I , J Schematic diagram showing the CpG islands of Mus musculus PRDM16 ( I ) and CIDEA ( J ) genes . The conditions to be met by CpG islands identified were : 200 - 300 bp length , CG content of > 50 % and observed CpG content vs expected CpG content > 0 . 6 . Methylation levels of each CpG islands in PRDM16 ( I ) and CIDEA ( J ) are presented as the fold change relative to the average value of the M - SCT floxed group and β - actin promoter was used as a negative control ( n = 3 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O O O O O O O B-ORGANISM I-ORGANISM 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 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 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 B-GENEPROD O O O B-GENEPROD I-GENEPROD I-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

I, J Schematic diagram showing the CpG islands of Mus musculus PRDM16 (I) and CIDEA (J) genes. The conditions to be met by CpG islands identified were: 200-300 bp length, CG content of > 50% and observed CpG content vs expected CpG content > 0.6. Methylation levels of each CpG islands in PRDM16 (I) and CIDEA (J) are presented as the fold change relative to the average value of the M-SCT floxed group and β-actin promoter was used as a negative control (n = 3 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


A Western blots analysis of DNMT1 , DNMT3a , DNMT3b and HSP90 in Ing from M - SCT KO and M - SCT floxed mice with HFD .

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

A Western blots analysis of DNMT1, DNMT3a, DNMT3b and HSP90 in Ing from M-SCT KO and M-SCT floxed mice with HFD.


B Relative mRNA levels of DNMT1 , DNMT3a and DNMT3b in Ing from M - SCT KO and M - SCT floxed mice with HFD ( n = 8 ) . Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . ns , not statistically significant .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O O B-GENEPROD O O O 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

B Relative mRNA levels of DNMT1, DNMT3a and DNMT3b in Ing from M-SCT KO and M-SCT floxed mice with HFD (n = 8). Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. ns, not statistically significant.


C Western blots analysis of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 and HSP90 in embryo of M - SCT KO and M - SCT floxed mice at E18 . 5 .

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

C Western blots analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in embryo of M-SCT KO and M-SCT floxed mice at E18.5.


D Western blot analysis of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 and HSP90 in mouse embryo fibroblast ( MEF ) cells treated with 10 nM or 100 nM SCT .

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

D Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in mouse embryo fibroblast (MEF) cells treated with 10 nM or 100 nM SCT.


E Western blot analysis of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 and HSP90 in MEF cells treated with 10 nM SCT or 20 μM compound C ( C . C . ) for 24 h .

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

E Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in MEF cells treated with 10 nM SCT or 20 μM compound C (C.C.) for 24 h.


F Western blot analysis of p - AMPKα , AMPKα , DNMT1 , SCTR and HSP90 . MEF cells were transfected with indicated siRNA and then treated with 1 mM AMPK agonist ( AICAR ) for 24 h .

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

F Western blot analysis of p-AMPKα, AMPKα, DNMT1, SCTR and HSP90. MEF cells were transfected with indicated siRNA and then treated with 1 mM AMPK agonist (AICAR) for 24 h.


G Western blot analysis of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 and HSP90 in AMPK + / + or AMPKα1 / α2 double knockout ( AMPK - / - ) MEF cells treated with 10 nM SCT for 24 h .

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

G Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in AMPK+/+ or AMPKα1/α2 double knockout (AMPK-/-) MEF cells treated with 10 nM SCT for 24 h.


H , I Western blot analysis of Flag - DNMT1 in Flag - DNMT1 - transfected HEK293T cells treated with AICAR with indicated dose or time periods .

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

H, I Western blot analysis of Flag-DNMT1 in Flag-DNMT1-transfected HEK293T cells treated with AICAR with indicated dose or time periods.


J HEK293T cells were transfected with indicated plasmids and then treated with 10 nM SCT for 24 h , followed by IP and IB analysis .

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

J HEK293T cells were transfected with indicated plasmids and then treated with 10 nM SCT for 24 h, followed by IP and IB analysis.


K HEK293T cells were transfected with indicated plasmids or siRNA and then lysed , followed by IP and IB analysis .

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

K HEK293T cells were transfected with indicated plasmids or siRNA and then lysed, followed by IP and IB analysis.


L AMPK + / + or AMPK - / - MEF cells were transfected with indicated plasmids and then lysed , followed by IP and IB analysis .

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

L AMPK+/+ or AMPK-/- MEF cells were transfected with indicated plasmids and then lysed, followed by IP and IB analysis.


A Serum SCT levels in M - HFD and M - HFD + SCT mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O B-TISSUE B-GENEPROD O O O O O O O O O O B-GENEPROD 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

A Serum SCT levels in M-HFD and M-HFD+SCT mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


B Change of body weight of M - HFD ( offspring of dams with high - fat pregnant diets ) and M - HFD + SCT ( offspring of dams with high - fat pregnant diets supplied with SCT ) mice upon HFD feeding ( n = 8 per group ) . Representative photograph of M - HFD and M - HFD + SCT mice after HFD feeding . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

B Change of body weight of M-HFD (offspring of dams with high-fat pregnant diets) and M-HFD+SCT (offspring of dams with high-fat pregnant diets supplied with SCT) mice upon HFD feeding (n = 8 per group). Representative photograph of M-HFD and M-HFD+SCT mice after HFD feeding. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


C , Time - resolved oxygen consumption ( C ) in 48 - hour light / dark cycle measured by CLAMS in M - HFD and M - HFD + SCT mice with HFD ( n = 5 per group ) , dark phase is marked as dark background . Regression - based absolute oxygen consumption ( C ) analysis of body weight are shown in the right part . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data in panel C have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable and body weight as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 B-GENEPROD B-ORGANISM 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 I-EXP_ASSAY 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 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, Time-resolved oxygen consumption (C) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (C) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel C have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


D Time - resolved heat production ( D ) in 48 - hour light / dark cycle measured by CLAMS in M - HFD and M - HFD + SCT mice with HFD ( n = 5 per group ) , dark phase is marked as dark background . Regression - based absolute heat production ( D ) analysis of body weight are shown in the right part . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data in panel D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable and body weight as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 B-GENEPROD B-ORGANISM 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 I-EXP_ASSAY 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 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 Time-resolved heat production (D) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute heat production (D) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


E GTT ( 1 g glucose / kg body weight ) in M - HFD and M - HFD + SCT mice with HFD ( n = 5 per group ) and AUCs were calculated in right part . Mice were fasted for 18h before assays . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GTT (1 g glucose/kg body weight) in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group) and AUCs were calculated in right part. Mice were fasted for 18h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


F ITT ( 0 . 75 U insulin / kg body weight ) in M - HFD and M - HFD + SCT mice with HFD , and AUCs were calculated in bottom ( n = 5 per group ) . Mice were fasted for 6h before assays . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 B-GENEPROD B-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ITT (0.75 U insulin/kg body weight) in M-HFD and M-HFD+SCT mice with HFD, and AUCs were calculated in bottom (n = 5 per group). Mice were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


G Serum TG , TC and NEFA levels in M - HFD and M - HFD + SCT mice with HFD . ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O B-TISSUE B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD 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

G Serum TG, TC and NEFA levels in M-HFD and M-HFD+SCT mice with HFD. (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


H Representative HE staining and UCP1 staining of iWAT from M - HFD and M - HFD + SCT mice with HFD . Scale bar , 100 μm ( n = 5 per group ) .

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

H Representative HE staining and UCP1 staining of iWAT from M-HFD and M-HFD+SCT mice with HFD. Scale bar, 100 μm (n = 5 per group).


I Relative mRNA levels of UCP1 , PGC1α , PRDM16 , CIDEA and DIO2 in iWAT from M - HFD and M - HFD + SCT mice with HFD ( n = 5 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O O O B-GENEPROD 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

I Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in iWAT from M-HFD and M-HFD+SCT mice with HFD (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


( F ) 5 - day - old Col - 0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 30 - 32 biological replicates from 2 pooled experiments ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 001 ) .

O O O O O O O O O O O O O B-GENEPROD I-GENEPROD B-ORGANISM O O O O O B-GENEPROD 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 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

(F) 5-day-old Col-0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=30-32 biological replicates from 2 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001).


( G ) Ethylene production was measured in Col - 0Cas9 WT and CRISPR glv 3 . 5 h after elicitation with 500 nM flg22 or co - treatment with 1 µM GLV2 . Ethylene production was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 11 - 12 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 01 ) .

O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O 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 O O O O O O O O O O O O O O O O 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) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22 or co-treatment with 1 µM GLV2. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=11-12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.01).


( H ) Mean cfu of Pto DC3000 COR - 4 days after spray inoculation of Col - 0Cas9 WT and CRISPR glv ; n = 12 biological replicates from 3 pooled experiments - / + SD ( Students T - Test , * p < 0 . 05 ) .

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

(H) Mean cfu of Pto DC3000 COR- 4 days after spray inoculation of Col-0Cas9 WT and CRISPR glv; n=12 biological replicates from 3 pooled experiments-/+ SD (Students T-Test, *p<0.05).


( B ) ROS burst in WT after elicitation with 10 nM flg22 , 1 μM GLV2 , co - treatment or pre - treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22 . Shown is the mean of total ROS production ( Total RLU ) over 40 minutes normalized to the flg22 response set as 100 % - / + SD , n = 96 - 120 biological replicates from 15 pooled experiments ( one - way ANOVA , Tukey post - hoc test , a - b / c / d , p < 0 . 001 ; b - c , p < 0 . 05 ; b - d , p < 0 .

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

(B) ROS burst in WT after elicitation with 10 nM flg22, 1 μM GLV2, co-treatment or pre-treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22. Shown is the mean of total ROS production (Total RLU) over 40 minutes normalized to the flg22 response set as 100% -/+ SD, n=96-120 biological replicates from 15 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b/c/d, p<0.001; b-c, p<0.05; b-d, p<0.001


( D ) PR1 expression was measured in 10 - day - old WT seedlings treated for 24 h with 100 nM flg22 , 1 μM GLV2 or co - treatment . Expression was normalized to the UBQ house - keeping gene . Shown is the mean of n = 4 . Symbol colors represent different biological

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

(D) PR1 expression was measured in 10-day-old WT seedlings treated for 24 h with 100 nM flg22, 1 μM GLV2 or co-treatment. Expression was normalized to the UBQ house-keeping gene. Shown is the mean of n=4. Symbol colors represent different biological replicates


( B ) Ethylene production was measured in the indicated genotypes 3 . 5 h after elicitation with mock or 500 nM flg22 . Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 18 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 001 ; a - c , p < 0 . 001 ; b - c , p < 0 . 001 ) .

O O O B-SMALL_MOLECULE O O O O O 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 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

(B) Ethylene production was measured in the indicated genotypes 3.5 h after elicitation with mock or 500 nM flg22. Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=18 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.001; b-c, p<0.001).


( E ) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3 : : RGI3 # 2 and # 3 3 . 5 h after elicitation with 500 nM flg22 . Ethylene production was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 8 biological replicates from 2 pooled experiments - / + SD ( one - way ANOVA , Dunnett post - hoc test , a - b p < 0 . 05 ) .

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

(E) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3::RGI3 #2 and #3 3.5 h after elicitation with 500 nM flg22. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=8 biological replicates from 2 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05).


( F ) 5 - day - old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 36 biological replicates from 3 pooled experiments ( Students T - test , * * * p < 0 . 001 ) .

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

(F) 5-day-old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=36 biological replicates from 3 pooled experiments (Students T-test, ***p<0.001).


( C ) 5 - day - old seedlings of WT , rgi5x and rgi5x pRGI3 : : RGI3 # 2 and # 3 were treated with 10 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 32 biological replicates pooled from 4 experiments ( Students T - test , * * p < 0 . 01 ) .

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

(C) 5-day-old seedlings of WT, rgi5x and rgi5x pRGI3::RGI3 #2 and #3 were treated with 10 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=32 biological replicates pooled from 4 experiments (Students T-test, **p<0.01).


( D ) Quantitative analysis of synthetic GLV2 or GLV2 - S peptide binding to RGI3ECD using microscale thermophoresis . Shown is the mean - / + SD , n = 3 technical replicates .

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

(D) Quantitative analysis of synthetic GLV2 or GLV2-S peptide binding to RGI3ECD using microscale thermophoresis. Shown is the mean -/+ SD, n=3 technical replicates.


( E ) Co - immunoprecipitation experiment upon co - expression of BAK1 - HA and RGI3 - GFP in N . benthamiana . IP was performed using GFP - TRAP agarose beads . Western blots for protein detection were probed with α - GFP or α - HA antibodies . Similar results were obtained in 3 independent experiments .

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

(E) Co-immunoprecipitation experiment upon co-expression of BAK1-HA and RGI3-GFP in N. benthamiana. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Similar results were obtained in 3 independent experiments.


( C ) Co - immunoprecipitation experiment upon co - expression of FLS2 - HA , BAK1 - HA and RGI3 - GFP in N . benthamiana . Protein extraction was performed 30 min after mock , 1 μM flg22 or 1 μM GLV2 treatment . IP was performed using GFP - TRAP agarose beads . Western blots for protein detection were probed with α - GFP or α - HA antibodies . Data information : Similar results were obtained in 3 independent experiments .

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

(C) Co-immunoprecipitation experiment upon co-expression of FLS2-HA, BAK1-HA and RGI3-GFP in N. benthamiana. Protein extraction was performed 30 min after mock, 1 μM flg22 or 1 μM GLV2 treatment. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Data information: Similar results were obtained in 3 independent experiments.


( B ) Quantitative real - time PCR of FLS2 transcripts from 12 - day - old seedlings treated with mock or 1 μM GLV2 for the indicated time , n = 3 biological replicates . UBQ was used as a house keeping gene . Students T - test revealed no statistical difference for each of the different timepoints . Data information : Similar results were obtained in 3 independent experiments .

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

(B) Quantitative real-time PCR of FLS2 transcripts from 12-day-old seedlings treated with mock or 1 μM GLV2 for the indicated time, n=3 biological replicates. UBQ was used as a house keeping gene. Students T-test revealed no statistical difference for each of the different timepoints. Data information: Similar results were obtained in 3 independent experiments.


( C ) Protein extraction was performed from 12 - day - old seedlings of the indicated genotypes . Western blot was probed with α - FLS2 antibodies . ( D ) Protein extraction was performed from 12 - day - old seedlings after 1 hour treatment with mock or 50 μM MG132 . Western blots were probed with α - FLS2 antibodies . Data information : Similar results were obtained in 3 independent experiments .

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

(C) Protein extraction was performed from 12-day-old seedlings of the indicated genotypes. Western blot was probed with α-FLS2 antibodies. (D) Protein extraction was performed from 12-day-old seedlings after 1 hour treatment with mock or 50 μM MG132. Western blots were probed with α-FLS2 antibodies. Data information: Similar results were obtained in 3 independent experiments.


C . Phage displaying specific peptides for mATG8 are identified by phage ELISA on immobilized GST - mATG8s and negative control proteins BSA and GST . Binding signal is shown as absorbance at 450 nm in arbitrary units [ AU ] .

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

C. Phage displaying specific peptides for mATG8 are identified by phage ELISA on immobilized GST-mATG8s and negative control proteins BSA and GST. Binding signal is shown as absorbance at 450 nm in arbitrary units [AU].


D . Pull - down of mTurquoise - peptide fusions from cell lysates on GST - mATG8s beads followed by immuno - blotting with α - GFP . As loading control , the protein staining with amido black is shown .

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

D. Pull-down of mTurquoise-peptide fusions from cell lysates on GST-mATG8s beads followed by immuno-blotting with α-GFP. As loading control, the protein staining with amido black is shown.


B , C , E . Annexin A4 based co - translocation assays of peptide binders and EGFP - mATG8s . In comparison to single peptides ( B ) , triplication of peptides ( C ) or introduction of negative charges adjacent to the binding motif ( E ) increases the co - translocation extent of the peptide - sensors with their target mATG8 . Control assays with non - target mATG8 are shown in expanded view figure 2 .

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

B, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2.


D . Biotinylated octameric peptides representing a single amino acid walk - through along the complete 16mer peptide AS _ 67 and AS _ M67 are immobilized on streptavidin . Binding of GST - LC3C fusion - protein to the immobilized peptides is detected by a GST specific antibody ( HRP - conjugate ) and shown as absorbance at 450 nm . AU = arbitrary

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

D. Biotinylated octameric peptides representing a single amino acid walk-through along the complete 16mer peptide AS_67 and AS_M67 are immobilized on streptavidin. Binding of GST-LC3C fusion-protein to the immobilized peptides is detected by a GST specific antibody (HRP-conjugate) and shown as absorbance at 450 nm. AU=arbitrary unit


F . Translocation efficiency of the unmodified peptide AS _ 67 , triplicate AS3 _ 67 and charge optimized peptide AS _ M67 is classified as % of cells that show no translocation ' - ' to very good ' + + + + ' translocation with EGFP - LC3C ; n = total number of analyzed cells . Data are presented as mean of three biological replicates ± SD .

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

F. Translocation efficiency of the unmodified peptide AS_67, triplicate AS3_67 and charge optimized peptide AS_M67 is classified as % of cells that show no translocation '-' to very good '++++' translocation with EGFP-LC3C; n = total number of analyzed cells. Data are presented as mean of three biological replicates ±SD.


A , B . The recruitment of the mCherry fused p62 derived sensor AS3 _ p62 to the autophagosomes positive for overexpressed EGFP - LC3B ( A ) or endogenous LC3B ( B ) visualized by immunofluorescence upon autophagy induction for 3h by KU + Baf treatment .

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

A, B. The recruitment of the mCherry fused p62 derived sensor AS3_p62 to the autophagosomes positive for overexpressed EGFP-LC3B (A) or endogenous LC3B (B) visualized by immunofluorescence upon autophagy induction for 3h by KU + Baf treatment.


C . Recruitment of AS3 _ p62 in Atg5 + / + or Atg5 - / - MEFs to endogenous Lc3b .

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

C. Recruitment of AS3_p62 in Atg5+/+ or Atg5-/- MEFs to endogenous Lc3b.


D . Lack of recruitment of the p62 - derived sensor in the absence of the hydrophobic core residues of the LIR motif ( ΔLIR ) to EGFP - LC3B positive autophagosomes .

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 B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O

D. Lack of recruitment of the p62-derived sensor in the absence of the hydrophobic core residues of the LIR motif (ΔLIR) to EGFP-LC3B positive autophagosomes.


E , F . ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 proteins .

O O O O O O O O O B-SUBCELLULAR O O O O O O O

E, F. ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 proteins.


A - C . Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry - fusion proteins of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored in basal conditions ( A ) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62 .

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

A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored in basal conditions (A) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.


A - C . Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry - fusion proteins of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored upon starvation ( EBSS ) ( B ) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62 .

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

A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored upon starvation (EBSS) (B) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.


A - C . Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry - fusion proteins of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored after autophagy induction by Torin I ( C ) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62 .

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

A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored after autophagy induction by Torin I (C) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.


D . FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA - Parkin , mt - mKeima , and a GFP fusion of the indicated sensor . Cells were treated with OAQ ( oligomycin , antimycin , Q - VD ) for indicated times and analyzed by FACS for lysosomal - positive mt - mKeima ( 561nm ) . The average of three independent experiments is presented . Representative FACS data from experiments ( GFP control and AS3 _ 67 ) are shown in the lower panels . Error bars indicate standard deviation . Significance calculation : 2 - way anova of one - way anova . n . s . = not significant . * * * indicates p - value of 0 . 0001 .

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

D. FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA-Parkin, mt-mKeima, and a GFP fusion of the indicated sensor. Cells were treated with OAQ (oligomycin, antimycin, Q-VD) for indicated times and analyzed by FACS for lysosomal-positive mt-mKeima (561nm). The average of three independent experiments is presented. Representative FACS data from experiments (GFP control and AS3_67) are shown in the lower panels. Error bars indicate standard deviation. Significance calculation: 2-way anova of one-way anova. n.s.= not significant. *** indicates p-value of 0.0001.


A . LC3C specific sensor mCh - AS3 _ 67 is recruited to Salmonella ( SFH2 ) escaping SCV . Salmonella SFH2 strain is carrying a GFP - reporter gene activated by availability of glucose - 6 - phosphate in the host ' s cytosol .

O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SUBCELLULAR O

A. LC3C specific sensor mCh-AS3_67 is recruited to Salmonella (SFH2) escaping SCV. Salmonella SFH2 strain is carrying a GFP-reporter gene activated by availability of glucose-6-phosphate in the host's cytosol.


B . Salmonella with prolonged cytosolic residence and strong activation of the GFP - reporter gene ( green arrow ) are not recognized by the LC3C specific sensor mCh - AS3 _ 67 .

O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

B. Salmonella with prolonged cytosolic residence and strong activation of the GFP-reporter gene (green arrow) are not recognized by the LC3C specific sensor mCh-AS3_67.


C . A subpopulation of hyper proliferative Salmonella in the cytosol is coated with endogenous LC3C . In contrast , endogenous LC3B is enriched in spots in close proximity to the cytosolic bacteria .

O O O O O O O B-ORGANISM O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O

C. A subpopulation of hyper proliferative Salmonella in the cytosol is coated with endogenous LC3C. In contrast, endogenous LC3B is enriched in spots in close proximity to the cytosolic bacteria.


D . Sensitivity of LC3C specific sensor mCh - AS3 _ 67 is increased upon fusion of membrane - tethering FYVE domain or PB1 oligomerization domain .

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

D. Sensitivity of LC3C specific sensor mCh-AS3_67 is increased upon fusion of membrane-tethering FYVE domain or PB1 oligomerization domain.


E . Introduction of the PB1 domain to mCh - AS3 _ p62 increases its performance in recognizing endogenous LC3B .

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

E. Introduction of the PB1 domain to mCh-AS3_p62 increases its performance in recognizing endogenous LC3B.


F . High - performance sensor mCh - PB1 - AS3 _ 67 was transiently expressed in Salmonella infected HeLa cells to monitor xenophagy events .

O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O B-CELL_LINE O O O O O O

F. High-performance sensor mCh-PB1-AS3_67 was transiently expressed in Salmonella infected HeLa cells to monitor xenophagy events.


A , B . LC3C specific sensor mCh - PB1 - AS3 _ 67 is recruited to a subset of mitochondria undergoing mitophagy . Mitochondria were labeled with MitoTracker Deep Red ( ThermoFisher ) . U2OS cells were transiently co - transfected for indicated sensor and mATG8 proteins . Mitophagy was induced by CCCP treatment over three hours . Lysosomal degradation of mitochondria undergoing mitophagy was blocked by the addition of BafilomycinA1 ( Baf ) .

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

A,B. LC3C specific sensor mCh-PB1-AS3_67 is recruited to a subset of mitochondria undergoing mitophagy. Mitochondria were labeled with MitoTracker Deep Red (ThermoFisher). U2OS cells were transiently co-transfected for indicated sensor and mATG8 proteins. Mitophagy was induced by CCCP treatment over three hours. Lysosomal degradation of mitochondria undergoing mitophagy was blocked by the addition of BafilomycinA1 (Baf).


C . Upon depolarization , endogenous LC3C is localized to mitochondria labeled with mt - mCherry in U2OS cells . An example of rare events with nearly complete overlap of signals for endogenous LC3C and mitochondria ( upper panel ) and a representative image ( lower panel ) is given .

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

C. Upon depolarization, endogenous LC3C is localized to mitochondria labeled with mt-mCherry in U2OS cells. An example of rare events with nearly complete overlap of signals for endogenous LC3C and mitochondria (upper panel) and a representative image (lower panel) is given.


D . Overlap of LC3C specific sensor signal with endogenous LC3C and endogenous LAMP1 in U2OS cells induced for mitophagy .

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

D. Overlap of LC3C specific sensor signal with endogenous LC3C and endogenous LAMP1 in U2OS cells induced for mitophagy.


( a ) Expression of ATG16L1 in a set of different tissues as detected by RT - PCR ( IEC , intestinal epithelial cells ) . The corresponding β - actin control ( with a 518 - bp amplicon size ) is given below .

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

(a) Expression of ATG16L1 in a set of different tissues as detected by RT-PCR (IEC, intestinal epithelial cells). The corresponding β-actin control (with a 518-bp amplicon size) is given below.


b ) Amounts of ATG16L1 in colonic mucosal biopsy specimens . Proteins ( 15 μg ) from rectal mucosal biopsies of Crohn disease patients and normal controls ( N ) were separated by denaturing SDS - PAGE , transferred onto polyvinylidine fluoride ( PVDF ) membranes and probed for the presence of ATG16L1 using a specific primary antibody and horseradish peroxidase - coupled secondary antibody . The abundant 68 . 2 - kDa band corresponds to the predicted molecular weight of the isoform encoded by GenBank AY398617 ( UniProt accession number Q676U5 ) . This major protein form was used for three - dimensional modeling of the ATG16L1 protein ( Fig . 2 ) . The 48 . 5 - kDa band corresponds to GenBank EF079890 , which contains exon 9 ( harboring rs2241880 ) in the same reading frame as the major form .

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

b) Amounts of ATG16L1 in colonic mucosal biopsy specimens. Proteins (15 μg) from rectal mucosal biopsies of Crohn disease patients and normal controls (N) were separated by denaturing SDS-PAGE, transferred onto polyvinylidine fluoride (PVDF) membranes and probed for the presence of ATG16L1 using a specific primary antibody and horseradish peroxidase-coupled secondary antibody. The abundant 68.2-kDa band corresponds to the predicted molecular weight of the isoform encoded by GenBank AY398617 (UniProt accession number Q676U5). This major protein form was used for three-dimensional modeling of the ATG16L1 protein (Fig. 2). The 48.5-kDa band corresponds to GenBank EF079890, which contains exon 9 (harboring rs2241880) in the same reading frame as the major form.


( c - i ) Representative immunohistochemistry results ( out of six normal controls and six individuals with Crohn disease ) from colonic mucosal tissue samples from normal controls for genotype AA ( c ) and GG ( f ) and individuals with Crohn disease for genotype AA ( d ) and GG ( g ) are shown to demonstrate the cellular localization of the ATG16L1 protein . Cellular expression patterns in mononuclear cells ( h ) and intestinal epithelial cells ( i ) of a Crohn disease sample ( rs2241880AA ) are shown at higher magnification . e shows a Crohn disease sample without the primary antibody , used as a control . Bar represents 10 μm in c - g .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O 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 B-CELL_TYPE I-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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

(c-i) Representative immunohistochemistry results (out of six normal controls and six individuals with Crohn disease) from colonic mucosal tissue samples from normal controls for genotype AA (c) and GG (f) and individuals with Crohn disease for genotype AA (d) and GG (g) are shown to demonstrate the cellular localization of the ATG16L1 protein. Cellular expression patterns in mononuclear cells (h) and intestinal epithelial cells (i) of a Crohn disease sample (rs2241880AA) are shown at higher magnification. e shows a Crohn disease sample without the primary antibody, used as a control. Bar represents 10 μm in c-g.


The 32 β - strands forming an eight - bladed β - propeller are numbered as in Supplementary Note . The location of the variant amino acid T300A in strand β3 , corresponding to rs2241880 , is marked in yellow .

O O O O O O O O O O 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

The 32 β-strands forming an eight-bladed β-propeller are numbered as in Supplementary Note. The location of the variant amino acid T300A in strand β3, corresponding to rs2241880, is marked in yellow.


( A - H ) Six to eight weeks old female outbred CD - 1 mice were infected via tail vein injection with 5x107 CFU of WT S . aureus Newman or isogenic mutant strains grown in TSB medium to an OD600 of 0 . 5 . Organs were harvested after ( A - D ) four days or ( E - H ) eight days post - infection , and bacterial burdens in the ( A and E ) liver , ( B and F ) heart , ( C and G ) spleen and ( D and H ) kidney were determined . Data represent mean ± SEM ( n = 7 biological replicates ) . . * Denotes p < 0 . 05 , * * denotes p < 0 . 005 and * * * denotes p < 0 . 0007 , using the Mann - Whitney test .

O O O O O O O O O O O O O O O B-ORGANISM O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM I-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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Six to eight weeks old female outbred CD-1 mice were infected via tail vein injection with 5x107 CFU of WT S. aureus Newman or isogenic mutant strains grown in TSB medium to an OD600 of 0.5. Organs were harvested after (A-D) four days or (E-H) eight days post-infection, and bacterial burdens in the (A and E) liver, (B and F) heart, (C and G) spleen and (D and H) kidney were determined. Data represent mean ± SEM (n=7 biological replicates).. * Denotes p < 0.05, ** denotes p < 0.005 and *** denotes p < 0.0007, using the Mann-Whitney test.


Cells were grown in MHB - ca medium without or with 20 mM glucose as indicated . ( A ) Bacterial growth was measured by optical density at 600 nm . Data are representative of five independent experiments .

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

Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (A) Bacterial growth was measured by optical density at 600 nm. Data are representative of five independent experiments.


Cells were grown in MHB - ca medium without or with 20 mM glucose as indicated . ( B ) PndhC and PndhF YFP reporter constructs were used for following gene expression and data was normalized by growth . Data are expressed as average ± SEM of three independent experiments . * * * Denotes p < 0 . 001 compared to ndhC expression in WT strain grown in MHB - ca medium via two - way ANOVA with Tukey ' s posttest .

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

Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (B) PndhC and PndhF YFP reporter constructs were used for following gene expression and data was normalized by growth. Data are expressed as average ± SEM of three independent experiments. *** Denotes p < 0.001 compared to ndhC expression in WT strain grown in MHB-ca medium via two-way ANOVA with Tukey's posttest.


( A ) Oxygen consumption of WT and isogenic mutant strains was measured with Clark oxygen electrode . Cells were grown in MHB - ca medium until exponential phase ( OD600 = 0 . 6 ) or until late stationary phase ( 24 h ) . Cells were washed with PBS and respiration initiated by the addition of 20 mM glycerol . Data are expressed as average ± SD of four independent experiments . * * * denotes p < 0 . 001 compared to WT via two - way ANOVA with Tukey ' s posttest .

O O O B-EXP_ASSAY I-EXP_ASSAY 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 O O O O 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 O O O O O O O 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) Oxygen consumption of WT and isogenic mutant strains was measured with Clark oxygen electrode. Cells were grown in MHB-ca medium until exponential phase (OD600 = 0.6) or until late stationary phase (24 h). Cells were washed with PBS and respiration initiated by the addition of 20 mM glycerol. Data are expressed as average ± SD of four independent experiments. *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest.


( B ) NADH / NAD + ratios were measured in cells grown aerobically in MHB - ca medium for 20 h were measured . 100 % corresponds to a NADH / NAD + ratio of 0 . 003 . Data are expressed as average ± SD of four independent experiments . * Denotes p < 0 . 05 , and * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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

(B) NADH/NAD+ ratios were measured in cells grown aerobically in MHB-ca medium for 20 h were measured. 100% corresponds to a NADH/NAD+ ratio of 0.003. Data are expressed as average ± SD of four independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


( C ) Acetate concentration was monitored in supernatants of WT and isogenic ndh strains at 7 and 24 h of growth in MHB - ca medium . Data are expressed as average ± SD of three independent experiments . * * Denotes p < 0 . 005 and * * * denotes p < 0 . 001 compared to WT via two - way ANOVA with Tukey ' s posttest .

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

(C) Acetate concentration was monitored in supernatants of WT and isogenic ndh strains at 7 and 24 h of growth in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest.


( D ) MKH2 / MK ratios were measured in cells grown aerobically for 20 h in MHB - ca medium . Data are expressed as average ± SD of three independent experiments . * Denotes p < 0 . 05 compared to WT via one - way ANOVA with Dunnett ' s posttest .

O O O B-SMALL_MOLECULE 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

(D) MKH2/MK ratios were measured in cells grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.


( A ) The carotenoid pigment of different S . aureus strains was extracted by methanol and quantified by the optical density at 465 nm , and the values were normalized to WT Newman strain , which was set at 100 . Data are expressed as the average ± SD of five independent experiments . * * Denotes p < 0 . 005 and * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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

(A) The carotenoid pigment of different S. aureus strains was extracted by methanol and quantified by the optical density at 465 nm, and the values were normalized to WT Newman strain, which was set at 100. Data are expressed as the average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


( B ) Hydrogen peroxide sensitivity was measured by incubating WT and isogenic strains , previously grown for 20 h in MHB - ca , with different concentrations of hydrogen peroxide . Serial dilutions were plated in TSB medium for measuring CFU . Data are expressed as the average ± SD of three independent experiments .

O O O B-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 B-SMALL_MOLECULE I-SMALL_MOLECULE 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

(B) Hydrogen peroxide sensitivity was measured by incubating WT and isogenic strains, previously grown for 20 h in MHB-ca, with different concentrations of hydrogen peroxide. Serial dilutions were plated in TSB medium for measuring CFU. Data are expressed as the average ± SD of three independent experiments.


( A ) PndhC and PndhF YFP reporter constructs were used to follow gene expression under the biofilm growth conditions . Data collected after 24 h of static incubation was normalized by optical density at 600 nm . Data are expressed as average ± SEM of three independent experiments . No significant differences were found when ndhC expression in WT strain was compared via one - way ANOVA with Dunnett ' s posttest .

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

(A) PndhC and PndhF YFP reporter constructs were used to follow gene expression under the biofilm growth conditions. Data collected after 24 h of static incubation was normalized by optical density at 600 nm. Data are expressed as average ± SEM of three independent experiments. No significant differences were found when ndhC expression in WT strain was compared via one-way ANOVA with Dunnett's posttest.


( B ) Biofilm formation of Newman WT and ndh deficient mutants after static incubation at 37 ° C for 20 h . The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose and 2 % NaCl . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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

(B) Biofilm formation of Newman WT and ndh deficient mutants after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


( C ) MKH2 / MK ratios were measured in cells grown under the same conditions as for biofilm formation . Data are expressed as average ± SD of three independent experiments . * Denotes p < 0 . 05 compared to WT via one - way ANOVA with Dunnett ' s posttest .

O O O B-SMALL_MOLECULE 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

(C) MKH2/MK ratios were measured in cells grown under the same conditions as for biofilm formation. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.


( D ) NADH / NAD + ratios were measured in cells grown under the same conditions as for biofilm formation . 100 % corresponds to a NADH / NAD + ratio of 0 . 09 . Data are expressed as average ± SD of four independent experiments performed by duplicates . * Denotes p < 0 . 05 , and * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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

(D) NADH/NAD+ ratios were measured in cells grown under the same conditions as for biofilm formation. 100% corresponds to a NADH/NAD+ ratio of 0.09. Data are expressed as average ± SD of four independent experiments performed by duplicates. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB - ca medium without and with 25 mM glucose , 40 mM sodium pyruvate or 60 mM sodium acetate . Lysis was monitored by the release of hemoglobin . Data are expressed as average ± SD of five independent experiments . * Denotes p < 0 . 05 , and * * * denotes p < 0 . 001 , ns denotes no significant compared to WT grown in MHB - ca and WT grown in MHB - ca + glucose via two - way ANOVA with Tukey ' s posttest .

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

Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium without and with 25 mM glucose, 40 mM sodium pyruvate or 60 mM sodium acetate. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of five independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001, ns denotes no significant compared to WT grown in MHB-ca and WT grown in MHB-ca + glucose via two-way ANOVA with Tukey's posttest.


( A ) Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB - ca medium . Lysis was monitored by the release of hemoglobin . Data are expressed as average ± SD of four independent experiments . * * * Denotes p < 0 . 001 , ns denotes no significant compared to WT via one - way ANOVA with Dunnett ' s posttest .

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

(A) Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of four independent experiments. *** Denotes p < 0.001, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.


( B ) Biofilm formation of Newman WT and isogenic mutant strains after static incubation at 37 ° C for 20 h . The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose and 2 % NaCl . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * Denotes p < 0 . 005 , ns denotes no significant compared to WT via one - way ANOVA with Dunnett ' s posttest .

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

(B) Biofilm formation of Newman WT and isogenic mutant strains after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. ** Denotes p < 0.005, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.


( C ) Hemolytic activity of the Newman WT and isogenic mutant strains grown aerobically for 20 h in MHB - ca medium , in the absence or presence of 10 mg / ml FA - deficient BSA and 0 . 01 % oleic acid . Data are expressed as average ± SD of five independent experiments . * * Denotes p < 0 . 005 and * * * denotes p < 0 . 001 compared to each strain without any BSA or oleic acid addition via two - way ANOVA with Tukey ' s posttest .

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

(C) Hemolytic activity of the Newman WT and isogenic mutant strains grown aerobically for 20 h in MHB-ca medium, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.


( D ) Biofilm formation of Newman WT and isogenic mutant strains after static incubation for 20 h in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose and 2 % NaCl , in the absence or presence of 10 mg / ml FA - deficient BSA and 0 . 01 % oleic acid . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to each strain without any BSA or oleic acid addition via two - way ANOVA with Tukey ' s posttest .

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 B-ORGANISM B-TISSUE O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O B-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 B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(D) Biofilm formation of Newman WT and isogenic mutant strains after static incubation for 20 h in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.


( E ) Hemolytic activity of Newman ∆ ndhC strain overexpressing the N . gonorrhea acyl - ACP synthetase and grown aerobically for 20 h in MHB - ca medium . Data are expressed as average ± SD of four independent experiments . * * Denotes p < 0 . 005 compared to ∆ ndhC strain with the empty plasmid via Student ' s t - test .

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

(E) Hemolytic activity of Newman ∆ndhC strain overexpressing the N. gonorrhea acyl-ACP synthetase and grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of four independent experiments. ** Denotes p < 0.005 compared to ∆ndhC strain with the empty plasmid via Student's t-test.


( F ) Biofilm formation of Newman ∆ ndhC strain overexpressing the N . gonorrhea acyl - ACP synthetase and grown under biofilm producing conditions . Data are expressed as average ± SD of three independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to ∆ ndhC strain with the empty plasmid via Student ' s t - test .

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

(F) Biofilm formation of Newman ∆ndhC strain overexpressing the N. gonorrhea acyl-ACP synthetase and grown under biofilm producing conditions. Data are expressed as average ± SD of three independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to ∆ndhC strain with the empty plasmid via Student's t-test.


Immunoblot analysis of phosphorylated ERK1 / 2 ( P - ERK1 / 2 ) , phosphorylated p38 ( P - P38 ) , phosphorylated JNK1 / 2 ( P - JNK1 / 2 ) , phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , MEKK2 , TRAF2 , TRAF3 , TRAF6 , NEDD4L , and actin in HeLa cells transfected with Nedd4l siRNA ( siNedd4l ) or control RNA ( siNC ) ( A ) , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown .

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

Immunoblot analysis of phosphorylated ERK1/2 (P-ERK1/2), phosphorylated p38 (P-P38), phosphorylated JNK1/2 (P-JNK1/2), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, TRAF2, TRAF3, TRAF6, NEDD4L, and actin in HeLa cells transfected with Nedd4l siRNA (siNedd4l) or control RNA (siNC) (A) , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.


Immunoblot analysis of phosphorylated ERK1 / 2 ( P - ERK1 / 2 ) , phosphorylated p38 ( P - P38 ) , phosphorylated JNK1 / 2 ( P - JNK1 / 2 ) , phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , MEKK2 , TRAF2 , TRAF3 , TRAF6 , NEDD4L , and actin in Nedd4l + / + and Nedd4l - / - MEFs , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown .

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

Immunoblot analysis of phosphorylated ERK1/2 (P-ERK1/2), phosphorylated p38 (P-P38), phosphorylated JNK1/2 (P-JNK1/2), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, TRAF2, TRAF3, TRAF6, NEDD4L, and actin in Nedd4l+/+ and Nedd4l-/- MEFs , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.


Immunoblot analysis of phosphorylated p38 ( P - P38 ) , phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , MEKK2 , NEDD4L , and actin in Nedd4l + / + and Nedd4l - / - primary lung epithelial cells ( C ) , or Nedd4l + / + and Nedd4l + / - astrocytes ( D ) , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown .

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

Immunoblot analysis of phosphorylated p38 (P-P38), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, NEDD4L, and actin in Nedd4l+/+ and Nedd4l-/- primary lung epithelial cells (C), or Nedd4l+/+ and Nedd4l+/- astrocytes (D) , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.


( E ) Immunoblot analysis of phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , phosphorylated p38 ( P - P38 ) , total P38 , MEKK2 , NEDD4L , and actin in HeLa cells transfected with Myc - tagged NEDD4L ， Myc - tagged NEDD4L - CA ( C942A ) , or Myc - tagged Null plasmids , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown .

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

(E) Immunoblot analysis of phosphorylated p65 subunit of NF-κB (P-P65), total P65, phosphorylated p38 (P-P38), total P38, MEKK2, NEDD4L, and actin in HeLa cells transfected with Myc-tagged NEDD4L，Myc- tagged NEDD4L-CA(C942A), or Myc- tagged Null plasmids, treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.


( F ) Immunoblot analysis of MEKK2 , TRAF2 , TRAF6 , NEDD4L , and actin in Nedd4l + / + , Nedd4l + / - , and Nedd4l - / - MEFs . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown .

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

(F)Immunoblot analysis of MEKK2, TRAF2, TRAF6, NEDD4L, and actin in Nedd4l+/+, Nedd4l+/-, and Nedd4l-/- MEFs. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.


( G ) Immunoblot analysis of MEKK2 in lysates of Nedd4l + / + and Nedd4l - / - MEFs treated with CHX ( 50 μg / ml ) for indicated time . ( H ) The intensity values were measured by Image J software to calculate half - life of MEKK protein ( n = 3 biological replicates ) . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown . Significant differences were tested using a two - tailed Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 . NS , no significance .

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

(G)Immunoblot analysis of MEKK2 in lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with CHX (50 μg/ ml) for indicated time. (H)The intensity values were measured by Image J software to calculate half-life of MEKK protein ( n = 3 biological replicates). Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown. Significant differences were tested using a two-tailed Student's t-test. *P<0.05, **P<0.01. NS, no significance.


( A ) Immunoblot analysis of NEDD4L , MEKK2 , and MEKK3 immunoprecipitated with NEDD4L - specific antibody from lysates of HeLa cells pre - treated with 20μM MG - 132 for 6 h and then treated with IL - 17 for the indicated time . Data information : All experiments were performed three times , and representative data are shown .

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

(A)Immunoblot analysis of NEDD4L, MEKK2, and MEKK3 immunoprecipitated with NEDD4L-specific antibody from lysates of HeLa cells pre-treated with 20μM MG-132 for 6 h and then treated with IL-17 for the indicated time. Data information: All experiments were performed three times, and representative data are shown.


( B ) Immunoblot analysis of NEDD4L precipitated with GST - tagged Null or GST - tagged MEKK2 recombinant protein . Data information : All experiments were performed three times , and representative data are shown .

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

(B)Immunoblot analysis of NEDD4L precipitated with GST-tagged Null or GST-tagged MEKK2 recombinant protein. Data information: All experiments were performed three times, and representative data are shown.


( C ) Immunoblot of HA - tagged MEKK2 co - immunoprecipitated with anti - Myc antibody from lysates of HEK293T cells co - transfected with plasmids expressing HA - tagged MEKK2 and Myc - tagged wild - type NEDD4L or mutant NEDD4L with C2 ( ∆ C2 ) , WW ( ∆ WW ) or HECT ( ∆ HECT ) domain deleted . Data information : All experiments were performed three times , and representative data are shown .

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

(C)Immunoblot of HA-tagged MEKK2 co-immunoprecipitated with anti-Myc antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted. Data information: All experiments were performed three times, and representative data are shown.


( D ) Immunoblot analysis of MEKK2 and NEDD4L in Nedd4l + / + , and Neddl4 - / - treated with DMSO , 20μM MG - 132 , or 0 . 2 μM bafilomycin A1 ( Baf A1 ) for 6 h . Densitometry of MEKK2 was relative to actin . Data information : All experiments were performed three times , and representative data are shown .

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

(D)Immunoblot analysis of MEKK2 and NEDD4L in Nedd4l+/+, and Neddl4- /- treated with DMSO, 20μM MG-132, or 0.2 μM bafilomycin A1 (Baf A1) for 6 h. Densitometry of MEKK2 was relative to actin. Data information: All experiments were performed three times, and representative data are shown.


( E ) Immunoblot analysis of MEKK2 incubated with NEDD4L or TRAF6 recombinant protein and the full complement of ubiquitination reaction components in vitro . Data information : All experiments were performed three times , and representative data are shown .

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

(E)Immunoblot analysis of MEKK2 incubated with NEDD4L or TRAF6 recombinant protein and the full complement of ubiquitination reaction components in vitro. Data information: All experiments were performed three times, and representative data are shown.


( F ) Immunoblot analysis of K48 - , K63 - linked , and total ubiquitination of MEKK2 following precipitation of MEKK2 with MEKK2 - specific antibody from lysates of Nedd4l + / + and Nedd4l - / - MEFs treated with MG - 132 . Data information : All experiments were performed three times , and representative data are shown .

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

(F)Immunoblot analysis of K48-, K63-linked, and total ubiquitination of MEKK2 following precipitation of MEKK2 with MEKK2-specific antibody from lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with MG-132. Data information: All experiments were performed three times, and representative data are shown.


( G ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following precipitation of MEKK2 with Flag - tagged specific antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 and Myc - tagged wild - type NEDD4L or mutant NEDD4L with C2 ( ∆ C2 ) , WW ( ∆ WW ) or HECT ( ∆ HECT ) domain deleted . Data information : All experiments were performed three times , and representative data are shown .

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

(G)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following precipitation of MEKK2 with Flag-tagged specific antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted. Data information: All experiments were performed three times, and representative data are shown.


( H ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , Myc - tagged NEDD4L , and HA - tagged wild - type or K6O , K11O , K27O , K29O , K33O , K48O , K63O mutant Ub . ( I ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , Myc - tagged NEDD4L , and HA - tagged wild - type or K6R , K11R , K27R , K29R , K33R , K48R , K63R mutant Ub . Data information : All experiments were performed three times , and representative data are shown .

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

(H)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6O, K11O, K27O, K29O, K33O, K48O, K63O mutant Ub. (I)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6R, K11R, K27R, K29R, K33R, K48R, K63R mutant Ub. Data information: All experiments were performed three times, and representative data are shown.


( J ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , increased dosage of Myc - tagged NEDD4L , and HA - tagged wild - type , K27O , or K63O mutant Ub . Data information : All experiments were performed three times , and representative data are shown .

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

(J)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, increased dosage of Myc-tagged NEDD4L, and HA-tagged wild-type, K27O, or K63O mutant Ub. Data information: All experiments were performed three times, and representative data are shown.


( A , Immunoblot analysis of phosphorylated p38 ( p - p38 ) , phosphorylated NF - κB p65 ( P - P65 ) , MEKK2 and actin in HeLa cells ( A ) transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 ( HeLa cells 50 ng / ml , for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown .

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

(A, Immunoblot analysis of phosphorylated p38 (p-p38), phosphorylated NF-κB p65 (P-P65), MEKK2 and actin in HeLa cells (A) transfected with Mekk2 siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 (HeLa cells 50 ng/ml, for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.


B ) Immunoblot analysis of phosphorylated p38 ( p - p38 ) , phosphorylated NF - κB p65 ( P - P65 ) , MEKK2 and actin in MEFs ( B ) transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 MEFs 100ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown .

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

B) Immunoblot analysis of phosphorylated p38 (p-p38), phosphorylated NF-κB p65 (P-P65), MEKK2 and actin in MEFs (B) transfected with Mekk2 siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 MEFs 100ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.


( C , Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells ( C ) transfected with siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 ( HeLa cells 50 ng / ml , for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (C) transfected with siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 (HeLa cells 50 ng/ml, for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


D ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in MEFs ( D ) transfected with siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 MEFs 100ng / ml ) for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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

D) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in MEFs (D) transfected with siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 MEFs 100ng/ml) for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


( E ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( P - P65 ) , NEDD4L , MEKK2 and actin in HeLa cells transfected with control RNA ( siNC ) , Nedd4l siRNA ( siNedd4l ) , Mekk2 siRNA ( siMekk2 ) or Nedd4l siRNA plus Mekk2 siRNA ( siNedd4l + siMekk2 ) , and stimulated with IL - 17 ( 50 ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown .

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

(E) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (P-P65), NEDD4L, MEKK2 and actin in HeLa cells transfected with control RNA (siNC), Nedd4l siRNA (siNedd4l), Mekk2 siRNA(siMekk2) or Nedd4l siRNA plus Mekk2 siRNA (siNedd4l+ siMekk2), and stimulated with IL-17(50 ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.


( F ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( p - p65 ) , NEDD4L , MEKK2 and actin in Nedd4l + / + and Nedd4l - / - MEFs transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNc ) , and stimulated with IL - 17 ( 100 ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown .

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

(F) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (p-p65), NEDD4L, MEKK2 and actin in Nedd4l+/+ and Nedd4l-/-MEFs transfected with Mekk2 siRNA (siMekk2) or control RNA (siNc), and stimulated with IL-17 (100 ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.


( G , Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells ( G ) treated as described in ( E ) ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (G) treated as described in (E) (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


H ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in MEFs ( H ) treated as described in ( F ) ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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

H) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in MEFs (H) treated as described in (F) (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


( A ) Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells stimulated with IL - 17 ( 50 ng / ml ) for indicated time , and then incubated with or without calf intestinal alkaline phosphatasetime ( CIAP ) for 30 mins . Data information : All experiments were performed two times , and representative data are shown .

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

(A)Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells stimulated with IL-17 (50 ng/ml) for indicated time, and then incubated with or without calf intestinal alkaline phosphatasetime (CIAP) for 30 mins. Data information: All experiments were performed two times, and representative data are shown.


( B ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells transfected with HA - tagged MEKK2 S520A or control vector , and stimulated with IL - 17 ( 50 ng / ml ) for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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

(B) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells transfected with HA-tagged MEKK2 S520A or control vector, and stimulated with IL-17 (50 ng/ml) for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.