A ) Native mass spectrum of NHE9 * purified in presence ( top panel ) or absence of additional brain lipids ( bottom panel ) . In presence of brain lipids , NHE9 * is detected as exclusively dimeric with poorly resolved peaks , indicating presence of multiple lipid adducts . Without brain lipid addition , NHE9 * appears as sharp peaks , revealing several adducts of approximately 1 kDa each , as well as a minor monomer population .

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

A) Native mass spectrum of NHE9* purified in the presence (top panel) or the absence of additional brain lipids (bottom panel). In the presence of brain lipids, NHE9* is detected as exclusively dimeric protein with poorly resolved peaks, indicating the presence of multiple lipid adducts. Without brain lipid addition, NHE9* appears as sharp peaks, revealing several adducts of approximately 1 kDa each, as well as a minor monomer population.


B ) Thermal stabilization of purified dimeric NHE9 * - GFP by lipids . Normalized mean FSEC peak before heating ( open bars ) , after heating centrifugation ( grey bars ) in presence of different lipids . Data presented are mean values ± data range of n = 4 experiments ( see Materials Methods ) .

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

B) Thermal stabilization of purified dimeric NHE9*-GFP by lipids. Normalized mean FSEC peak fluorescence before heating (open bars), and after heating and centrifugation (grey bars) in presence of different lipids. Data presented are mean values ± data range of n = 4 experiments (see Materials and Methods).


C ) Thermal shift stabilization of purified dimeric NHE9 * - GFP in presence of PIP2 ( red ) compared to PIP2 - free ( black ) . Data presented are normalized mean FSEC peak as mean values ± data range of n = 2 technical repeats ; apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; average ΔTm presented is calculated from n = 2 independent titrations .

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

C) Thermal shift stabilization of purified dimeric NHE9*-GFP in the presence of PIP2 (red) compared to PIP2-free (black). Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.


E ) Thermal shift stabilization of dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; average ΔTm presented is calculated from n = 2 independent titrations . ( F ) Native MS of triple mutant reveals monomeric NHE9 with no notable lipid adducts ; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained .

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

E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations. (F) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained.


E ) Thermal shift stabilization of dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; average ΔTm presented is calculated from n = 2 independent titrations .

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

E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.


A . Expression heat map of netrin - 3 netrin - 1 . Receptors of netrin - 1 putative receptors of netrin - 3 : DCC , UNC5 - A , - B , - C , - D , Neogenin , APP , CD146 are presented . color legend ( right - side ) describes types included in heat map . Asterisks indicate two main pathologies in which netrin - 3 expression is detectable . C . of lines positive for netrin - 1 / or - 3 expression . Co - occurrence of expression was calculated using Fisher ' s exact . P - value is indicated in table .

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

A. Expression heat map of netrin-3 and netrin-1. Receptors of netrin-1 and putative receptors of netrin-3: DCC, UNC5-A, -B, -C, -D, Neogenin, APP, CD146 are presented. The color legend (right-hand side) describes all tumor types included in the heat map. Asterisks indicate the two main pathologies in which netrin-3 expression is detectable. C. Percentage of cell lines positive for netrin-1 and/or -3 expression. Co-occurrence of expression was calculated using Fisher's exact test. P-value is indicated in the table.


B . of netrin - 1 - 3 expression in lines ranked according to netrin - 1 expression

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

B. Analysis of netrin-1 and -3 expression in tumor cell lines and ranked according to netrin-1 expression


B . of netrin - 1 - 3 expression in lines ranked according to netrin - 1

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


A . of netrin - 3 gene expression by q - RT - PCR in a panel of 181 human NB stages 1 , 2 , 3 , 4 4S . Number of cases is indicated on graph . Error bars indicate s . e . m . Statistical treatment of data was performed using a two - sided Student t - .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM B-DISEASE O O O O O O O O O O O O O O O O O O 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. Quantification of netrin-3 gene expression by q-RT-PCR in a panel of 181 human NB stages 1, 2, 3, 4 and 4S. Number of cases is indicated on the graph. Error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Student t-test.


B . of netrin - 3 gene expression by q - RT - PCR in a panel of 181 human neuroblastoma , defined as low - high - risk NB . number of cases is indicated on graph . Error bars indicate s . e . m . Statistical treatment of data was performed using a two - sided Student t - .

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

B. Quantification of netrin-3 gene expression by q-RT-PCR in a panel of 181 human neuroblastoma patients, defined as low- and high-risk NB. The number of cases is indicated on the graph. Error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Student t-test.


C . Netrin - 3 high expression is a marker of poor prognosis in NB . 280 months overall Kaplan - Meier survival curves in a panel of 181 of NB stages . cohort was dichotomized base on netrin - 3 median expression ( in green red ) . Statistical treatment of data : Mantel - Cox ; p value is indicated below graph .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O B-DISEASE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-DISEASE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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. Netrin-3 high expression is a marker of poor prognosis in NB. 280 months overall Kaplan-Meier survival curves in a panel of 181 patients of all NB stages. The cohort was dichotomized base on netrin-3 median expression (in green and red). Statistical treatment of the data: Mantel-Cox; p value is indicated below the graph.


A . Gene Set Enrichment ( GSEA ) in high netrin - 3 NB . cohort was dichotomized with n = 50 lowest vs 50 highest : amplification MYCN pathway ( p = 0 . 039 ; fdqr = 0 . 025 ) .

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

A. Gene Set Enrichment Analysis (GSEA) in high netrin-3 NB patients. The cohort was dichotomized with the n=50 lowest vs 50 highest: amplification the MYCN pathway (p=0.039; fdqr=0.025).


C . Q - RT - PCR of netrin - 3 gene expression in IMR32 ( n = 5 ) IGRN91 ( n = 5 ) NB lines after MYCN silencing by siRNA ( U - ) . Error bars indicate s . e . m .

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

C. Q-RT-PCR analysis of netrin-3 gene expression in IMR32 (n=5) and IGRN91 (n=5) NB cell lines after MYCN silencing by siRNA (U-test). Error bars indicate s.e.m.


D . ChIP - seq of MYCN binding , active enhancer epigenetic marks H3K27ac , H3K4me1 , active promoter epigenetic mark H3K4me3 , on netrin - 3 locus ( red ) . An enrichment of MYCN , associated with active enhancer marks , was detected in three different neuroblastoma lines NB - 1643 , COGN415 LAN5 .

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

D. ChIP-seq analysis of MYCN binding, active enhancer epigenetic marks H3K27ac, H3K4me1, and active promoter epigenetic mark H3K4me3, on netrin-3 locus (red line). An enrichment of MYCN, associated with active enhancer marks, was detected in three different neuroblastoma cell lines NB-1643, COGN415 and LAN5.


E . Quantitative showing of IGRN91 tumors implanted on CAM silenced or not for netrin - 3 ( n = 11 siScr , n = 9 siNetrin - 3 ; U - ) . Error bars indicate s . e . m . F . Quantitative showing of IMR32 tumors silenced or not for netrin - 3 ( n = 5 siScr , n = 7 siNetrin - 3 ; U - ) . Error bars indicate s . e . m .

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

E. Quantitative analysis showing the size of IGRN91 primary tumors implanted on CAM and silenced or not for netrin-3 (n=11 siScr, n=9 siNetrin-3; U-test). Error bars indicate s.e.m. F. Quantitative analysis showing the size of IMR32 primary tumors silenced or not for netrin-3 (n=5 siScr, n=7 siNetrin-3; U-test). Error bars indicate s.e.m.


A . Deep of netrin - 3 gene expression in lung cancer lines ( n = 76 ; - Seq ) .

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

A. Deep analysis of netrin-3 gene expression in lung cancer cell lines (n=76; RNA-Seq).


B . Q - RT - PCR of netrin - 1 - 3 expression , in lung cancer Lines ( Error bars indicate SD , n = 3 ) .

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

B. Q-RT-PCR analysis of netrin-1 and -3 expression, in lung cancer cell Lines (Error bars indicate SD, n=3).


C . Representative netrin - 3 - 1 mRNA detection , using RNAscope on SCLC paraffin embedded sections . Negative control DAPB , positive control PPiB . Each brown dot is a unique molecule of mRNA of each targeted gene ( n = 10 , scale bars 20µm ) . D . of netrin - 3 - 1 expression in human SCLC paraffin embedded sections ( High ≥ 50 % marked ; Low ≤ 5 % ) .

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

C. Representative netrin-3 and -1 mRNA detection, using RNAscope on SCLC paraffin embedded tumor sections. Negative control DAPB, positive control PPiB. Each brown dot is a unique molecule of mRNA of each targeted gene (n=10, scale bars 20µm). D. Quantification of netrin-3 and -1 expression in human SCLC paraffin embedded tumor sections (High≥ 50% marked cells; Low≤ 5%).


C . Representative netrin - 3 - 1 mRNA detection , using RNAscope on SCLC paraffin embedded sections . Negative control DAPB , positive control PPiB . Each brown dot is a unique molecule of mRNA of each targeted gene ( n = 10 , scale bars 20µm ) .

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

C. Representative netrin-3 and -1 mRNA detection, using RNAscope on SCLC paraffin embedded tumor sections. Negative control DAPB, positive control PPiB. Each brown dot is a unique molecule of mRNA of each targeted gene (n=10, scale bars 20µm).


A . ChIP - seq of NeuroD1 ASCL - 1 binding on netrin - 3 gene promoter . A major binding site was detected ( red blue peaks for NeuroD1 ASCL1 respectively ) . Input controls are shown below ( black ) .

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

A. ChIP-seq analysis of NeuroD1 and ASCL-1 binding on the netrin-3 gene promoter. A major binding site was detected (red and blue peaks for NeuroD1 and ASCL1 respectively). Input controls are shown below (black).


B . ChIP - seq of NeuroD1 ASCL - 1 enrichment , which was associated with broad H3K27ac - enriched regions at netrin - 3 locus ( red ) , indicating a transcriptionally active region in SCLC lines . ( rep = experimental replicates ) .

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

B. ChIP-seq analysis of NeuroD1 and ASCL-1 chromatin enrichment, which was associated with broad H3K27ac-enriched regions at the netrin-3 locus (red line), indicating a transcriptionally active region in SCLC cell lines. (rep= experimental replicates).


C . Q - RT - PCR of netrin - 3 gene expression in NCI - H82 ( n = 6 ) NCI - H69 ( n = 4 ) SCLC lines after Neurod - 1 ASCL - 1 silencing by siRNAs ( U - ) . Error bars indicate s . e . m .

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

C. Q-RT-PCR analysis of netrin-3 gene expression in NCI-H82 (n=6) and NCI-H69 (n=4) SCLC cell lines after Neurod-1 and ASCL-1 silencing by siRNAs (U-test). Error bars indicate s.e.m.


A . Bio - layer interferometry of netrin - 2L / receptor interactions . Other members of netrin family were also tested : netrin - 1 , - 4 - G2 . ( red : netrin - 2L ; bleu : netrin - 1 ; green netrin - 4 )

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

A. Bio-layer interferometry analysis of netrin-2L/receptor interactions. Other members of the netrin family were also tested: netrin-1, -4 and -G2. (red: netrin-2L; bleu: netrin-1; green netrin-4)


C . NCI - H82 CRISPR / Cas9 were subjected to immunoblots . sGx , designates number of guide RNAs , control ( sGc ) .

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

C. NCI-H82 CRISPR/Cas9 were subjected to immunoblots. sGx, designates the number of guide RNAs, control cells (sGc).


D . Netrin - 3 silencing delays or inhibits SCLC engraftment in vivo . NMRI nude mice were subcutaneously engrafted with either NCI - H82 - CRIPSR / cas9 - NTN3 with three different guides RNAs ( sG1 p = 0 . 0022 ; sG2 p ≤ 0 . 001 ; sG3 p ≤ 0 . 001 ) or control ( sGc ) n = 6 / group . Positive engraftment was report when tumors reached 50 mm3 ( Mantel Cox ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-DISEASE O O O O O O B-ORGANISM O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Netrin-3 silencing delays or inhibits SCLC engraftment in vivo. NMRI nude mice were subcutaneously engrafted with either NCI-H82-CRIPSR/cas9-NTN3 with three different guides RNAs (sG1 p=0.0022; sG2 p≤0.001; and sG3 p≤0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 50 mm3 (Mantel Cox).


E . NCI - H69 - CRISPR / Cas9 were subjected to immunoblots . sGx , designates number of guide RNAs , control ( sGc )

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

E. NCI-H69-CRISPR/Cas9 were subjected to immunoblots. sGx, designates the number of guide RNAs, control cells (sGc)


F . NCI - H69 - CRISPR / Cas9 presented in E . were subcutaneously engrafted with either NCI - H69 - CRIPSR / cas9 - NTN3 with three different guide RNAs ( sG1 p = 0 . 017 ; sG2 p = 0 . 028 ; sG3 p = 0 . 001 ) or control ( sGc ) n = 6 / group . Positive engraftment was report when tumors reached 20 mm3 ( Mantel Cox ) .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. NCI-H69-CRISPR/Cas9 cells presented in E. were subcutaneously engrafted with either NCI-H69- CRIPSR/cas9-NTN3 with three different guide RNAs (sG1 p=0.017; sG2 p=0.028; and sG3 p=0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 20 mm3 (Mantel Cox).


B . of NP137 binding on netrin - 2L , netrin - 1 ; netrin - 4 or netrin - G2 by bio - layer interferometry assays . Kd rhNTN1 : 0 . 96 ± 0 . 30nM ; Kd for rchNTN2 : 1 . 53 ± 0 . 96nM

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

B. Analysis of NP137 binding on netrin-2L, netrin-1; netrin-4 or netrin-G2 by bio-layer interferometry assays. Kd rhNTN1: 0.96 ± 0.30nM; Kd for rchNTN2: 1.53 ± 0.96nM


B . of NP137 binding on netrin - 2L , netrin - 1 ; netrin - 4 or netrin - G2 by bio - layer interferometry assays . Kd rhNTN1 : 0 . 96 ± 0 . 30nM ; Kd for rchNTN2 : 1 . 53 ± 0 .

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


C . NP137 inhibits NTN1 / UNC5B or NTN - 2L / UNC5B interaction , detected by Elisa assays . Here recombinant are murine forms ( Control IgG = mouse IgG2A , mNP137 = murine Fc , UNCB = rat UN5H2 ) bars indicate SD , n = 3 .

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

C. NP137 inhibits NTN1/UNC5B or NTN-2L/UNC5B interaction, detected by Elisa assays. Here the recombinant proteins are the murine forms (Control IgG= mouse IgG2A, mNP137= murine Fc, UNCB=rat UN5H2) bars indicate SD, n=3.


D . NMRI nude mice were engrafted with NCI - H82 by subcutaneous injection of 2 million . When mean volume reached approximately 80 mm3 , were treated 3 x time / weekly by intra - abdominal injection of PBS ; NP001 ( Human IgG1 , isotype ) ; NP137 ( anti - netrin1 / 3 ) during 20 days . n = 10 / group ( * p < 0 . 05 , two - way Anova , Error bars indicate s . e . m . ) . E . NP137 enhances survival of mice engrafted with NCI - H82 ( see D ) . of Kaplan - Meier survival curves of mice treated or not with NP137 . Mantel Cox ; n = 10 / group .

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

D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.). E. NP137 enhances survival of mice engrafted with NCI-H82 cell line (see D). Analysis of Kaplan-Meier survival curves of mice treated or not with NP137. Mantel Cox test; n=10 animals/group.


D . NMRI nude mice were engrafted with NCI - H82 by subcutaneous injection of 2 million . When mean volume reached approximately 80 mm3 , were treated 3 x time / weekly by intra - abdominal injection of PBS ; NP001 ( Human IgG1 , isotype ) ; NP137 ( anti - netrin1 / 3 ) during 20 days . n = 10 / group ( * p < 0 . 05 , two - way Anova , Error bars indicate s . e . m . ) .

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

D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.).


F . of ( PARP ) in H82 treated tumors in D . Tumors were analyzed 7 days after first treatment ( ie : 400mm3 ; n = 3 / group . U - , bars indicate SD ) .

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F. Quantification of cell death (cleaved PARP) in H82 treated tumors in D. Tumors were analyzed 7 days after the first treatment (ie: 400mm3; n=3/group. U-test, bars indicate SD).


( A ) ROS burst response in wild - type ( WT ) map4k4 lines in response to 1 μM flg22 . Values are means ± , n = 16 ( biological replicates ) . Results are representative of three independent experiments . Statistical was performed using a one - way ANOVA with Tukey post - , p < 0 . 01 . Data information Different letters indicate statistical significant base on one - way ANOVA with Tukey post - , samples sharing letters are not significantly different .

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

(A) ROS burst response in wild-type (WT) and map4k4 lines in response to 1 μM flg22. Values are means ± SEM, n = 16 (biological replicates). Results are representative of three independent experiments. Statistical analysis was performed using a one-way ANOVA with Tukey post-test, p < 0.01. Data information Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( B ) qRT - PCR of expression of PTI marker gene FRK1 in wild - type map4k4 - 1 seedlings 1 h after treatment with 1 μM flg22 . Bars represent means ± SD on three biological replicates , p < 0 . 01 . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - , samples sharing letters are not significantly different .

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

(B) qRT-PCR analysis of the expression of PTI marker gene FRK1 in wild-type and map4k4-1 seedlings 1 h after treatment with 1 μM flg22. Bars represent means ± SD based on three biological replicates, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( D ) Heat map of gene expression response to flg22 in wild - type map4k4 - 1 , red blue indicate high low expression .

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(D) Heat map of gene expression response to flg22 in wild-type and map4k4-1, red and blue indicate high and low expression levels.


( F ) qRT - PCR of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour . Values are means of three biological replicates ± SD , p < 0 . 01 . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - , samples sharing letters are not significantly different .

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

(F) qRT-PCR analysis of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour. Values are the means of three biological replicates ± SD, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( A ) left panel , map4k4 - 1 shows enhanced resistance to Pseudomonas syringae pv . tomato DC3000 . growth of Pst DC3000 after inoculation in indicated genotypes ( Col - 0 , bars , map4k4 - 1 , green bars ) . titers were determined 2 hours ( day 0 ) 3 days after inoculation . Right panel , disease symptoms of wild - type map4k4 - 1 at 3 days after inoculation . Values are means ± SD , n = 6 ( biological replicates ) , p < 0 . 01 . Data information : Statistical were performed using a one - way ANOVA with Newman - Keuls multiple comparison or t - . n . s . indicate non - significant .

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

(A) left panel, map4k4-1 shows enhanced resistance to Pseudomonas syringae pv. tomato DC3000. The growth of Pst DC3000 after inoculation in the indicated genotypes (Col-0, white bars, map4k4-1, green bars). Bacterial titers were determined 2 hours (day 0) and 3 days after inoculation. Right panel, disease symptoms of wild-type and map4k4-1 at 3 days after inoculation. Values are the means ± SD, n = 6 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( B ) map4k4 - 1 disease resistance phenotype was recovered to wild - type in two independent complementation lines , c867 c872 . titers were determined as described above . Values are means ± SD , n = 6 ( biological replicates ) , p < 0 . 01 . Data information : Statistical were performed using a one - way ANOVA with Newman - Keuls multiple comparison or t - . n . s . indicate non - significant .

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

(B) map4k4-1 disease resistance phenotype was recovered to wild-type levels in two independent complementation lines, c867 and c872. Bacterial titers were determined as described above. Values are the means ± SD, n = 6 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( C ) qRT - PCR of PR1 expression in Col - 0 , map4k4 - 1 c867 after mock ( MgCl2 ) or Pst DC3000 inoculation , values are means ± SD , n = 3 ( biological replicates ) , p < 0 . 01 . Data information : Statistical were performed using a one - way ANOVA with Newman - Keuls multiple comparison or t - . n . s . indicate non - significant .

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

(C) qRT-PCR analysis of PR1 expression levels in Col-0, map4k4-1 and c867 after mock (MgCl2) or Pst DC3000 inoculation, values are the means ± SD, n = 3 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( D ) Flg22 - protection . Col - 0 , map4k4 - 1 c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation . population was measured 3 days after infection . Values are means ± SD , n = 6 . * * p < 0 . 01 , Results are representative of three independent experiments . Data information : Statistical were performed using a one - way ANOVA with Newman - Keuls multiple comparison or t - . n . s . indicate non - significant .

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

(D) Flg22-protection assay. Col-0, map4k4-1 and c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation. The bacterial population was measured 3 days after infection. Values are the means ± SD, n = 6. **p < 0.01, Results are representative of three independent experiments. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( A ) Subcellular localization of MAP4K4 - GFP in Arabidopsis mesophyll protoplasts . MAP4K4 - GFP ( green ) partially co - localized with plasma dye FM4 - 64 ( red ) . 14 hours after transformation , were taken when plasma of mesophyll protoplasts was stained by FM4 - 64 ( < 3 min ) . Scale bars = 10 µm .

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(A) Subcellular localization of MAP4K4-GFP in Arabidopsis mesophyll protoplasts. MAP4K4-GFP (green) partially co-localized with the plasma membrane dye FM4-64 (red). 14 hours after transformation, photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4-64 (< 3 min). Scale bars = 10 µm.


( B ) Auto - phosphorylation of recombinant HisMBP - MAP4K4 wild - type , no auto - phosphorylation in kinase - inactive HisMBP - MAP4K4 - D139A . phosphorylation level was determined by Pro - Q Diamond phosphoprotein gel stain ( upper panel ) , loading amounts were visualized by Coomassie G - 250 ( lower panel ) . predicted molecular of HisMBP - MAP4K4 is 118 . 4 kDa .

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

(B) Auto-phosphorylation of recombinant HisMBP-MAP4K4 wild-type, no auto-phosphorylation in the kinase-inactive HisMBP-MAP4K4-D139A protein. The phosphorylation level was determined by Pro-Q Diamond phosphoprotein gel stain (upper panel), protein loading amounts were visualized by Coomassie G-250 staining (lower panel). The predicted molecular weight of HisMBP-MAP4K4 is 118.4 kDa.


( C ) BiFC visualization of interaction between MAP4K4 BIK1 in Arabidopsis mesophyll protoplasts . BIK1 - nYFP MAP4K4 - cYFP were co - expressed in protoplasts to show of complemented YFP signal ( green ) , 14 h after transformation , results were photographed by confocal scanning microscopy , scale bars = 10 µm . Chl indicates Chloroplast , BF indicate Bright field . nYFP cYFP constructs were used as empty controls . MAP3K5 - K375M MKK4 were used as control .

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

(C) BiFC visualization of interaction between MAP4K4 and BIK1 in Arabidopsis mesophyll protoplasts. BIK1-nYFP and MAP4K4-cYFP were co-expressed in protoplasts to show fluorescence of complemented YFP signal (green), 14 h after transformation, the results were photographed by confocal laser scanning microscopy, scale bars = 10 µm. Chl indicates Chloroplast, BF indicate Bright field. nYFP and cYFP constructs were used as empty controls. MAP3K5-K375M and MKK4 were used as control.


( D ) MAP4K4 associates with BIK1 in vivo by Co - IP . BIK1 - HA MAP4K4 - GFP were co - expressed in by crossing of 35S : : BIK1 - HA 35S : : MAP4K4 - GFP transgenic lines . 14 days old were treated ( + ) with 1 µM flg22 or H2O ( - ) for 10 min . GFP trap beads were used for immunoprecipitation , target were detected by western blots using anti - GFP or anti - HA antibodies . predicted molecular of BIK1 - HA , MAP4K4 - GFP is 49 . 3 kDa 103 . 8 kDa respectively .

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

(D) MAP4K4 associates with BIK1 in vivo by Co-IP. BIK1-HA and MAP4K4-GFP were co-expressed in plants by crossing of 35S::BIK1-HA and 35S::MAP4K4-GFP transgenic lines. 14 days old plants were treated (+) with 1 µM flg22 or H2O (-) for 10 min. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of BIK1-HA, MAP4K4-GFP is 49.3 kDa and 103.8 kDa respectively.


( A ) Decreased BIK1 abundance in map4k4 - 1 . 35S : : BIK1 - HA was introduced into map4k4 - 1 by crossing , sibling lines carrying either map4k4 - 1 or MAP4K4 genotype were isolated from F3 generation . BIK1 were detected by western blot using anti - HA antibodies .

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

(A) Decreased BIK1 abundance in map4k4-1. 35S::BIK1-HA was introduced into map4k4-1 by crossing, sibling lines carrying either map4k4-1 or MAP4K4 genotype were isolated from F3 generation. BIK1 levels were detected by western blot analysis using anti-HA antibodies.


( B ) MAP4K4 regulates BIK1 accumulation through proteasome pathway . 10 days old 35S : : BIK1 - HA seedlings of WT or map4k4 - 1 were treated with DMSO ( - ) or proteasome inhibitor MG132 ( 50 µM ) for 5h , then were collected for immunoblot .

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(B) MAP4K4 regulates BIK1 accumulation through the proteasome pathway. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with DMSO (-) or the proteasome inhibitor MG132 (50 µM) for 5h, then the plants were collected for immunoblot analysis.


( C ) Flg22 - induced BIK1 phosphorylation . 10 days old 35S : : BIK1 - HA seedlings of WT or map4k4 - 1 were treated with H2O ( - ) or 1 µM flg22 for 15 min , BIK1 phosphorylation were detected by mobility - shift using anti - HA immunoblot .

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

(C) Flg22-induced BIK1 phosphorylation assay. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with H2O (-) or 1 µM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot.


( D , E F ) Identification of phosphorylation sites by LC - MS / MS . Five residues in GST - BIK1 - K105E were phosphorylated in vitro by HisMBP - MAP4K4 . peptide sequences are shown above spectrum with PH designating phosphorylated residue . In spectrum , matched fragment ions shows in red for terminal series ions in orange for immonium ions modified ions . ( G ) S233 T242 were required for BIK1 hyper - phosphorylation . BIK1 - HA , BIK1S233 - HA BIK1S242 - HA mutants were expressed in Col - 0 protoplasts treated with 100 nM flg22 for 15 min , BIK1 phosphorylation were detected by mobility - shift using anti - HA immunoblot . Each experiment was performed at least three times with similar results .

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

(D, E and F) Identification of phosphorylation sites by LC-MS/MS analysis. Five residues in GST-BIK1-K105E were phosphorylated in vitro by HisMBP-MAP4K4. The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions. (G) S233 and T242 were required for BIK1 hyper-phosphorylation. BIK1-HA, BIK1S233-HA and BIK1S242-HA mutants were expressed in Col-0 protoplasts and treated with 100 nM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot. Each experiment was performed at least three times with similar results.


( A ) MAP4K4 associates with PP2C38 in vivo by Co - IP . MAP4K4 - GFP PP2C38 - HA were co - expressed in protoplasts . GFP trap beads were used for immunoprecipitation , target were detected by western blots using anti - GFP or anti - HA antibodies . predicted molecular of MAP4K4 - GFP PP2C38 - HA is 103 . 8kDa 48 . 1 kDa respectively .

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

(A) MAP4K4 associates with PP2C38 in vivo by Co-IP. MAP4K4-GFP and PP2C38-HA were co-expressed in protoplasts. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of MAP4K4-GFP and PP2C38-HA is 103.8kDa and 48.1 kDa respectively.


( B ) Identification of phosphorylation sites by LC - MS / MS . In vitro phosphorylation site of HisMBP - PP2C38 by HisMBP - MAP4K4 . peptide is shown above spectrum with PH designating phosphorylated residue . In spectrum , matched fragment ions shows in red for terminal series ions in orange for immonium ions modified ions .

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

(B) Identification of phosphorylation sites by LC-MS/MS analysis. In vitro phosphorylation site of HisMBP-PP2C38 by HisMBP-MAP4K4. The peptide sequence is shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions.


( C ) S77 is required for flg22 - induced PP2C38 band shift . PP2C38 - HA PP2C38 - S77A - HA were expressed in Col - 0 map4k4 protoplasts treated with 1µM flg22 for 30 min . phosphorylated PP2C38 was detected by western blot mobility shift .

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(C) S77 is required for flg22-induced PP2C38 band shift. PP2C38-HA and PP2C38-S77A-HA were expressed in Col-0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min. The phosphorylated PP2C38 was detected by western blot mobility shift assay.


( D ) flg22 - induced PP2C38 band shift was compromised in map4k4 mutants . PP2C38 - HA were expressed in Col - 0 map4k4 protoplasts treated with 1µM flg22 for 30 min .

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(D) flg22-induced PP2C38 band shift was compromised in map4k4 mutants. PP2C38-HA were expressed in Col-0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min.


( E ) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity . HisMBP - MAP4K4 was incubated with HisMBP - PP2C38 or HisMBP - PP2C38D88N D289N ( phosphatase - inactive ) or alone , phosphorylation level of MAP4K4 was determined by Pro - Q Diamond phosphoprotein gel stain . experiments were performed three times with similar results .

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

(E) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity. HisMBP-MAP4K4 was incubated with HisMBP-PP2C38 or HisMBP-PP2C38D88N D289N (phosphatase-inactive) or alone, and the phosphorylation level of MAP4K4 was determined by Pro-Q Diamond phosphoprotein gel stain. All the experiments were performed three times with similar results.


( A ) BIK1 transgene restores flg22 - induced ROS burst in map4k4 - 1 . 35S : : BIK1 - HA WT or 35S : : BIK1 - HA map4k4 - 1 were generated by crossing as described before . ROS burst response in indicated genotypes in response to 1 μM flg22 . Values are means ± , n = 16 . Results are representative of three independent experiments . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - , samples sharing letters are not significantly different , p < 0 . 05 .

O O O B-GENEPROD O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE 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) BIK1 transgene restores flg22-induced ROS burst in map4k4-1. 35S::BIK1-HA WT or 35S::BIK1-HA map4k4-1 were generated by crossing as described before. ROS burst response in the indicated genotypes in response to 1 μM flg22. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


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

(B) Morphological phenotype of 6-week-old of Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants.


( C ) ROS burst response in Col - 0 , map4k4 - 1 , bik1 map4k4 - 1 bik1 mutants . Values are means ± , n = 16 . Results are representative of three independent experiments . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - , samples sharing letters are not significantly different , p < 0 . 05 .

O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O 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 O O O O O O O O O O O O O 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) ROS burst response in Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


( D ) Relative expression level of PR1 in Col - 0 , map4k4 - 1 , bik1 map4k4 - 1 bik1mutants by qRT - PCR . Values are means ± SD , n = 3 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - , samples sharing letters are not significantly different , p < 0 . 05 .

O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative expression level of PR1 in Col-0, map4k4-1, bik1 and map4k4-1 bik1mutants by qRT-PCR analysis. Values are means ± SD, n = 3 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


(E) Morphological phenotype of 6-week-old of Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants.


( F ) Relative expression level of PR1 in Col - 0 , map4k4 - 1 , map4k3 map4k4 - 1 map4k3 mutants by qRT - PCR . Values are means ± SD , n = 3 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - , samples sharing letters are not significantly different , p < 0 . 05 .

O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative expression level of PR1 in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants by qRT-PCR analysis. Values are means ± SD, n = 3 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


( G ) ROS burst response in Col - 0 , map4k4 - 1 , map4k3 map4k4 - 1 map4k3 mutants . Values are means ± , n = 16 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - , samples sharing letters are not significantly different , p < 0 . 05 .

O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O 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 O O O O O O O O O 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) ROS burst response in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants. Values are means ± SEM, n = 16 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


ACE2 - Ig potently neutralizes pseudotyped with S glycoproteins from SARS CoV 2019 nCoV . HIVs pseudotyped with S glycoprotein from CoVs were incubated with different fusion for 1 h before infection . Luciferase activities in target were measured , neutralization was calculated .

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

ACE2-Ig potently neutralizes viruses pseudotyped with S glycoproteins from the SARS CoV and 2019 nCoV.HIVs pseudotyped with the S glycoprotein from CoVs were incubated with different fusion proteins for 1 h before infection. Luciferase activities in target cells were measured, and the percent neutralization was calculated.


Inhibition of fusion by ACE2 - Ig . Potent inhibition of fusion mediated by SARS - CoV ( left ) or 2019 nCoV ( right ) spike ( S ) glycoprotein . expressing diffferent S glycoprotein were incubated with indicated fusion mixed with ACE2 - expressing . activity of reporter gene , β - gal , was measured as a correlate of fusion . curves represent best fit to experimental data were used for calculation of IC50 . Bars indicate S . E .

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

Inhibition of cell fusion by ACE2-Ig.Potent inhibition of cell fusion mediated by the SARS-CoV (left) or 2019 nCoV (right) spike (S) glycoprotein. Cells expressing diffferent S glycoprotein were incubated with indicated fusion proteins and mixed with ACE2-expressing cells. The activity of the reporter gene, β-gal, was measured as a correlate of fusion. The curves represent the best fit to the experimental data and were used for calculation of the IC50. Bars indicate the S.E.


A , B Whole blood or PBMC were treated with increasing concentrations of 24F4A stimulated with CpG - A for 16 h at 37 ° C . Anti - BDCA2 - mediated IFNα inhibition in whole - blood assays ( A ) . IFNα were detected using human IFNα ELISA . Graph depicts average of duplicate wells of one representative donor ( n = 10 ) . IC50 of 24F4A - mediated IFNα inhibition ( B ) in whole blood ( circles ) ( n = 10 ) PBMC ( n = 18 ) from healthy human donors ( squares ) or SLE ( triangles ) ( n = 11 ) . Horizontal bar represents mean IC50 for each sample type . Error bars represent SD of IC50 between donors .

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

A, B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG-A for 16 h at 37°C. Anti-BDCA2-mediated IFNα inhibition in whole-blood assays (A). IFNα levels were detected using human IFNα ELISA. Graph depicts average of duplicate wells of one representative donor (n = 10). IC50 of 24F4A-mediated IFNα inhibition (B) in whole blood (circles) (n = 10) and PBMC (n = 18) from healthy human donors (squares) or SLE patients (triangles) (n = 11). Horizontal bar represents the mean IC50 for each sample type. Error bars represent SD of IC50 between donors.


C Anti - BDCA2 - mediated internalization in whole - blood assays . Whole blood was treated with increasing concentrations of 24F4A for 16 h . Mean ( MFI ) of BDCA2 was determined with a non - cross - blocking anti - BDCA2 mAb ( 2D6 ) . Shown is a representative plot of 10 experiments conducted .

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

C Anti-BDCA2-mediated internalization in whole-blood assays. Whole blood was treated with increasing concentrations of 24F4A for 16 h. Mean fluorescence intensity (MFI) of BDCA2 was determined with a non-cross-blocking anti-BDCA2 mAb (2D6). Shown is a representative plot of 10 experiments conducted.


A Whole blood was treated with indicated concentrations of 24F4A or IgG1 isotype control antibody for 0 , 1 , 3 , 6 , 9 , 24 h . FACS was performed to determine BDCA2 on pDCs . minus one ( FMO ) represents background of BDCA2 . Shown is a representative plot of three independent experiments .

O O B-TISSUE O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O

A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0, 1, 3, 6, 9, and 24 h. FACS analysis was performed to determine BDCA2 levels on pDCs. Fluorescence minus one (FMO) represents background staining of BDCA2. Shown is a representative plot of three independent experiments.


B Isolated pDCs were treated with 10 μg / ml 24F4A - AF647 at 4 ° C or at 37 ° C analyzed at indicated time points . Subcellular localization of BDCA2 ( red ) compared to LAMP1 ( green ) was assessed using confocal microscopy . Phalloidin was used to delineate . Yellow represents co - localization of BDCA2 LAMP1 . Shown is a representative image of four experiments conducted .

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

B Isolated pDCs were treated with 10 μg/ml 24F4A-AF647 at 4°C or at 37°C and analyzed at the indicated time points. Subcellular localization of BDCA2 (red) compared to LAMP1 (green) was assessed using confocal microscopy. Phalloidin was used to delineate the cell membrane. Yellow represents co-localization of BDCA2 and LAMP1. Shown is a representative image of four experiments conducted.


C , D Whole blood was either treated with 24F4A for 1 h at 37 ° C ( pre - incubation ) or left untreated . PBMC were isolated from each condition . PBMC from untreated whole blood were subsequently treated with 10 μg / ml 24F4A , from conditions were stimulated with CpG - A for 16 h . Plot represents mean of duplicate wells . Flow cytometry was used to measure BDCA2 ( MFI ) . Shown is a representative donor of eight donors tested .

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

C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested.


E Whole blood was pre - treated for 0 , 1 , 3 , 6 , 9 h with 10 μg / ml of 24F4 or isotype control then stimulated with CpG - A for additional 16 h . IFNα were measured by ELISA . Shown is a representative plot of two independent experiments .

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

E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , flow cytometry was used to measure BDCA2 expression receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , HLADR + . A - C Prior to in vivo dosing , baseline surface of BDCA2 for both vehicle ( Ai ) 1 mg / kg ( Bi ) ( red , dotted ) were established by with fluorescently labeled 24F4A ( direct method ) . Maximal binding of 24F4A to BDCA2 was also established pre - dose in vehicle ( Aii ) 1 mg / kg ( Bii ) ( red , solid ) by treating whole blood with 10 μg / ml of 24F4A at 4 ° C then detecting bound 24F4A with a fluorescently labeled anti - human IgG1 ( indirect method ) . direct method was used to stain whole blood from both vehicle ( Aiii ) 1 mg / kg 24F4A ( Biii ) 6 h post - dose ( red , dotted ) . In a separate stain , indirect method was used to detect bound 24F4A in vehicle ( Aiv ) 1 mg / kg ( Biv ) treated ( black , solid ) . ( C ) BDCA2 internalization relative to pre - dose BDCA2 6 h post - dose with vehicle , 10 mg / kg , or 1 mg / kg 24F4A . Graph shows mean ± standard deviation for each group ( n = 3 ) .

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

Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A-C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , flow cytometry was used to measure BDCA2 expression receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , HLADR + . D PK / PD relationship between 24F4Aserum concentrations ( red triangle , left axis ) BDCA2 expression on pDCs ( black squares , right axis , normalized to pre - dose ) from 1 mg / kg group ( i - iii ) . Serum 24F4A was measured by ELISA . ( iv ) BDCA2 internalization versus serum of 24F4A for dosed cynomolgus monkeys at time points tested .

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

Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.D PK/PD relationship between 24F4Aserum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i-iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , flow cytometry was used to measure BDCA2 expression receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , HLADR + . E Whole blood from vehicle - or 1 mg / kg 24F4A - treated monkeys was stimulated with CpG - A , induction of IFN - I was measured by MxA bioassay at various time points pre - post - treatment . Horizontal black lines represent model - estimates of geometric mean of IFN - I in pre - post - dose samples . Duplicate symbols represent independent replicates of MxA bioassay for that time point . Statistical was performed using a two - way mixed - effects of variance ( ANOVA ) .

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

Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).


A - F Isolated human pDCs were treated with increasing concentrations of 24F4A ( red circles ) , 24F4A - ef ( black squares ) , or 10 μg / ml of isotype control ( black triangle ) stimulated with 5 μM of R848 ( A B ) , 1 μM of CpG - A ( C D ) , or immune complexes ( E F ) for 16 h at 37 ° C . IFNα concentrations in cultured supernatants were determined using ELISA . Representative plots for each stimulation ( A , C E ) average IC50 of 24F4A 24F4A - ef for each stimulation condition ( B , D F ) are shown ( n = 3 ) . Horizontal bars represent mean IC50 , error bars represent SD of mean from three independent experiments .

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

A-F Isolated human pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (black squares), or 10 μg/ml of the isotype control (black triangle) and stimulated with 5 μM of R848 (A and B), 1 μM of CpG-A (C and D), or immune complexes (E and F) for 16 h at 37°C. IFNα concentrations in cultured supernatants were determined using ELISA. Representative plots for each stimulation (A, C and E) and the average IC50 of 24F4A and 24F4A-ef for each stimulation condition (B, D and F) are shown (n = 3). Horizontal bars represent mean IC50, and error bars represent SD of the mean from three independent experiments.


G Isolated human pDCs were treated with 10 μg / ml of 24F4A ( black ) , 24F4A - ef ( gray shaded histogram ) , or isotype control ( gray dotted ) for 10 min at 37 ° C . Flow cytometry was used to evaluate pSYK pPLCγ2 . Shown is a representative plot of two independent experiments .

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

G Isolated human pDCs were treated with 10 μg/ml of 24F4A (black line), 24F4A-ef (gray shaded histogram), or the isotype control (gray dotted line) for 10 min at 37°C. Flow cytometry was used to evaluate pSYK and pPLCγ2. Shown is a representative plot of two independent experiments.


H Isolated pDCs treated with 10 μg / ml of anti - CD32a ( AT10 ) stimulated with CpG - A , R848 , or immune complexes . Error bars represent SD of inhibition of IFNα from three independent experiments .

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

H Isolated pDCs treated with 10 μg/ml of anti-CD32a (AT10) and stimulated with CpG-A, R848, or immune complexes. Error bars represent SD of percent inhibition of IFNα from three independent experiments.


A - F Isolated pDCs were treated with increasing concentrations of 24F4A ( red circles ) , 24F4A - ef ( squares ) , Chi - 6G6 ( triangle ) , or isotype control ( diamond ) for 16 h at 37 ° C stimulated with CpG - A . Flow cytometry was performed to evaluate MFI of ( A ) BDCA2 ( C ) CD32a on surface of pDCs . internalization of BDCA2 ( B ) CD32a ( D ) on pDCs treated with 10 μg / ml of 24F4A , 24F4A - ef , Chi - 6G6 , or isotype control . Error bars represent SD of internalization of five experiments conducted . ( E ) CD32a downmodulation on isolated pDCs treated with 10 μg / ml of an anti - CD40 mAb . Error bars represent SD of downmodulation of two independent experiments . FMO represents background of CD32a ( inverted triangle ) . ( F ) of maximal expression of BDCA2 or CD32a in presence of increasing concentrations of 24F4A or Chi - 6G6 . Maximal expression was defined by MFI of both BDCA2 CD32a on untreated . Shown is a representative plot of five independent experiments conducted .

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

A-F Isolated pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (squares), Chi-6G6 (triangle), or isotype control (diamond) for 16 h at 37°C and stimulated with CpG-A. Flow cytometry was performed to evaluate the MFI of (A) BDCA2 and (C) CD32a on the surface of pDCs. Percent internalization of BDCA2 (B) and CD32a (D) on pDCs treated with 10 μg/ml of 24F4A, 24F4A-ef, Chi-6G6, or isotype control. Error bars represent SD of percent internalization of five experiments conducted. (E) Percent CD32a downmodulation on isolated pDCs treated with 10 μg/ml of an anti-CD40 mAb. Error bars represent SD of percent downmodulation of two independent experiments. FMO represents background staining of CD32a (inverted triangle). (F) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi-6G6. Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells. Shown is a representative plot of five independent experiments conducted.


( A - C ) Distribution of spindle pole factors in centrosomal acentrosomal spindles . DMSO - treated control mitotic spindles ( 2 - centrosomes ) centrinone - treated acentrosomal spindles ( 0 - centrosome ) of HeLa . ( A ) Red , gray , green , blue represent NuMA , α - tubulin , Cep152 , , respectively . Z - projections of eight sections , every 0 . 3 μm . Scale bar , 5 μm . ( B ) Red , gray , blue represent NuMA , Katanin p60 , , respectively . Z - projections of five sections , every 0 . 3 μm . Scale bar , 5 μm . ( C ) Red , gray , blue represent p150glued , ASPM , , respectively . Z - projections of 10 ( 2 - centrosomes ) or five ( 0 - centrosome ) sections , every 0 . 3 μm . Scale bar , 5 μm .

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

(A-C) Distribution of spindle pole factors in centrosomal and acentrosomal spindles. DMSO-treated control mitotic spindles (2-centrosomes) and centrinone-treated acentrosomal spindles (0-centrosome) of HeLa cells. (A) Red, gray, green, and blue represent NuMA, α-tubulin, Cep152, and DNA, respectively. Z-projections of eight sections, every 0.3 μm. Scale bar, 5 μm. (B) Red, gray, and blue represent NuMA, Katanin p60, and DNA, respectively. Z-projections of five sections, every 0.3 μm. Scale bar, 5 μm. (C) Red, gray, and blue represent p150glued, ASPM, and DNA, respectively. Z-projections of 10 (2-centrosomes) or five (0-centrosome) sections, every 0.3 μm. Scale bar, 5 μm.


( D ) structure of NuMA microtubules in centrinone - treated acentrosomal spindles of HeLa . Two - way arrows indicate bipolarity of elongated NuMA structures . Gray , red , green blue represent α - tubulin , NuMA , Cep152 , respectively . Scale bar , 5 μm .

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

(D) The structure of NuMA and microtubules in centrinone-treated acentrosomal spindles of HeLa cells. Two-way arrows indicate the bipolarity of elongated NuMA structures. Gray, red, green and blue represent α-tubulin, NuMA, Cep152 and DNA, respectively. Scale bar, 5 μm.


( E ) localization of p150glued in centrinone - treated acentrosomal spindles of HeLa . Red , gray , green blue represent NuMA , p150glued , Cep152 , respectively . Scale bar , 5 μm .

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

(E) The localization of p150glued in centrinone-treated acentrosomal spindles of HeLa cells. Red, gray, green and blue represent NuMA, p150glued, Cep152 and DNA, respectively. Scale bar, 5 μm.


( F ) NuMA distribution in 3D . of NuMA ( Red ) chromosomes ( blue ) were reconstructed in 3D surface - rendered .

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

(F) NuMA distribution in 3D. Images of NuMA (Red) and chromosomes (blue) were reconstructed in 3D and surface-rendered.


( G ) Representative of NuMA ( arrowheads ) in centrosomal acentrosomal before after photobleaching . DMSO - or centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were observed . Time after photobleaching ( sec ) is indicated . Scale bar , 5 μm .

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

(G) Representative images of NuMA (arrowheads) in centrosomal and acentrosomal cells before and after photobleaching. DMSO- or centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed. Time after photobleaching (sec) is indicated. Scale bar, 5 μm.


( H ) Normalized intensities of NuMA - mClover at centrosomal poles ( control , 2 - centrosomes ) or at acentrosomal spindle pole ( 0 - centrosome ) during FRAP time course ( N = 20 for each condition ) . first three time points are before photobleaching . Error bars show standard deviation ( SD ) .

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

(H) Normalized fluorescence intensities of NuMA-mClover at centrosomal poles (control, 2-centrosomes) or at acentrosomal spindle pole (0-centrosome) during the FRAP time course (N=20 cells for each condition). The first three time points are before photobleaching. Error bars show standard deviation (SD).


( I ) Mobile fractions from normalized recovery curves of NuMA - mClover at centrosomal spindle poles ( control , 2 - centrosomes ) or at acentrosomal spindle poles ( 0 - centrosome ) . Mobile fractions ( roughly corresponds to normalized intensities at last time point were determined by single exponential fitting . error bars represent median interquartile range ( N = 20 for each condition ) . Mann - Whitney U ( two - ) was used to obtain a P value .

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

(I) Mobile fractions from the normalized recovery curves of NuMA-mClover at centrosomal spindle poles (control, 2-centrosomes) or at acentrosomal spindle poles (0-centrosome). Mobile fractions (roughly corresponds to normalized fluorescence intensities at the last time point were determined by single exponential fitting. Line and error bars represent the median and interquartile range (N=20 cells for each condition). The Mann-Whitney U test (two-tailed) was used to obtain a P value.


( A ) Time - lapse observation of establishment of bipolarity in acentrosomal . HeLa expressing EGFP - centrin1 mCherry - NuMA were observed with a 63 × objective . Magenta green represent mCherry - NuMA EGFP - centrin1 , respectively . Arrowheads indicate assembling NuMA after NEBD . Two - way arrows indicate bipolarity . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD .

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

(A) Time-lapse observation of the establishment of bipolarity in acentrosomal cells. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads indicate the assembling NuMA after NEBD. Two-way arrows indicate the bipolarity. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.


( B ) Time - lapse observation of dynamics of NuMA chromosomes . Centrinone - treated HeLa expressing mCover - NuMA were observed with a 63 × objective . Magenta green represent SiR - mClover - NuMA , respectively . Arrowheads two - way arrows indicate assembling NuMA after NEBD bipolarity , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD .

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

(B) Time-lapse observation of the dynamics of NuMA and chromosomes. Centrinone-treated HeLa cells expressing mCover-NuMA were observed with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads and two-way arrows indicate the assembling NuMA after NEBD and bipolarity, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.


( C ) timing of bipolarity establishment in acentrosomal pole , chromosome alignment , anaphase onset Each plot shows cumulative of each event at time points ( N = 49 from two independent experiments ) .

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

(C) The timing of bipolarity establishment in the acentrosomal pole, chromosome alignment, and anaphase onset Each plot shows the cumulative percentage of each event at individual time points (N=49 cells from two independent experiments).


( D ) distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed . Two - way arrow indicates bipolarity of elongated NuMA structures . Red , gray , green , blue represent NuMA , CENP - C , GT335 , , respectively . Scale bar , 5 μm .

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

(D) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells. Two-way arrow indicates the bipolarity of elongated NuMA structures. Red, gray, green, and blue represent NuMA, CENP-C, GT335, and DNA, respectively. Scale bar, 5 μm.


( E , F ) Time - lapse observation of structure of NuMA microtubules upon centrosome removal . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were observed with a 60 × objective . Red green represent NuMA SiR - tubulin , respectively . Z - projections of 17 sections , every 1 μm . Scale bar , 5 μm . Time zero corresponds to NEBD . ( F ) An example of NuMA formed several asters ( arrowheads ) at time of NEBD .

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

(E, F) Time-lapse observation of the structure of NuMA and microtubules upon centrosome removal. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed with a 60× objective. Red and green represent NuMA and SiR-tubulin, respectively. Z-projections of 17 sections, every 1 μm. Scale bar, 5 μm. Time zero corresponds to NEBD. (F) An example of NuMA formed several asters (arrowheads) at the time of NEBD.


( A ) NuMA structure upon microtubule depolymerization . Nocodazole - treated HCT116 CMV - OsTIR1 CEP152 - mClover - mAID were treated with 100 ng / ml nocodazole . Gray , red , green blue represent α - tubulin , NuMA , CEP152 ( mClover ) , respectively . Scale bar , 5 μm .

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

(A) NuMA structure upon microtubule depolymerization. Nocodazole-treated HCT116 CMV-OsTIR1 CEP152-mClover-mAID cells were treated with 100 ng/ml nocodazole. Gray, red, green and blue represent α-tubulin, NuMA, CEP152 (mClover) and DNA, respectively. Scale bar, 5 μm.


( B ) Immunostaining of DHC1 NuMA in centrinone - treated HCT116 TetOsTIR1 DHC1 - 3X - mAID - mClover . Gray , green , red blue represent DHC1 ( mClover ) , GT335 , NuMA , , respectively . Scale bar , 5 μm .

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

(B) Immunostaining of DHC1 and NuMA in centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells. Gray, green, red and blue represent DHC1 (mClover), GT335, NuMA, and DNA, respectively. Scale bar, 5 μm.


( D ) NuMA structure upon DHC1 depletion . Centrinone - treated HCT116 TetOsTIR1 DHC1 - 3X - mAID - mClover were treated with 1 μg / ml doxycycline ( Dox ) 500 μM indole - 3 - acetic acid ( IAA ) . Arrowhead indicates spindle pole . Green , red , gray blue represent DHC1 ( mClover ) , NuMA , GT335 , respectively . Scale bar , 5 μm . ( E ) Frequency of acentrosomal pole patterns upon DHC1 depletion in ( D ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 24 spindles in each experiment ) . (

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

(D) NuMA structure upon DHC1 depletion. Centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM indole-3-acetic acid (IAA). Arrowhead indicates the spindle pole. Green, red, gray and blue represent DHC1 (mClover), NuMA, GT335 and DNA, respectively. Scale bar, 5 μm. (E) Frequency of acentrosomal pole patterns upon DHC1 depletion in (D). Values are mean percentages ± SD. from three independent experiments (N≧24 spindles in each experiment). (


( F ) distribution of NuMA upon DHC1 depletion . Arrowheads indicate presence of NuMA on microtubules . Green , red , blue represent α - tubulin , NuMA , , respectively . Scale bars , 5 μm .

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

(F) The distribution of NuMA upon DHC1 depletion. Arrowheads indicate the presence of NuMA on microtubules. Green, red, and blue represent α-tubulin, NuMA, and DNA, respectively. Scale bars, 5 μm.


( G ) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA - mAID - mClover - FLAG with either mCherry - NuMA WT or 5A - 3 . Arrowheads indicate assembled NuMA structure . Green , red , gray blue represent endogenous NuMA ( mClover ) , expressed NuMA ( mCherry ) , GT335 , respectively . Z - projections of 21 sections , every 1 μm . Scale bar , 5 μm . ( H ) number of NuMA structure in ( G ) . Values are mean percentages ± SD . from four independent experiments ( N ≧ 15 spindles in each experiment ) . One - way ANOVA with Tukey ' s multiple comparisons was used to obtain a P value .

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

(G) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA-mAID-mClover-FLAG with either mCherry-NuMA WT or 5A-3. Arrowheads indicate the assembled NuMA structure. Green, red, gray and blue represent endogenous NuMA (mClover), expressed NuMA (mCherry), GT335 and DNA, respectively. Z-projections of 21 sections, every 1 μm. Scale bar, 5 μm. (H) The number of NuMA structure in (G). Values are mean percentages ± SD. from four independent experiments (N≧15 spindles in each experiment). One-way ANOVA with Tukey's multiple comparisons test was used to obtain a P value.


( A , B ) Structure of spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were treated with 1 μg / ml doxycycline ( Dox ) 500 μM IAA . Two - way arrow indicates bipolarity . Gray , green , red , blue represent α - tubulin , NuMA ( mClover ) , GT335 , , respectively . Z - projections of 10 sections , every 0 . 3 μm . Scale bar , 5 μm . ( C ) Frequency of spindle structure patterns upon NuMA depletion in ( A , B ) . Values are mean percentages ± SD . from three independent experiments ( N = 30 spindles in each experiment ) . (

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

(A, B) Structure of the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM IAA. Two-way arrow indicates the bipolarity. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (C) Frequency of spindle structure patterns upon NuMA depletion in (A, B). Values are mean percentages ± SD. from three independent experiments (N=30 spindles in each experiment). (


( D ) localization of Eg5 in centrinone - treated acentrosomal HeLa . Red , gray , green blue represent NuMA , Eg5 , GT335 , respectively . Scale bar , 5 μm .

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

(D) The localization of Eg5 in centrinone-treated acentrosomal HeLa cells. Red, gray, green and blue represent NuMA, Eg5, GT335 and DNA, respectively. Scale bar, 5 μm.


( E ) localization of Eg5 in spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were treated with Dox 500 μM IAA . Gray , green , red , blue represent Eg5 , NuMA ( mClover ) , CP110 , , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm .

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

(E) The localization of Eg5 in the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with Dox and 500 μM IAA. Gray, green, red, and blue represent Eg5, NuMA (mClover), CP110, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.


( F ) Structure of spindle upon Eg5 inhibition . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG were treated with 30 μM monastrol . Gray , green , red , blue represent α - tubulin , NuMA ( mClover ) , GT335 , , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm . ( G ) Frequency of spindle structure patterns upon Eg5 inhibition in ( F ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 23 spindles in each experiment ) .

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

(F) Structure of the spindle upon Eg5 inhibition. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 30 μM monastrol. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm. (G) Frequency of spindle structure patterns upon Eg5 inhibition in (F). Values are mean percentages ± SD. from three independent experiments (N≧23 spindles in each experiment).


( A ) Time - lapse observation of establishment of bipolarity within acentrosomal spindle pole upon inhibition of Eg5 . HeLa expressing EGFP - centrin1 mCherry - NuMA were observed in presence of 50 μM of monastrol with a 63 × objective . Magenta green represent mCherry - NuMA EGFP - centrin1 , respectively . Arrowheads two - way arrows indicate monopolar bipolar states , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( B ) time of bipolarity establishment being stuck in monopolar - like state of acentrosomal spindle poles in ( A ) . Each plot shows cumulative of each event at each time point ( N = total 75 from two independent experiments ) . (

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

(A) Time-lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed in the presence of 50 μM of monastrol with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads and two-way arrows indicate the monopolar and bipolar states, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (B) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (A). Each plot shows the cumulative percentage of each event at each time point (N=total 75 cells from two independent experiments). (


( C ) Time - lapse observation of dynamics of mClover - NuMA chromosomes . HeLa expressing mClover - NuMA were observed in presence of SiR - with a 63 × objective . Magenta green represent SiR - mClover - NuMA , respectively . Arrowheads indicate initial bipolarity in acentrosomal spindle pole ( 8 min ) , dispersed chromosomes ( 328 min ) , monopolar - like state of acentrosomal spindle pole ( 332 min ) , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( D ) time of bipolarity establishment being stuck in monopolar - like state of acentrosomal spindle poles in ( C ) . Each plot shows cumulative of each event at each time point ( N = total 35 from three independent experiments ) .

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

(C) Time-lapse observation of the dynamics of mClover-NuMA and chromosomes. HeLa cells expressing mClover-NuMA were observed in the presence of SiR-DNA with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads indicate initial bipolarity in the acentrosomal spindle pole (8 min), dispersed chromosomes (328 min), and the monopolar-like state of the acentrosomal spindle pole (332 min), respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (D) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (C). Each plot shows the cumulative percentage of each event at each time point (N=total 35 cells from three independent experiments).