( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O 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 O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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-MEASURED_VAR O O O O 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 .

O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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-MEASURED_VAR O O O O 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 .

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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-MEASURED_VAR O O O O 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 O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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-MEASURED_VAR O O O O 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 O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O 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-MEASURED_VAR O O O O 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 O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR 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 O O O O O B-MEASURED_VAR O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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(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 .

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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 .

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(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 .

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(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 .

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(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 .

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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 .

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(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 .

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(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.


( C ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( P - P65 ) , HA , and actin in HeLa cells treated as in ( B ) . Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Data information : All experiments were performed two times , and representative data are shown .

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(C) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (P-P65), HA, and actin in HeLa cells treated as in (B). Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Data information: All experiments were performed two times, and representative data are shown.


( D ) Immunoblot analysis of MEKK2 and TAK1 co - immunoprecipitated with anti - TAK1 antibody from lysates of HeLa cells treated with 50ng / ml IL - 17 for indicated time . Data information : All experiments were performed two times , and representative data are shown .

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(D)Immunoblot analysis of MEKK2 and TAK1 co-immunoprecipitated with anti-TAK1 antibody from lysates of HeLa cells treated with 50ng/ml IL-17 for indicated time. Data information: All experiments were performed two times, and representative data are shown.


( E ) Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells transfected with Tak1 - specific ( siTak1 ) or control RNA ( siNC ) , and then stimulated with IL - 17 ( 50 ng / ml ) for indicated time . Data information : All experiments were performed two times , and representative data are shown .

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(E)Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells transfected with Tak1-specific (siTak1) or control RNA (siNC), and then stimulated with IL-17 (50 ng/ml) for indicated time. Data information: All experiments were performed two times, and representative data are shown.


( F ) Immunoblot analysis of Myc - tagged NEDD4L co - immunoprecipitated with anti - HA antibody from lysates of HEK293T cells co - transfected with plasmids expressing Myc - tagged NEDD4L and HA - tagged wild - type MEKK2 or mutant MEKK2 ( S520A ) . Data information : All experiments were performed two times , and representative data are shown .

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(F)Immunoblot analysis of Myc-tagged NEDD4L co-immunoprecipitated with anti-HA antibody from lysates of HEK293T cells co-transfected with plasmids expressing Myc-tagged NEDD4L and HA-tagged wild-type MEKK2 or mutant MEKK2 (S520A). Data information: All experiments were performed two times, and representative data are shown.


( G ) Immunoblot analysis of total ubiquitination of HA - tagged MEKK2 and HA - tagged MEKK2 ( S520A ) following precipitation of MEKK2 with anti - HA antibody from lysates of HEK293T cells co - transfected with plasmids expressing HA - tagged MEKK2 or HA - tagged MEKK2 ( S520A ) together with Myc - tagged NEDD4L and Flag - tagged Ub . Data information : All experiments were performed two times , and representative data are shown .

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(G)Immunoblot analysis of total ubiquitination of HA-tagged MEKK2 and HA-tagged MEKK2(S520A) following precipitation of MEKK2 with anti-HA antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 or HA-tagged MEKK2(S520A) together with Myc-tagged NEDD4L and Flag-tagged Ub. Data information: All experiments were performed two times, and representative data are shown.


( H ) Immunoblot analysis of HA - tagged MEKK2 , HA - tagged MEKK2 ( S520A ) , Myc - tagged NEDD4L , and actin in HEK293T cells transfected with the indicated plasmids with anti - HA , anti - Myc , or anti - actin antibody . Data information : All experiments were performed two times , and representative data are shown .

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(H)Immunoblot analysis of HA-tagged MEKK2, HA-tagged MEKK2 (S520A), Myc-tagged NEDD4L, and actin in HEK293T cells transfected with the indicated plasmids with anti-HA, anti-Myc, or anti-actin antibody. Data information: All experiments were performed two times, and representative data are shown.


FACS gating strategy representative of 5 independent experiments adopted to sort CD11b + CD45int microglia distinctly from CD11b + CD45high resident macrophages and CD11b - CD45high lymphocytes .

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FACS gating strategy representative of 5 independent experiments adopted to sort CD11b+CD45int microglia distinctly from CD11b+CD45high resident macrophages and CD11b-CD45high lymphocytes.


Analysis of relative transcript levels of CD11b + CD45int FACS - sorted microglia compared with whole brain tissue by qPCR . Gene expression levels of microglia ( Olfml3 , Fcrls , Tmem119 , Siglech , Gpr34 , P2ry12 ) , astrocyte ( Gfap , Gjb6 , Ntsr2 , Aldh1l1 ) , oligodendrocyte ( Mobp , Mog , Cldn1 ) , and neuron ( Tubb3 , Vglut1 , NeuN ) markers . Bars represent mean ( n = 4 ; pool of 1 female and 1 male per biological replicate ) of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * p < 0 . 05 ; * * p < 0 . 01 by two - tailed Student ' s test ) . N . D . not detected .

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Analysis of relative transcript levels of CD11b+CD45int FACS-sorted microglia compared with whole brain tissue by qPCR. Gene expression levels of microglia (Olfml3, Fcrls, Tmem119, Siglech, Gpr34, P2ry12), astrocyte (Gfap, Gjb6, Ntsr2, Aldh1l1), oligodendrocyte (Mobp, Mog, Cldn1), and neuron (Tubb3, Vglut1, NeuN) markers. Bars represent mean (n=4; pool of 1 female and 1 male per biological replicate) of relative expression (Gapdh as housekeeping gene) ± SEM (*p < 0.05; **p < 0.01 by two-tailed Student's test). N.D. not detected.


Representative quantification of CD206 expression in CD11b + CD45int microglia and CD11b + CD45high resident macrophages . Values denote the percentage of the mean ± SEM of 5 independent experiments .

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Representative quantification of CD206 expression in CD11b+CD45int microglia and CD11b+CD45high resident macrophages. Values denote the percentage of the mean ± SEM of 5 independent experiments.


Representative images of 2 independent experiments showing microglia , resident macrophages and lymphocytes acquired with ImageStream imaging cytometer ( Amnis ) based on CD45 , CD11b and CD206 expression levels ( scale bar represents 7 µm ) .

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Representative images of 2 independent experiments showing microglia, resident macrophages and lymphocytes acquired with ImageStream imaging cytometer (Amnis) based on CD45, CD11b and CD206 expression levels (scale bar represents 7 µm).


Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Gene expression levels of microglia homeostatic ( Olfml3 , Fcrls , Tmem119 , Siglech , Gpr34 , P2ry12 , Mef2c ) , phagocytic ( Tyrobp , Trem2 ) , and inflammatory ( Il1β , Tnf , Ccl2 , Mrc1 , Arg1 ) markers were analyzed by qPCR . Bars represent mean of relative expression ( % of saline ; Gapdh as housekeeping gene ) ± SEM ( * p < 0 . 05 ; * * p < 0 . 01 by two - tailed Student ' s test ; n = 4 ) .

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Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of microglia homeostatic (Olfml3, Fcrls, Tmem119, Siglech, Gpr34, P2ry12, Mef2c), phagocytic (Tyrobp, Trem2), and inflammatory (Il1β, Tnf, Ccl2, Mrc1, Arg1) markers were analyzed by qPCR. Bars represent mean of relative expression (% of saline; Gapdh as housekeeping gene) ± SEM (*p < 0.05; **p < 0.01 by two-tailed Student's test; n=4).


Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS - injected mouse brains .

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Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS-injected mouse brains.


Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Representative multicolour flow cytometry analysis showing the percentage of the mean ± SEM of 5 independent experiments of Ly6C and CD206 expressing cells in CD11b + CD45int cells from saline or LPS - injected mice .

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Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis showing the percentage of the mean ± SEM of 5 independent experiments of Ly6C and CD206 expressing cells in CD11b+CD45int cells from saline or LPS-injected mice.


Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Gene expression levels of the monocytic markers Ly6c1 and Ccr2 in purified microglia ( n = 4 ) and isolated bone marrow monocytes ( n = 2 ) by qPCR . Bars represent mean of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * * p < 0 . 01 by two - tailed Student ' s test ) .

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Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of the monocytic markers Ly6c1 and Ccr2 in purified microglia (n=4) and isolated bone marrow monocytes (n=2) by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test).


Primary adult microglia were cultivated in the presence of TGF - β ( 50 μg / ml ) and M - CSF ( 10 ng / ml ) , while neonatal cells were stimulated for 24 hours with TGF - β ( 50 μg / ml ) followed by 6 hours stimulation with LPS ( 1 ng / mL ) or left untreated . Expression levels of microglia homeostatic ( Olfml3 , Tmem119 , Gpr34 ) and inflammatory ( Il1β , Tnf , Ccl2 ) genes were analysed by qPCR . Bars represent mean of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * * p < 0 . 01 by two - tailed Student ' s test ; n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary adult microglia were cultivated in the presence of TGF-β (50 μg/ml) and M-CSF (10 ng/ml), while neonatal cells were stimulated for 24 hours with TGF-β (50 μg/ml) followed by 6 hours stimulation with LPS (1 ng/mL) or left untreated. Expression levels of microglia homeostatic (Olfml3, Tmem119, Gpr34) and inflammatory (Il1β, Tnf, Ccl2) genes were analysed by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test; n=3).


Heatmap showing clustering analysis of 1 , 247 single cells , featuring 100 most variable genes ( FDR < 0 . 05 ) . Single - cell RNA - seq results are obtained from 2 mice per group ( 1 female and 1 male each ) .

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Heatmap showing clustering analysis of 1,247 single cells, featuring 100 most variable genes (FDR<0.05). Single-cell RNA-seq results are obtained from 2 mice per group (1 female and 1 male each).


Expression of specific homeostatic ( Tmem119 , P2ry12 , Siglech ) and inflammatory ( Ccl2 , Gpr84 ) genes overlaid on the 2D - tSNE space . Bars represent log2 ( Count + 1 ) .

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Expression of specific homeostatic (Tmem119, P2ry12, Siglech) and inflammatory (Ccl2, Gpr84) genes overlaid on the 2D-tSNE space. Bars represent log2 (Count+1).


Representative multicolour flow cytometry analysis of 2 independent experiments showing TMEM119 and P2RY12 expression levels in CD11b + CD45int microglia of saline or LPS - injected mouse brains . For the unconjugated TMEM119 antibody , negative stands for primary antibody without secondary antibody . For P2RY12 antibody , negative represents isotype PE control .

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Representative multicolour flow cytometry analysis of 2 independent experiments showing TMEM119 and P2RY12 expression levels in CD11b+CD45int microglia of saline or LPS-injected mouse brains. For the unconjugated TMEM119 antibody, negative stands for primary antibody without secondary antibody. For P2RY12 antibody, negative represents isotype PE control.


Heatmap showing examples of specific genes mainly up - regulated in " main LPS " ( Manf ) or " subset LPS " ( Ash1l ) and down - regulated in " main LPS " ( Mef2c ) or " subset LPS " ( Lamp1 ) overlaid on the 2D - tSNE space . Bars represent log2 ( Count + 1 ) .

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Heatmap showing examples of specific genes mainly up-regulated in "main LPS" (Manf) or "subset LPS" (Ash1l) and down-regulated in "main LPS" (Mef2c) or "subset LPS" (Lamp1) overlaid on the 2D-tSNE space. Bars represent log2 (Count+1).


Pseudotime dynamics of inflammatory ( Ccl12 , Ccl2 , Gpr84 ) and homeostatic ( Mef2c , P2ry12 , Siglech ) genes in dependence on inferred cell states .

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Pseudotime dynamics of inflammatory (Ccl12, Ccl2, Gpr84) and homeostatic (Mef2c, P2ry12, Siglech) genes in dependence on inferred cell states.


( A ) HeLa cells stably expressing GFP - Parkin ( HeLa - GFP - Parkin cells ) were treated with 1 µM valinomycin and immunostained with anti - NDP52 and anti - Tom20 antibodies . Boxed areas in the images are shown in the next panels on the right . Colocalization of GFP - Parkin , NDP52 , and Tom20 were determined using Line scan . Fluorescence intensities of each channels were measured along the dotted white arrow . Scale bars , 10 µm . Images are representative of three independent

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(A) HeLa cells stably expressing GFP-Parkin (HeLa-GFP-Parkin cells) were treated with 1 µM valinomycin and immunostained with anti-NDP52 and anti-Tom20 antibodies. Boxed areas in the images are shown in the next panels on the right. Colocalization of GFP-Parkin, NDP52, and Tom20 were determined using Line scan. Fluorescence intensities of each channels were measured along the dotted white arrow. Scale bars, 10 µm. Images are representative of three independent experiments


( B ) HeLa - GFP - Parkin cells were transfected with siNDP52 or scrambled siRNA . Seventy - two hours after transfection , cells were incubated with 1 µM valinomycin for the indicated times . Western blotting was performed using the indicated antibodies . Data are representative of three independent experiments

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(B) HeLa-GFP-Parkin cells were transfected with siNDP52 or scrambled siRNA. Seventy-two hours after transfection, cells were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments


( C ) Mitophagy flux assay using Mtphagy dye . Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment . Results are a summary of three independent experiments . Values are the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 compared with scrambled oligo - treated cells , determined with one - way ANOVA followed by the Student ' s t test

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(C) Mitophagy flux assay using Mtphagy dye. Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment. Results are a summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test


( D ) Fluorescence images of NDP52 KD and control cells expressing mCherry - LC3 and MTS - TagBFP . Arrows indicate mCherry - LC3 puncta colocalized with MTS - TagBFP . Scale bars , 10 µm . Boxed areas in images are shown in the next panels on the right . Images are representative of four independent experiments

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(D) Fluorescence images of NDP52 KD and control cells expressing mCherry-LC3 and MTS-TagBFP. Arrows indicate mCherry-LC3 puncta colocalized with MTS-TagBFP. Scale bars, 10 µm. Boxed areas in images are shown in the next panels on the right. Images are representative of four independent experiments


( E ) The number of mCherry - LC3 puncta colocalized with MTS - TagBFP ( representing mitophagosomes ) per cell was counted . Results were from at least six microscopic fields of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 compared with scrambled oligo - treated cells , determined with one - way ANOVA followed by the Student ' s t test

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(E) The number of mCherry-LC3 puncta colocalized with MTS-TagBFP (representing mitophagosomes) per cell was counted. Results were from at least six microscopic fields of three independent experiments. Values are the means ± SEM. **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test


( F , G ) Electron micrographs of NDP52 KD and control cells . Asterisks indicate mitochondria sequestered by autophagosomes ( mitophagosomes , yellow dotted line ) . The ratios of mitophagosomes per mitochondrial area are shown in ( G ) . Ten cells from three independent experiments were counted . Values are the means ± SD . * p < 0 . 05 compared with scrambled - oligo treated cells , determined with one - way ANOVA followed by the Student ' s t

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(F, G) Electron micrographs of NDP52 KD and control cells. Asterisks indicate mitochondria sequestered by autophagosomes (mitophagosomes, yellow dotted line). The ratios of mitophagosomes per mitochondrial area are shown in (G). Ten cells from three independent experiments were counted. Values are the means ± SD. *p < 0.05 compared with scrambled-oligo treated cells, determined with one-way ANOVA followed by the Student's t test


( Images of HeLa GFP - Parkin cells expressing mCherry - NDP52 ( A under valinomycin treatment . The images are from Movie EV 2 and 3 , respectively . Cyan , GFP - Parkin ; magenta , mCherry - NDP5 Scale bars , 10 µm . Images are representative of five independent

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( Images of HeLa GFP-Parkin cells expressing mCherry-NDP52 (A under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta, mCherry-NDP5 Scale bars, 10 µm. Images are representative of five independent experiments


( B ) Time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 under valinomycin treatment . The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring

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(B) Time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin treatment. The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures


Images o HeLa GFP - Parkin cells expressin mCherry - OPTN ( C ) under valinomycin treatment . The images are from Movie EV 2 and 3 , respectively . Cyan , GFP - Parkin ; magenta OPTN . Scale bars , 10 µm . Images are representative of five independent

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Images o HeLa GFP-Parkin cells expressin mCherry-OPTN (C) under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta OPTN. Scale bars, 10 µm. Images are representative of five independent experiments


( D ) mCherry - NDP52 or OPTN signals colocalized with Parkin - ring structures and existing inside of Parkin - ring structures ( irrupted ) were quantified . Data were from five independent experiments in cells incubated with valinomycin for 2 h . Values are the means ± SEM . * * p < 0 . 01 compared with mCherry - OPTN expressing cells , determined with one - way ANOVA followed by the Student " s

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(D) mCherry-NDP52 or OPTN signals colocalized with Parkin-ring structures and existing inside of Parkin-ring structures (irrupted) were quantified. Data were from five independent experiments in cells incubated with valinomycin for 2 h. Values are the means ± SEM. **p < 0.01 compared with mCherry-OPTN expressing cells, determined with one-way ANOVA followed by the Student"s t


( E ) Mitophagic flux in NDP52 KD cells expressing siRNA - resistant NDP52 or NDP52 mutants . Cells were incubated with valinomycin for 3 h . Results are the summary of three independent experiments . Values are the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 vs scrambled oligo - transfected cells ; † p < 0 . 05 , † † p < 0 . 01 vs empty vector - transfected cells , determined with one - way ANOVA followed by the Tukey - Kramer post hoc

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(E) Mitophagic flux in NDP52 KD cells expressing siRNA-resistant NDP52 or NDP52 mutants. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05, ††p < 0.01 vs empty vector-transfected cells, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test


( F ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

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(F) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


( G ) Images of HeLa GFP - Parkin cells expressing mCherry - NDP52 ∆ LIM - L or mCherry - NDP52 ∆ SKICH treated with valinomycin for 2h . The images are from Movie EV 4 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 mutant . Scale bars , 10 µm . Images are representative of three independent

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(G) Images of HeLa GFP-Parkin cells expressing mCherry-NDP52∆LIM-L or mCherry-NDP52∆SKICH treated with valinomycin for 2h. The images are from Movie EV 4. Cyan, GFP-Parkin; magenta, mCherry-NDP52 mutant. Scale bars, 10 µm. Images are representative of three independent experiments


( A ) Mitophagic flux in cells transfected with siNDP52 , siTBK1 , siOPTN or a combination of siRNAs . Results are the summary of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 vs scrambled oligo - transfected cells ; † p < 0 . 05 vs siNDP52 - transfected cells ; § p < 0 . 05 , § § p < 0 . 01 vs siTBK1 ; NS : not significant , determined with one - way ANOVA followed by the Tukey - Kramer post hoc

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(A) Mitophagic flux in cells transfected with siNDP52, siTBK1, siOPTN or a combination of siRNAs. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05 vs siNDP52-transfected cells; §p < 0.05, §§p < 0.01 vs siTBK1; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test


( B ) HeLa - GFP - Parkin cells transfected with siNDP52 , siOPTN , siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times . Cell lysates were subjected to SDS - PAGE or Phos - tag - PAGE prior to western blotting using the indicated antibodies . Data are representative of two independent experiments . The position of phosphorylated forms of NDP52 or OPTN are indicated by

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(B) HeLa-GFP-Parkin cells transfected with siNDP52, siOPTN, siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times. Cell lysates were subjected to SDS-PAGE or Phos-tag-PAGE prior to western blotting using the indicated antibodies. Data are representative of two independent experiments. The position of phosphorylated forms of NDP52 or OPTN are indicated by arrowheads


( C ) Images of TBK1 KD cells expressing mCherry - NDP52 or mCherry - OPTN under valinomycin treatment from Movie EV 5 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . Images are representative of two independent experiments . Scale bars , 10

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(C) Images of TBK1 KD cells expressing mCherry-NDP52 or mCherry-OPTN under valinomycin treatment from Movie EV 5. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm


( D ) TBK1 KD and control cells expressed mCherry - NDP52 . Lysates were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

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(D) TBK1 KD and control cells expressed mCherry-NDP52. Lysates were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


Anti - RFP immunoprecipitates from mCherry - NDP52 and mCherry - NDP52 ∆ SKICH - transfected cells were subjected to SDS - PAGE followed by silver staining . Four bands specific for mCherry - NDP52 are numbered and were identified by mass spectrometric analysis . Asterisk ( * ) and double asterisk ( * * ) indicate mCherry - NDP52 and mCherry - NDP52 ∆ SKICH bands ,

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Anti-RFP immunoprecipitates from mCherry-NDP52 and mCherry-NDP52 ∆SKICH-transfected cells were subjected to SDS-PAGE followed by silver staining. Four bands specific for mCherry-NDP52 are numbered and were identified by mass spectrometric analysis. Asterisk (*) and double asterisk (**) indicate mCherry-NDP52 and mCherry-NDP52∆SKICH bands, respectively


( C ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

(C) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


( D ) HeLa - GFP - Parkin cells transfected with siMTPAP were incubated with 1 µM valinomycin for the indicated times . Western blotting was performed using the indicated antibodies . Data are representative of three independent

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(D) HeLa-GFP-Parkin cells transfected with siMTPAP were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments


( E ) Mitophagic flux in cells transfected with siNDP52 , siMTPAP and combinations of siRNAs . Cells were incubated with valinomycin for 3 h . Results are the summary of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 vs scrambled oligo - transfected cells ; NS : not significant , determined with one - way ANOVA followed by the Tukey - Kramer post hoc

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(E) Mitophagic flux in cells transfected with siNDP52, siMTPAP and combinations of siRNAs. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test


( F ) Images of MTPAP KD cells expressing mCherry - NDP52 under valinomycin treatment . The images are from Movie EV 7 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . Images are representative of two independent experiments . Scale bars , 10

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(F) Images of MTPAP KD cells expressing mCherry-NDP52 under valinomycin treatment. The images are from Movie EV 7. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm


( A ) HeLa - GFP - Parkin cells were incubated with valinomycin for 0 or 2 h . Immunostaining was performed with anti - MTPAP and anti - Tom20 antibodies . Images are representative of two independent experiments . Scale bar , 10

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(A) HeLa-GFP-Parkin cells were incubated with valinomycin for 0 or 2 h. Immunostaining was performed with anti-MTPAP and anti-Tom20 antibodies. Images are representative of two independent experiments. Scale bar, 10 µm


( B ) Mitochondrial - rich fraction prepared from HeLa - GFP - Parkin cells treated with or without valinomycin and epoxomicin for 2 h , was incubated with various concentrations of trypsin ( 0 - 1000 µg / ml ) for 30 min on ice . Protease protection was analyzed by western blotting using antibodies for mitochondrial marker proteins ( Tom20 for OMM , Tim23 for intermembrane space , UQCRC1 and TFAM for mitochondrial matrix ) . Data are representative of five independent

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O

(B) Mitochondrial-rich fraction prepared from HeLa-GFP-Parkin cells treated with or without valinomycin and epoxomicin for 2 h, was incubated with various concentrations of trypsin (0-1000 µg/ml) for 30 min on ice. Protease protection was analyzed by western blotting using antibodies for mitochondrial marker proteins (Tom20 for OMM, Tim23 for intermembrane space, UQCRC1 and TFAM for mitochondrial matrix). Data are representative of five independent experiments


( C ) HeLa - GFP - Parkin cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 0 - 3 h in the presence or absence of 10 µM epoxomicin . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody or control IgG . Immunoprecipitates were analyzed by western blotting . Data are representative of five independent experiments . ( D ) Quantification of MTPAP co - immunoprecipitated with NDP52 in ( C ) . Results are a summary of five independent experiments . Values are the means ± SEM . * * p < 0 . 01 compared to cells treated with valinomycin and bafilomycin A1 for 3 h , determined with one - way ANOVA followed by the Tukey - Kramer post hoc

O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa-GFP-Parkin cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 0-3 h in the presence or absence of 10 µM epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of five independent experiments. (D) Quantification of MTPAP co-immunoprecipitated with NDP52 in (C). Results are a summary of five independent experiments. Values are the means ± SEM. **p < 0.01 compared to cells treated with valinomycin and bafilomycin A1 for 3 h, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test


( E ) HeLa - GFP - Parkin cells were incubated with 100 µM DFO and 100 nM bafilomycin A1 for 0 , 4 and 8 h in the presence or absence of epoxomicin . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent

O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O

(E) HeLa-GFP-Parkin cells were incubated with 100 µM DFO and 100 nM bafilomycin A1 for 0, 4 and 8 h in the presence or absence of epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments


( F ) HeLa cells and HEK293T cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h or 100 µM DFO and 100 nM bafilomycin A1 for 8 h . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent

(F) HeLa cells and HEK293T cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h or 100 µM DFO and 100 nM bafilomycin A1 for 8 h. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments


( G ) Immunoelectron micrographs of HeLa - GFP - Parkin cells expressing FLAG - NDP52 . Cells treated for 2 h with valinomycin and bafilomycin A1 with or without epoxomicin , were processed for immune - electron microscopy with anti - FLAG antibody . Images are representative of ten cells . Arrow , immunogold labeled FLAG - NDP52 . Mito , mitochondrion ; MP , mitophagosome . Scale bars , 200

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O

(G) Immunoelectron micrographs of HeLa-GFP-Parkin cells expressing FLAG-NDP52. Cells treated for 2 h with valinomycin and bafilomycin A1 with or without epoxomicin, were processed for immune-electron microscopy with anti-FLAG antibody. Images are representative of ten cells. Arrow, immunogold labeled FLAG-NDP52. Mito, mitochondrion; MP, mitophagosome. Scale bars, 200 nm


( H ) Selected frames from time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 under valinomycin and bafilomycin A1 treatment . The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 6 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring structures . Scale bars , 1

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(H) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 6. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm


( A , B ) mCherry - LC3 and MTS - TagBFP were expressed in MTPAP KD and control cells . Cells were incubated with valinomycin for the indicated times and immunostained with anti - NDP52 antibody . Images are representative of two independent experiments . Yellow arrows indicate co - localization of MTS - TagBFP , mCherry - LC3 and NDP52 . White arrows indicate co - localization of MTS - TagBFP and NDP52 ( not mCherry - LC3 ) . Scale bars , 10 µm . Co - localization of mCherry - LC3 and NDP52 on mitochondria is quantified in ( B ) . At least ten cells from two independent experiments were quantified . Values are the means ± SEM . * * p < 0 . 01 vs control , determined with one - way ANOVA followed by the Student " s

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) mCherry-LC3 and MTS-TagBFP were expressed in MTPAP KD and control cells. Cells were incubated with valinomycin for the indicated times and immunostained with anti-NDP52 antibody. Images are representative of two independent experiments. Yellow arrows indicate co-localization of MTS-TagBFP, mCherry-LC3 and NDP52. White arrows indicate co-localization of MTS-TagBFP and NDP52 (not mCherry-LC3). Scale bars, 10 µm. Co-localization of mCherry-LC3 and NDP52 on mitochondria is quantified in (B). At least ten cells from two independent experiments were quantified. Values are the means ± SEM. **p < 0.01 vs control, determined with one-way ANOVA followed by the Student"s t


MTPAP KD and control cells expressed mCherry - NDP52 or empty vector ( C Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MTPAP KD and control cells expressed mCherry-NDP52 or empty vector (C Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


MTPAP KD and control cells expresse mCherry - LC3 ( D ) . Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MTPAP KD and control cells expresse mCherry-LC3 (D). Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


( E ) MTPAP KD and control cells were incubated with valinomycin and bafilomycin A1 for 2 h . Cell lysates were subjected to immunoprecipitation using an anti - NDP52 antibody or control IgG . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

O O O B-CONTROLLED_VAR O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(E) MTPAP KD and control cells were incubated with valinomycin and bafilomycin A1 for 2 h. Cell lysates were subjected to immunoprecipitation using an anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


( F ) GST - NDP52 conjugated with glutathione - Sepharose resin was incubated with purified recombinant LC3 and MTPAP ∆ MTS ∆ C . The pulled down complex was subjected to western blotting . Data are representative of three independent

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(F) GST-NDP52 conjugated with glutathione-Sepharose resin was incubated with purified recombinant LC3 and MTPAP∆MTS∆C. The pulled down complex was subjected to western blotting. Data are representative of three independent experiments


( A ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 D439R / C443K mutant or WT NDP52 were incubated with valinomycin and nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent experiments

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(A) HeLa-GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant or WT NDP52 were incubated with valinomycin and nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments


( B ) Selected frames from time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 D439R / C443K mutant under valinomycin and bafilomycin A1 treatment . The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 9 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 D439R / C443K . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring structures . Scale bars , 1

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(B) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 9. Cyan, GFP-Parkin; magenta, mCherry-NDP52 D439R/C443K. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm


( A ) Confocal images of ALIX ( red ) , Asl ( white ) , α - tubulin ( green ) and Hoechst ( blue ) in wild type ( w1118 ) , Sas - 6c02901 , DSpdG02143 , cnnHK21 and γ - Tub23CPI NBs ( scale bars , 5 μm ) . The insets show higher magnification of the boxed

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(A) Confocal images of ALIX (red), Asl (white), α-tubulin (green) and Hoechst (blue) in wild type (w1118), Sas-6c02901, DSpdG02143, cnnHK21 and γ-Tub23CPI NBs (scale bars, 5 μm). The insets show higher magnification of the boxed centrosomes


( B ) The centrosomal fluorescence intensity of ALIX was measured and the graph shows the average intensity ( relative to w1118 intensity ) of three separate experiments ( ± S . E ) , where in total 78 w1118 , 73 Sas - 6c02901 , 45 DSpdG02143 , 61 cnnHK21 and 26 γ - Tub23CPI NBs were analyzed . Statistically significant differences are indicated ( * p < 0 . 05 and * * p < 0 . 01 , Student ' s t - test )

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(B) The centrosomal fluorescence intensity of ALIX was measured and the graph shows the average intensity (relative to w1118 intensity) of three separate experiments (±S.E), where in total 78 w1118, 73 Sas-6c02901, 45 DSpdG02143, 61 cnnHK21 and 26 γ-Tub23CPI NBs were analyzed. Statistically significant differences are indicated (*p<0.05 and **p<0.01, Student's t-test)


( C ) GST or GST - ALIX were incubated with in vitro translated 35S - labelled Cnn or DSpd - 2 proteins . Binding was detected by autoradiography ( upper panel ) , and expression of GST and GST - ALIX was visualized by Ponceau S - staining ( lower panel )

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(C) GST or GST-ALIX were incubated with in vitro translated 35S-labelled Cnn or DSpd-2 proteins. Binding was detected by autoradiography (upper panel), and expression of GST and GST-ALIX was visualized by Ponceau S-staining (lower panel)


( D ) HeLa cells were transfected with control , Cep192 or Cep215 siRNA . Efficient depletion of Cep192 and Cep215 was confirmed by Western blotting analysis , which also showed unchanged expression levels of ALIX . Unspecific bands , below Cep192 and above Cep215 , are indicated with *

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(D) HeLa cells were transfected with control, Cep192 or Cep215 siRNA. Efficient depletion of Cep192 and Cep215 was confirmed by Western blotting analysis, which also showed unchanged expression levels of ALIX. Unspecific bands, below Cep192 and above Cep215, are indicated with *


* . ( E ) Control , Cep192 or Cep215 siRNA - transfected HeLa cells were immunostained with anti - ALIX ( red ) , anti - α - tubulin ( green ) and Hoechst ( blue ) . Confocal micrographs are presented and the insets show the indicated centrosomes ( arrows ) at higher magnification ( scale bars , 5 μm )

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*. (E) Control, Cep192 or Cep215 siRNA-transfected HeLa cells were immunostained with anti-ALIX (red), anti-α-tubulin (green) and Hoechst (blue). Confocal micrographs are presented and the insets show the indicated centrosomes (arrows) at higher magnification (scale bars, 5 μm)


( F ) The centrosomal ALIX fluorescence intensity was measured and the graph shows the average intensity ( relative to control siRNA cells ) of three independent experiments ( ± S . E ) , where in total 41 control siRNA , 40 Cep192 siRNA and 39 Cep215 siRNA - transfected cells were analyzed , * * p < 0 . 01 , Student ' s t -

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(F) The centrosomal ALIX fluorescence intensity was measured and the graph shows the average intensity (relative to control siRNA cells) of three independent experiments (±S.E), where in total 41 control siRNA, 40 Cep192 siRNA and 39 Cep215 siRNA-transfected cells were analyzed, **p< 0.01, Student's t-test