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

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(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 RGI3 - GFP in N . benthamiana . IP was performed using GFP - TRAP agarose beads . Western blots for detection were probed with α - GFP or α - HA antibodies . Similar results were obtained in 3 independent experiments .

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(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 RGI3 - GFP in N . benthamiana . 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 detection were probed with α - GFP or α - HA antibodies . Data information : Similar results were obtained in 3 independent experiments .

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(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 indicated time , n = 3 biological replicates . UBQ was used as a house keeping gene . Students T - revealed no statistical difference for each of different timepoints . Data information : Similar results were obtained in 3 independent experiments .

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(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 ) extraction was performed from 12 - day - old seedlings of indicated genotypes . Western blot was probed with α - FLS2 antibodies . ( D ) 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 .

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(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 negative control BSA GST . Binding signal is shown as absorbance at 450 nm in arbitrary units [ AU ] .

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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 lysates on GST - mATG8s beads followed by immuno - blotting with α - GFP . As loading control , with amido black is shown .

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

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

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

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

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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 .

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


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

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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 .

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


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

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

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

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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 mitophagy flux performed in HeLa stably expressing HA - Parkin , mt - mKeima , a GFP fusion of indicated sensor . were treated with OAQ ( oligomycin , antimycin , Q - VD ) for indicated times analyzed by FACS for lysosomal - positive mt - mKeima ( 561nm ) . average of three independent experiments is presented . Representative FACS data from experiments ( GFP control AS3 _ 67 ) are shown in 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 .

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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 is carrying a GFP - reporter gene activated by availability of glucose - 6 - phosphate in host ' s cytosol .

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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 residence strong activation of GFP - reporter gene ( green arrow ) are not recognized by LC3C specific sensor mCh - AS3 _ 67 .

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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 cytosol is coated with endogenous LC3C . In contrast , endogenous LC3B is enriched in spots in close proximity to .

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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 - tethering FYVE domain or PB1 oligomerization domain .

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

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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 to monitor xenophagy events .

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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 were transiently co - transfected for indicated sensor mATG8 . Mitophagy was induced by CCCP treatment over three hours . Lysosomal degradation of mitochondria undergoing mitophagy was blocked by addition of BafilomycinA1 ( Baf ) .

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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 . An example of rare events with nearly complete overlap of signals for endogenous LC3C mitochondria ( upper panel ) a representative image ( lower panel ) is given .

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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 endogenous LAMP1 in U2OS induced for mitophagy .

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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 ) . corresponding β - actin control ( with a 518 - bp amplicon ) is given below .

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

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

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(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.


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

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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 old 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 , burdens in ( A E ) liver , ( B F ) heart , ( C G ) spleen ( D H ) kidney were determined . Data represent mean ± ( n = 7 biological replicates ) . . * Denotes p < 0 . 05 , * * denotes p < 0 . 005 * * * denotes p < 0 . 0007 , using Mann - Whitney .

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(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.


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

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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.


were grown in MHB - ca medium without or with 20 mM glucose as indicated . ( B ) PndhC PndhF YFP reporter constructs were used for following gene expression data was normalized by growth . Data are expressed as average ± of three independent experiments . * * * Denotes p < 0 . 001 compared to ndhC expression in WT 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 B-SMALL_MOLECULE 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 B-EXP_ASSAY 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

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 isogenic mutant strains was measured with Clark oxygen electrode . were grown in MHB - ca medium until exponential phase ( OD600 = 0 . 6 ) or until late stationary phase ( 24 h ) . were washed with PBS respiration initiated by 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 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 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 grown aerobically in MHB - ca medium for 20 h were measured . 100 % corresponds to a NADH / NAD + of 0 . 003 . Data are expressed as average ± SD of four independent experiments . * Denotes p < 0 . 05 , * * * 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 B-SMALL_MOLECULE 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 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 was monitored in supernatants of WT isogenic ndh strains at 7 24 h of growth in MHB - ca medium . Data are expressed as average ± SD of three independent experiments . * * Denotes p < 0 . 005 * * * 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

(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 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

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

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

(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 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 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 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

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

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 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-GENEPROD B-EXP_ASSAY 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 ndh deficient mutants after static incubation at 37 ° C for 20 h . histogram shows amount of biofilm biomass attached to microtiter plate when were grown in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose 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 B-EXP_ASSAY I-EXP_ASSAY 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 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 grown under 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

(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 grown under same conditions as for biofilm formation . 100 % corresponds to a NADH / NAD + of 0 . 09 . Data are expressed as average ± SD of four independent experiments performed by duplicates . * Denotes p < 0 . 05 , * * * 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 B-SMALL_MOLECULE 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 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 Newman WT isogenic mutant strains was measured by incubation of rabbit blood with serially diluted supernatants from 20 h cultures grown in MHB - ca medium without with 25 mM glucose , 40 mM sodium pyruvate or 60 mM sodium acetate . Lysis was monitored by release of hemoglobin . Data are expressed as average ± SD of five independent experiments . * Denotes p < 0 . 05 , * * * denotes p < 0 . 001 , ns denotes no significant compared to WT grown in MHB - ca 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 B-CELL_TYPE 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 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 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 Newman WT isogenic mutant strains was measured by incubation of rabbit blood with serially diluted supernatants from 20 h cultures grown in MHB - ca medium . Lysis was monitored by 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 B-CELL_TYPE 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 isogenic mutant strains after static incubation at 37 ° C for 20 h . histogram shows amount of biofilm biomass attached to microtiter plate when were grown in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose 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 B-EXP_ASSAY I-EXP_ASSAY 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 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 Newman WT isogenic mutant strains grown aerobically for 20 h in MHB - ca medium , in absence or presence of 10 mg / ml FA - deficient BSA 0 . 01 % oleic acid . Data are expressed as average ± SD of five independent experiments . * * Denotes p < 0 . 005 * * * denotes p < 0 . 001 compared to each 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 B-GENEPROD 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 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 isogenic mutant strains after static incubation for 20 h in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose 2 % NaCl , in absence or presence of 10 mg / ml FA - deficient BSA 0 . 01 % oleic acid . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to each 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 B-ORGANISM B-TISSUE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD 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 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 overexpressing N . gonorrhea acyl - ACP synthetase 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 with empty plasmid via Student ' s t - .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD 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 B-GENEPROD 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 overexpressing N . gonorrhea acyl - ACP synthetase 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 with empty plasmid via Student ' s t - .

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

(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 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 , actin in HeLa transfected with Nedd4l siRNA ( siNedd4l ) or control ( siNC ) ( A ) , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated relative to that of total , respectively . Densitometry of MEKK2 was relative to actin . experiments were performed three times , representative data are shown .

B-EXP_ASSAY 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 B-GENEPROD O B-CELL_LINE O O B-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD 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 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 , actin in Nedd4l + / + Nedd4l - / - MEFs , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated relative to that of total , respectively . Densitometry of MEKK2 was relative to actin . experiments were performed three times , representative data are shown .

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

B-EXP_ASSAY 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 B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD 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 of phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , phosphorylated p38 ( P - P38 ) , total P38 , MEKK2 , NEDD4L , actin in HeLa 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 relative to that of total , respectively . Densitometry of MEKK2 was relative to actin . experiments were performed three times , representative data are shown .

O O O B-EXP_ASSAY 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 B-GENEPROD O B-CELL_LINE 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD 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 of MEKK2 , TRAF2 , TRAF6 , NEDD4L , actin in Nedd4l + / + , Nedd4l + / - , Nedd4l - / - MEFs . Data information : Numbers between two blots indicate densitometry of phosphorylated relative to that of total , respectively . Densitometry of MEKK2 was relative to actin . experiments were performed three times , representative data are shown .

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

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

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

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

(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 of NEDD4L precipitated with GST - tagged Null or GST - tagged MEKK2 recombinant . Data information : experiments were performed three times , representative data are shown .

O O O B-EXP_ASSAY 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

(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 co - transfected with plasmids expressing HA - tagged MEKK2 Myc - tagged wild - type NEDD4L or mutant NEDD4L with C2 ( ∆ C2 ) , WW ( ∆ WW ) or HECT ( ∆ HECT ) domain deleted . Data information : experiments were performed three times , 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 B-GENEPROD O O B-GENEPROD I-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

(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 of MEKK2 NEDD4L in Nedd4l + / + , 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 : experiments were performed three times , representative data are shown .

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

(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 of MEKK2 incubated with NEDD4L or TRAF6 recombinant full complement of ubiquitination reaction components in vitro . Data information : experiments were performed three times , representative data are shown .

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

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

O O O B-EXP_ASSAY 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 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

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

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

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

O O O B-EXP_ASSAY 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 B-GENEPROD O O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O O O O B-EXP_ASSAY 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 B-GENEPROD O O B-GENEPROD O B-GENEPROD 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 B-GENEPROD 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 of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T co - transfected with plasmids expressing Flag - tagged MEKK2 , increased dosage of Myc - tagged NEDD4L , HA - tagged wild - type , K27O , or K63O mutant Ub . Data information : experiments were performed three times , representative data are shown .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

O O O B-EXP_ASSAY 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-CELL_LINE 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

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

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

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

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

(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 of Myc - tagged NEDD4L co - immunoprecipitated with anti - HA antibody from lysates of HEK293T co - transfected with plasmids expressing Myc - tagged NEDD4L HA - tagged wild - type MEKK2 or mutant MEKK2 ( S520A ) . Data information : experiments were performed two times , 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 B-GENEPROD O O B-GENEPROD I-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

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

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

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

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

(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 CD11b - CD45high lymphocytes .

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

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


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

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

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 of CD206 expression in CD11b + CD45int microglia CD11b + CD45high resident macrophages . Values denote of mean ± of 5 independent experiments .

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

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

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

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 ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 1 ) were FACS - sorted 24 hours later . Gene expression of microglia homeostatic ( Olfml3 , Fcrls , Tmem119 , Siglech , Gpr34 , P2ry12 , Mef2c ) , phagocytic ( Tyrobp , Trem2 ) , inflammatory ( Il1β , Tnf , Ccl2 , Mrc1 , Arg1 ) markers were analyzed by qPCR . Bars represent mean of relative expression ( % of saline ; Gapdh as housekeeping gene ) ± ( * p < 0 . 05 ; * * p < 0 . 01 by two - Student ' s ; n = 4 ) .

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

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 ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 1 ) were FACS - sorted 24 hours later . Representative multicolour flow cytometry of 5 independent experiments showing CD11b CD45 positive populations in single viable in saline or LPS - injected mouse brains .

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

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 ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 1 ) were FACS - sorted 24 hours later . Representative multicolour flow cytometry showing of mean ± of 5 independent experiments of Ly6C CD206 expressing in CD11b + CD45int from saline or LPS - injected mice .

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

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 ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 1 ) were FACS - sorted 24 hours later . Gene expression of monocytic markers Ly6c1 Ccr2 in purified microglia ( n = 4 ) isolated bone marrow monocytes ( n = 2 ) by qPCR . Bars represent mean of relative expression ( Gapdh as housekeeping gene ) ± ( * * p < 0 . 01 by two - Student ' s ) .

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

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).


adult microglia were cultivated in presence of TGF - β ( 50 μg / ml ) M - CSF ( 10 ng / ml ) , while neonatal 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 of microglia homeostatic ( Olfml3 , Tmem119 , Gpr34 ) inflammatory ( Il1β , Tnf , Ccl2 ) genes were analysed by qPCR . Bars represent mean of relative expression ( Gapdh as housekeeping gene ) ± ( * * p < 0 . 01 by two - Student ' s ; n = 3 ) .

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

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).


showing clustering of 1 , 247 single , featuring 100 most variable genes ( FDR < 0 . 05 ) . Single - - seq results are obtained from 2 mice per group ( 1 1 each ) .

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

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 ) inflammatory ( Ccl2 , Gpr84 ) genes overlaid on 2D - tSNE space . Bars represent log2 ( + 1 ) .

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

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 of 2 independent experiments showing TMEM119 P2RY12 expression in CD11b + CD45int microglia of saline or LPS - injected mouse brains . For unconjugated TMEM119 antibody , negative stands for antibody without secondary antibody . For P2RY12 antibody , negative represents isotype PE control .

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

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.


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

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

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 ) homeostatic ( Mef2c , P2ry12 , Siglech ) genes in dependence on inferred states .

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

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


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

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

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

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

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

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

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

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

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

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

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

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

(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


( of HeLa GFP - Parkin expressing mCherry - NDP52 ( A under valinomycin treatment . are from Movie EV 2 3 , respectively . Cyan , GFP - Parkin ; magenta , mCherry - NDP5 Scale bars , 10 µm . 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 of HeLa GFP - Parkin expressing mCherry - NDP52 under valinomycin treatment . are representative of five independent experiments correspond to boxed regions of Figure 2A Movie EV 2 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . arrowheads indicate colocalization of GFP - Parkin mCherry - NDP52 . yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring

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

(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


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

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

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

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

(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