub ‐ G1 DNA content ( right ) . The values represent mean ± s . d . of a minimum of three independent experiments except for the right panel in E ( two experiments ) . * P ‐ value 0 . 05 and * * P ‐ value 0 . 01 as compared with cells treated in a same way , but transfected without siRNA .

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(D, E) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h, treated as in Figure 3C and analysed for LC3 translocation (left) and sub‐G1 DNA content (right). The values represent mean±s.d. of a minimum of three independent experiments except for the right panel in E (two experiments). *P‐value 0.05 and **P‐value 0.01 as compared with cells treated in a same way, but transfected without siRNA.


( A ) Protein lysates from MCF10A cells left untreated ( 0 ) or treated with 50 ng / ml TNF , 500 ng / ml TRAIL ( positive control ) or 10 ng / ml IL ‐ 1β for indicated times were analysed by immunoblotting for phosphorylated ACC ( P ‐ ACC ) , phosphorylated IκB ( P ‐ IκB ) and GAPDH ( loading control ) . The values show the P ‐ ACC / GAPDH and P ‐ IkB / GAPDH ratios as percentages of the ratios in untreated cells ( left lanes ) and are representative of two independent experiments .

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(A) Protein lysates from MCF10A cells left untreated (0) or treated with 50 ng/ml TNF, 500 ng/ml TRAIL (positive control) or 10 ng/ml IL‐1β for indicated times were analysed by immunoblotting for phosphorylated ACC (P‐ACC), phosphorylated IκB (P‐IκB) and GAPDH (loading control). The values show the P‐ACC/GAPDH and P‐IkB/GAPDH ratios as percentages of the ratios in untreated cells (left lanes) and are representative of two independent experiments.


( B , C ) MCF10A - eGFP - LC3 cells left untreated or treated with 500 ng / ml TRAIL , 50 ng / ml TNF or 10 ng / ml IL ‐ 1β for 24 h were analysed for LC3 translocation ( B ) and the DNA content ( C ) . The values represent mean ± s . d . for two independent experiments . * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with untreated cells .

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(B, C) MCF10A-eGFP-LC3 cells left untreated or treated with 500 ng/ml TRAIL, 50 ng/ml TNF or 10 ng/ml IL‐1β for 24 h were analysed for LC3 translocation (B) and the DNA content (C). The values represent mean±s.d. for two independent experiments. **P‐value 0.01 and ***P‐value 0.001 as compared with untreated cells.


( A ) The lysates of hTERT - RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng / ml TRAIL for 2 h were analysed by immunoblotting .

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(A) The lysates of hTERT-RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting.


( B ) hTERT - RPE1 - eGFP - LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng / ml TRAIL for 24 h and analysed for LC3 translocation ( left ) and sub ‐ G1 DNA content ( middle ) and AMPKα expression ( right ) .

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(B) hTERT-RPE1-eGFP-LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (left) and sub‐G1 DNA content (middle) and AMPKα expression (right).


( C ) hTERT - RPE1 - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs ( top ) . After 48 h , cells were left untreated or treated with 500 ng / ml TRAIL for 2 h , and analysed for P ‐ ACC and GAPDH ( loading control ) expression by immunoblotting . Similar results were obtained in two independent experiments .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

(C) hTERT-RPE1-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top). After 48 h, cells were left untreated or treated with 500 ng/ml TRAIL for 2 h, and analysed for P‐ACC and GAPDH (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.


( D ) Cells transfected as described in ( C ) and left untreated or treated with 500 ng / ml TRAIL , 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation .

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(D) Cells transfected as described in (C) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation.


( E ) Lysates of wild ‐ type ( WT ) MEFs and MEFs with an inactive TAK1 knock ‐ in ( TAK1Δ ) treated with 500 ng / ml TRAIL were analysed by immunoblotting . Similar results were obtained in two independent experiments .

O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O

(E) Lysates of wild‐type (WT) MEFs and MEFs with an inactive TAK1 knock‐in (TAK1Δ) treated with 500 ng/ml TRAIL were analysed by immunoblotting. Similar results were obtained in two independent experiments.


( F ) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng / ml TRAIL were analysed for cell death by the LDH release assay . Similar results were obtained in two independent experiments ( A , C and E ) . The values represent mean ± s . d . for two ( D ) or a minimum of three independent experiments ( B , F ) . * P ‐ value 0 . 05 , * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with cells treated in the same way , but transfected without siRNA ( B , D ) or the wild ‐ type cells ( F ) .

O O O O O B-GENEPROD B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng/ml TRAIL were analysed for cell death by the LDH release assay. Similar results were obtained in two independent experiments (A, C and E). The values represent mean±s.d. for two (D) or a minimum of three independent experiments (B, F). *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with cells treated in the same way, but transfected without siRNA (B, D) or the wild‐type cells (F).


( a ) Instant Blue stained SDS - polyacrylamide gel of Scc1wt cohesin tetramers used in ( b ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O

(a) Instant Blue stained SDS-polyacrylamide gel of Scc1wt cohesin tetramers used in (b).


( b ) Cohesin loading assay . Scc1wt cohesin tetramer was incubated with nicked circular ( CN ) or 1x , 2x or 4x concentration of linearised ( L ) plasmid DNA , immunoprecipitated with anti - Scc1 antibodies , washed with high salt buffer and then eluted with Scc1 peptide . Recovered DNA was separated by agarose gel electrophoresis and stained with GelRedDNA stain . Input DNA = 7 % .

O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O

(b) Cohesin loading assay. Scc1wt cohesin tetramer was incubated with nicked circular (CN) or 1x, 2x or 4x concentration of linearised (L) plasmid DNA, immunoprecipitated with anti-Scc1 antibodies, washed with high salt buffer and then eluted with Scc1 peptide. Recovered DNA was separated by agarose gel electrophoresis and stained with GelRedDNA stain. Input DNA = 7 %.


( c ) Silver stainedSDS - polyacrylamide gel of non - cleaved and cleaved Scc1Halo - TEVcohesin tetramers used in ( d ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

(c) Silver stainedSDS-polyacrylamide gel of non-cleaved and cleaved Scc1Halo-TEVcohesin tetramers used in (d).


( d ) Non - cleaved and cleaved Scc1Halo - TEVcohesin tetramer was incubated with nicked circular plasmid DNA and processed as in ( b ) . Input DNA = 4 % .

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(d) Non-cleaved and cleaved Scc1Halo-TEVcohesin tetramer was incubated with nicked circular plasmid DNA and processed as in (b). Input DNA = 4 %.


( e ) Kymograph of Scc1GFP - TEVcohesin bound to doubly tethered λ - DNA in cohesin binding buffer and washed with 750 mM NaCl buffer + Sytox Orange . XhoI flow in induced DNA cleavage and cohesin release . Flow in from top and scale bar = 5 µm in this and all subsequent kymographs .

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(e) Kymograph of Scc1GFP-TEVcohesin bound to doubly tethered λ-DNA in cohesin binding buffer and washed with 750 mM NaCl buffer + Sytox Orange. XhoI flow in induced DNA cleavage and cohesin release. Flow in from top and scale bar = 5 µm in this and all subsequent kymographs.


( f ) Kymographs of Scc1GFP - TEVcohesin and Scc1GFPcohesin bound to doubly tethered λ - DNA and washed with 750 mM NaCl buffer . TEV protease flow in released Scc1GFP - TEVbut not Scc1GFP from DNA .

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(f) Kymographs of Scc1GFP-TEVcohesin and Scc1GFPcohesin bound to doubly tethered λ-DNA and washed with 750 mM NaCl buffer. TEV protease flow in released Scc1GFP-TEVbut not Scc1GFP from DNA.


( a ) Kymograph of a single DNA - bound Scc1Halo - TMR - TEVcohesin complex after 750 mM NaCl buffer wash . ( b ) Distribution of background subtracted EMCCD counts of Scc1Halo - TMR - TEVcohesin complexes immobilized on the coverslip . Peaks at ~ 4 x 103 and ~ 8 x 103 EMCCD counts correspond to single fluorophores and a small fraction of double fluorophores respectively . n = 290 regions with fluorescent molecules , 228 regions with background . ( c ) Photobleaching kinetics of a single DNA - bound Scc1Halo - TMR - TEVcohesin complex . ( d ) Diffusion coefficients of Smc1 / 3 wild type or K38A ATP binding deficient ' KA ' forms of Scc1Halo - TMR - TEV - cohesin in the presence or absence of ATP . Diffusion coefficients were calculated based on the linear fit of the average mean square displacement of ≥ 13 freely diffusing molecules per condition over a 1 second time period .

O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Kymograph of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex after 750 mM NaCl buffer wash. (b) Distribution of background subtracted EMCCD counts of Scc1Halo-TMR-TEVcohesin complexes immobilized on the coverslip. Peaks at ~ 4 x 103 and ~ 8 x 103 EMCCD counts correspond to single fluorophores and a small fraction of double fluorophores respectively. n = 290 regions with fluorescent molecules, 228 regions with background. (c) Photobleaching kinetics of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex. (d) Diffusion coefficients of Smc1/3 wild type or K38A ATP binding deficient 'KA' forms of Scc1Halo-TMR-TEV-cohesin in the presence or absence of ATP. Diffusion coefficients were calculated based on the linear fit of the average mean square displacement of ≥ 13 freely diffusing molecules per condition over a 1 second time period.


( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( a ) TetO - DNA - bound TetRHalo - TMR .

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(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (a) TetO-DNA-bound TetRHalo-TMR.


( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( b ) DNA - bound Halo - TMREcoRIE111Q .

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(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (b) DNA-bound Halo-TMREcoRIE111Q.


( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( c ) a DNA - bound TMR - labelled nucleosome .

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(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (c) a DNA-bound TMR-labelled nucleosome.


( d ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin failing to bypass DNA - bound QDotEcoRIE111Q . DNA was stained with Sytox Orange .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O

(d) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound QDotEcoRIE111Q. DNA was stained with Sytox Orange.


( a ) Kymograph of two Halo - TMRT7 RNAP transcription elongation events . ( b , c ) Kymographs showing ( b ) Halo - TMRT7 RNAP transcription stalling following removal of NTPs and ( c ) subsequent resumption after NTP flow in .

O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

(a) Kymograph of two Halo-TMRT7 RNAP transcription elongation events. (b, c) Kymographs showing (b) Halo-TMRT7 RNAP transcription stalling following removal of NTPs and (c) subsequent resumption after NTP flow in.


( d ) Kymograph of Halo - TMRT7 RNAP ( diameter ~ 8 nm excluding HaloTag ) displacing Scc1GFP - TEV - cohesin following resumption of transcription in T7 reaction buffer + NTPs .

O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O

(d) Kymograph of Halo-TMRT7 RNAP (diameter ~ 8 nm excluding HaloTag) displacing Scc1GFP-TEV-cohesin following resumption of transcription in T7 reaction buffer + NTPs.