E . Titrated TF - mediated inhibition ( 0 - 6 µM range ) of proOmpA - stimulated translocation ATPase activity ( as in C ) . Values were normalized ( effectmax = 100 % ; effectmin = 0 % ) and plotted ( n = 1 - 4 biological replicates ; mean values ± SEM ) versus the log10 [ TF2 ] . The Ki was determined using a variable slope fit ( log [ inhibitor ] versus normalized response ; GraphPad Prism ) .

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

E. Titrated TF-mediated inhibition (0-6 µM range) of proOmpA-stimulated translocation ATPase activity (as in C). Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 1-4 biological replicates; mean values ± SEM) versus the log10[TF2]. The Ki was determined using a variable slope fit (log[inhibitor] versus normalized response; GraphPad Prism).


F . SecB - mediated relief of TF - mediated inhibition of proOmpA translocation ATPase activity ( as in C ) . TF2 ( 1μM ) and SecB4 ( 0 - 6 µM ) were used . The T / M ratios were calculated , normalized ( as in D ) and plotted ( n = 2 - 3 biological replicates ; mean values ± SEM ) versus the log10 [ SecB4 ] . An apparent relief constant ( Krel ) was determined ( as in E ) .

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

F. SecB-mediated relief of TF-mediated inhibition of proOmpA translocation ATPase activity(as in C). TF2 (1μM) and SecB4 (0-6 µM) were used. The T/M ratios were calculated, normalized (as in D) and plotted (n= 2-3 biological replicates; mean values ± SEM) versus the log10[SecB4]. An apparent relief constant (Krel) was determined (as in E).


A . TF - proOmpA physical interaction in solution . proOmpA1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 37 ° C ; 60 min ) with the indicated molar excess of TF2 . Soluble proteins were analyzed on 10 % native - PAGE ( 4 mA ; 16h ; 4 ° C ) and Coomassie - blue stained . A representative experiment is shown ; n = 4 biological replicates

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

A. TF-proOmpA physical interaction in solution. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) with the indicated molar excess of TF2. Soluble proteins were analyzed on 10% native-PAGE (4 mA; 16h; 4°C) and Coomassie-blue stained. A representative experiment is shown; n= 4 biological replicates


C . TF solubilizes proOmpA * . proOmpA * 1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 37 ° C ; 60 min ) alone or in the presence of TF . Proteins in the soluble fraction were analyzed on 15 % SDS - PAGE and Coomassie - blue stained . Lane 2 : 2 . 84 μg proOmpA * . Representative experiment shown ; n = 5 biological replicates

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

C. TF solubilizes proOmpA*. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) alone or in the presence of TF. Proteins in the soluble fraction were analyzed on 15% SDS-PAGE and Coomassie-blue stained. Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 5 biological replicates


D . proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity . proOmpA ( 0 . 5 μM ) was either diluted directly ( lane 1 ) or after preincubation alone ( 10 min ; buffer D ; < 0 . 2 M urea ; either at 4 ° C or 37 ° C ) into reactions containing SecYEG - SecA alone or together with TF2 or SecB4 ( 2 and 4 x molar excess over proOmpA , respectively ) . T / M ratios ( as in Fig . 1D ) are shown ( n = 8 biological replicates ; mean values ± SEM ; Unpaired parametric t - test , 95 % confidence interval : * * : p = 0 . 0017 ; * * * * : p < 0 . 0001 )

O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity. proOmpA (0.5 μM) was either diluted directly (lane 1) or after preincubation alone (10 min; buffer D; < 0.2 M urea; either at 4°C or 37°C) into reactions containing SecYEG-SecA alone or together with TF2 or SecB4 (2 and 4 x molar excess over proOmpA, respectively). T/M ratios (as in Fig. 1D) are shown (n= 8 biological replicates; mean values ± SEM; Unpaired parametric t-test, 95% confidence interval: **: p = 0.0017; ****: p < 0.0001)


E . proOmpA pre - incubated at 37oC does not interact with TF . proOmpA1 ( 5 μM ) was preincubated ( Buffer D ; < 0 . 2 M urea ; 37 ° C ; 10 min ) or not ( lane 3 ) , prior to addition of the indicated molar excess of TF2 ( analyzed as in A ) . Representative experiment shown ; n = 4 biological replicates

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

E. proOmpA pre-incubated at 37oC does not interact with TF. proOmpA1 (5 μM) was preincubated (Buffer D; < 0.2 M urea; 37°C; 10 min) or not (lane 3), prior to addition of the indicated molar excess of TF2 (analyzed as in A). Representative experiment shown; n= 4 biological replicates


B . SecB protects proOmpA * from aggregation . proOmpA * 1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; 37 ° C ; 60 min ) alone or in the presence of the indicated molar excess of SecB4 , and proOmpA * in the soluble fraction ( after centrifugation ) was analyzed by SDS - PAGE ( as in Fig . 2C ) . Lane 2 : 2 . 84 μg proOmpA * . Representative experiment shown ; n = 4 biological

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

B. SecB protects proOmpA* from aggregation. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; 37°C; 60 min) alone or in the presence of the indicated molar excess of SecB4, and proOmpA* in the soluble fraction (after centrifugation) was analyzed by SDS-PAGE (as in Fig. 2C). Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 4 biological replicates


C . SecB physically interacts with proOmpA . proOmpA1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 4 ° C ; 60 min ) , with the indicated molar excess of SecB4 ( filled arrow ) , analyzed by native - PAGE as in Fig . 2A , and stained as indicated . brace : higher order proOmpA species . Representative experiment shown ; n = 5 biological replicates

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

C. SecB physically interacts with proOmpA. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 4°C; 60 min), with the indicated molar excess of SecB4 (filled arrow), analyzed by native-PAGE as in Fig. 2A, and stained as indicated. brace: higher order proOmpA species. Representative experiment shown; n= 5 biological replicates


B . Blow up of the ESI - mass spectrum of the TF : proOmpA : SecB4 super - complex from A ( complete dataset in Fig . EV3 ) . Left : charge state distributions , at the indicated m / z range . Right : masses deconvoluted from the mass spectra ( as in Fig . 2B ) . Inset cartoon depicts stoichiometry . Representative spectrum shown ; n = 2 biological replicates .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Blow up of the ESI-mass spectrum of the TF:proOmpA:SecB4 super-complex from A (complete dataset in Fig. EV3). Left: charge state distributions, at the indicated m/z range. Right: masses deconvoluted from the mass spectra (as in Fig. 2B). Inset cartoon depicts stoichiometry. Representative spectrum shown; n=2 biological replicates.


C . TF : proOmpA : SecB supercomplex analysis using GPC - MALS in Buffer A . TF1 : proOmpA1 ( 10 µM each ; green ) , SecB4 ( 40 µM ; blue ) and TF1 : proOmpA1 : SecB4 ( 10 , 10 and 40 µM , respectively ; black ) . n = 5 biological replicates ; mean mass : 137 kDa ( ± 6 kDa ) representative experiment shown ; red circles : determined mass ( kDa ) ; AU : arbitrary units .

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

C. TF:proOmpA:SecB supercomplex analysis using GPC-MALS in Buffer A. TF1:proOmpA1 (10 µM each; green), SecB4 (40 µM; blue) and TF1:proOmpA1:SecB4 (10, 10 and 40 µM, respectively; black). n= 5 biological replicates; mean mass: 137 kDa (± 6 kDa) representative experiment shown; red circles: determined mass (kDa); AU: arbitrary units.


A . SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA ( noC - tail ) in the presence of TF . proOmpA translocation ATPase activity stimulation ( as in Fig . 1C ; SecB molar excess over proOmpA indicated ) . T / M ratio of SecA ( noC - tail ) in the absence of TF or SecB = 100 % ; lane 1 ; all other values were expressed as a % of this value . n = 4 biological replicates ; mean values ± SEM . Unpaired parametric t - test , 95 % confidence interval : * * : p = 0 . 0048 ; * * * * : p < 0 . 0001 .

O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA(noC-tail) in the presence of TF. proOmpA translocation ATPase activity stimulation (as in Fig. 1C; SecB molar excess over proOmpA indicated). T/M ratio of SecA(noC-tail) in the absence of TF or SecB= 100%; lane 1; all other values were expressed as a % of this value. n= 4 biological replicates; mean values ± SEM. Unpaired parametric t-test, 95% confidence interval: **: p= 0.0048; ****: p < 0.0001.


B . Excess of SecB inhibits the proOmpA - stimulated translocation ATPase activity of SecA ( noCtail ) . ATPase measured as in Fig . 1C ; SecB concentration range : 0 - 6 µM . Values were normalized ( effectmax = 100 % ; effectmin = 0 % ) and plotted ( n = 3 biological replicates ; mean values ± SEM ) versus the log10 [ SecB4 ] . The Ki was determined as in Fig . 1E .

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

B. Excess of SecB inhibits the proOmpA-stimulated translocation ATPase activity of SecA(noCtail). ATPase measured as in Fig. 1C; SecB concentration range: 0-6 µM. Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 3 biological replicates; mean values ± SEM) versus the log10[SecB4]. The Ki was determined as in Fig. 1E.


C . TF and SecB - regulated , SecA - dependent in vitro translocation of proOmpA into SecYEG / A - IMVs , analyzed after proteolytic treatment . Translocated , protease - resistant proOmpA was visualized using immunostaining ( α - His ) in the absence or presence of TF2 or / and SecB4 ( as indicated ) . Translocation in the absence of TF or SecB was considered 100 % ( lane 3 ) ; all other values were expressed as a percentage of this . A summary of all repeats is presented ( mean ± SEM ; n = 6 biological replicates ; ns : not significant ( p = 0 . 1568 for lane 3 vs 4 ; p = 0 . 4835 for lane 3 vs 6 ) ; Unpaired parametric t - test , 95 % confidence interval : * * * * : p < 0 . 0001 ) with a representative western blot ( bottom ) .

O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. TF and SecB-regulated, SecA-dependent in vitro translocation of proOmpA into SecYEG/A-IMVs, analyzed after proteolytic treatment. Translocated, protease-resistant proOmpA was visualized using immunostaining (α-His) in the absence or presence of TF2 or/and SecB4 (as indicated). Translocation in the absence of TF or SecB was considered 100% (lane 3); all other values were expressed as a percentage of this. A summary of all repeats is presented (mean ± SEM; n= 6 biological replicates; ns: not significant (p = 0.1568 for lane 3 vs 4; p = 0.4835 for lane 3 vs 6); Unpaired parametric t-test, 95% confidence interval: ****: p < 0.0001) with a representative western blot (bottom).


Immunofluorescence staining of apoE44 ( n = 3 ) and apoE23 ( n = 2 ) genotyped iNPH patient right frontal cortex biopsy samples Two representative microscopy images from six separate images show : ( A ) Colocalization of ( red ) apoE44 and ( green ) FH in the presence of ( white ) Aβ plaques . Scale bar = 50 μm . ( B ) Colocalization of ( red ) apoE23 and ( green ) FH around brain capillaries . The blue nuclei were detected using DAPI staining . ( C ) Colocalization of ( red ) apoE44 and ( green ) FH in the presence of ( white ) Aβ plaques from a biopsy sample obtained from an iNPH patient diagnosed with Alzheimer ' s clinical syndrome ( ACS ) . Colocalization of apoE on Aβ is shown with an arrow . The negative staining control is shown . ( D ) ApoE - FH colocalization analysis of all detected colocalized foci in six microscope images from the apoE44 ( n = 3 ) and apoE23 ( n = 2 ) genotyped iNPH patient biopsy samples . Six microscope images from Alzheimer ' s clinical syndrome ( ACS ) biopsy sample are included in the apoE44 dataset ( red dots ) .

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

Immunofluorescence staining of apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples Two representative microscopy images from six separate images show: (A) Colocalization of (red) apoE44 and (green) FH in the presence of (white) Aβ plaques. Scale bar = 50 μm. (B) Colocalization of (red) apoE23 and (green) FH around brain capillaries. The blue nuclei were detected using DAPI staining. (C) Colocalization of (red) apoE44 and (green) FH in the presence of (white) Aβ plaques from a biopsy sample obtained from an iNPH patient diagnosed with Alzheimer's clinical syndrome (ACS). Colocalization of apoE on Aβ is shown with an arrow. The negative staining control is shown. (D) ApoE-FH colocalization analysis of all detected colocalized foci in six microscope images from the apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient biopsy samples. Six microscope images from Alzheimer's clinical syndrome (ACS) biopsy sample are included in the apoE44 dataset (red dots).


Immunofluorescence staining of apoE44 ( n = 3 ) and apoE23 ( n = 2 ) genotyped iNPH patient right frontal cortex biopsy samples ( I ) MST showing binding of NT647 - labeled full - length FH to different apoE isoforms , ( J ) NT647 - labeled apoE2 ( 1 - 165 ) fragment to increasing concentrations of FH402Y and FH402H variants and FH domains 1 - 4 and 19 - 20 and ( K ) NT647 - labeled recombinant N - terminal apoE2 ( 1 - 165 ) to increasing concentrations of FH5 - 7 402Y and FH5 - 7 402H fragments .

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

Immunofluorescence staining of apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples (I) MST showing binding of NT647-labeled full-length FH to different apoE isoforms, (J) NT647-labeled apoE2(1-165) fragment to increasing concentrations of FH402Y and FH402H variants and FH domains 1-4 and 19-20 and (K) NT647-labeled recombinant N-terminal apoE2(1-165) to increasing concentrations of FH5-7 402Y and FH5-7 402H fragments.


( B ) Different mixtures of apoE isoforms , FH , FH19 - 20 and Aβ1 - 42 were incubated for 72 h , and the presence of stable complexes was analyzed by running the sample in Tris - glycine PAGE for ( left and middle ) 90 or ( right ) 60 min at 100 V in the presence of 1 Bolt ™ LDS Sample Buffer ( Thermo Fisher ) . Gels were subjected to silver staining ( left ) or WB using anti - Aβ antibody ( middle and right ) . ApoE isoform and incubation time are shown on the top . The presence of each protein is indicated with arrows . Aβ1 - 42 aggregate and the stable complexes formed between apoE , Aβ1 - 42 and FH are shown near the top of the gel , while apoE2 / Aβ1 - 42 formed in the absence of FH are shown in several bands below apoE / Aβ1 - 42 / FH complexes .

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

(B) Different mixtures of apoE isoforms, FH, FH19-20 and Aβ1-42 were incubated for 72 h, and the presence of stable complexes was analyzed by running the sample in Tris-glycine PAGE for (left and middle) 90 or (right) 60 min at 100 V in the presence of 1 Bolt™ LDS Sample Buffer (Thermo Fisher). Gels were subjected to silver staining (left) or WB using anti-Aβ antibody (middle and right). ApoE isoform and incubation time are shown on the top. The presence of each protein is indicated with arrows. Aβ1-42 aggregate and the stable complexes formed between apoE, Aβ1-42 and FH are shown near the top of the gel, while apoE2/Aβ1-42 formed in the absence of FH are shown in several bands below apoE/Aβ1-42/FH complexes.


( C ) Single - molecule TIRF micrograph of Hylite 488 labeled Aβ1 - 42 and Alexa Fluor 568 C5 Maleimide labeled apoE2 . Scale bar = 5 μm . ( D ) Jitter plot of proportion of colocalized Aβ1 - 42 / apoE2 and Aβ1 - 42 / apoE3 over multiple fields of view in the presence and absence of FH . Statistics was calculated using Student ' s t - test from multiple images ( n = 12 ) in each sample . Error bars indicate SD values . (

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

(C) Single-molecule TIRF micrograph of Hylite 488 labeled Aβ1-42 and Alexa Fluor 568 C5 Maleimide labeled apoE2. Scale bar = 5 μm. (D) Jitter plot of proportion of colocalized Aβ1-42/apoE2 and Aβ1-42/apoE3 over multiple fields of view in the presence and absence of FH. Statistics was calculated using Student's t-test from multiple images (n=12) in each sample. Error bars indicate SD values. (


( E ) Jitter plot of Aβ1 - 42 foci stoichiometry in number of fluorophores colocalized or not with apoE2 and apoE3 and in the presence and absence of FH . Statistics of multiple images was calculated using two - sided Student ' s t - test . Statistics was calculated using Student ' s t - test from all detected colocalized foci in multiple images ( n = 12 ) in each sample . Error bars indicate SD values .

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

(E) Jitter plot of Aβ1-42 foci stoichiometry in number of fluorophores colocalized or not with apoE2 and apoE3 and in the presence and absence of FH. Statistics of multiple images was calculated using two-sided Student's t-test. Statistics was calculated using Student's t-test from all detected colocalized foci in multiple images (n=12) in each sample. Error bars indicate SD values.


( A and B ) Immunofluorescence staining of apoE44 ( n = 2 ) and apoE23 ( n = 2 ) genotyped iNPH patient right frontal cortex biopsy samples showing localization of FH ( green ) with Iba - 1 stained cells ( red ) and clustering around cells and brain capillaries . Presence of ( white ) Aβ plaques in apoE44 samples and ( arrow ) FH in both samples are shown . . C - E ( C ) Iba - 1 , ( D ) Aβ and ( E ) FH intensity analysis was calculated from all detected foci in six microscope images from the apoE44 ( n = 2 ) and apoE23 ( n = 2 ) genotyped iNPH patient biopsy samples .

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

(A and B) Immunofluorescence staining of apoE44 (n=2) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples showing localization of FH (green) with Iba-1 stained cells (red) and clustering around cells and brain capillaries. Presence of (white) Aβ plaques in apoE44 samples and (arrow) FH in both samples are shown.. C-E (C) Iba-1, (D) Aβ and (E) FH intensity analysis was calculated from all detected foci in six microscope images from the apoE44 (n=2) and apoE23 (n=2) genotyped iNPH patient biopsy samples.


( F ) Flow cytometric experiments showing binding of ( left ) 405 - labeled apoE4 and ( right ) 405 - labeled apoE2 to activated or inactivated CR3 on U937 empty and U937 - CR3 cells in the presence of FH , FH fragments 19 - 20 and 5 - 7 , BSA and Aβ1 - 42 .

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

(F) Flow cytometric experiments showing binding of (left) 405-labeled apoE4 and (right) 405-labeled apoE2 to activated or inactivated CR3 on U937 empty and U937-CR3 cells in the presence of FH, FH fragments 19-20 and 5-7, BSA and Aβ1-42.


( I ) Binding of 488 - labeled Aβ1 - 42 to ( left ) activated or inactivated CR3 on U937 empty and U937 - CR3 cells in the presence of FH19 - 20 , BSA , with combinations of apoE4 and FH , apoE2 and FH or apoE3 and FH . The fluorescence intensities of 488 - Aβ1 - 42 in each experiment was normalized against activated U937 empty cells sample .

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

(I) Binding of 488-labeled Aβ1-42 to (left) activated or inactivated CR3 on U937 empty and U937-CR3 cells in the presence of FH19-20, BSA, with combinations of apoE4 and FH, apoE2 and FH or apoE3 and FH. The fluorescence intensities of 488-Aβ1-42 in each experiment was normalized against activated U937 empty cells sample.


( A ) Immunofluorescence staining of iNPH patient biopsy samples ( apoE33 ) showing ( arrow ) colocalization of complement activator , ( red ) C1q , and complement regulator ( green ) FH . ( B ) ( arrow ) Colocalization of ( green ) apoE and ( red ) C1q on ( white ) Aβ plaques . Colocalization analysis is calculated from colocalized foci in two separate microscope images .

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

(A) Immunofluorescence staining of iNPH patient biopsy samples (apoE33) showing (arrow) colocalization of complement activator, (red) C1q, and complement regulator (green) FH. (B) (arrow) Colocalization of (green) apoE and (red) C1q on (white) Aβ plaques. Colocalization analysis is calculated from colocalized foci in two separate microscope images.


( D ) C3a formation in supernatant of C3 , factor B ( fB ) , factor D ( fD ) and factor I ( fI ) incubated beads coupled with apoE2 / 488 - Aβ1 - 42 , apoE4 / 488 - Aβ1 - 42 , apoE2 / 488 - Aβ1 - 42 / FH , apoE4 / 488 - Aβ1 - 42 / FH , 488 - Aβ1 - 42 / FH complexes . Beads with488 - Aβ1 - 42 / BSA complexes shows the level of C3a formation without inhibitor . Release of C3a is calculated as relative C3a increase % compared to the absorbance measured from 488 - Aβ1 - 42 / FH sample .

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

(D) C3a formation in supernatant of C3, factor B (fB), factor D (fD) and factor I (fI) incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH complexes. Beads with488-Aβ1-42/BSA complexes shows the level of C3a formation without inhibitor. Release of C3a is calculated as relative C3a increase % compared to the absorbance measured from 488-Aβ1-42/FH sample.


( G ) ( left ) Phagocytosis of complement C3 , fB , fD and fI ( C ) or only buffer ( no C ) incubated beads coupled with apoE2 / 488 - Aβ1 - 42 , apoE4 / 488 - Aβ1 - 42 , apoE2 / 488 - Aβ1 - 42 / FH , apoE4 / 488 - Aβ1 - 42 / FH , 488 - Aβ1 - 42 / FH and 488 - Aβ1 - 42 / BSA ( oligomerized Aβ = OAβ ) or with non - oligomerized 488 - Aβ1 - 42 ( monomeric Aβ = MAβ ) . Also , samples without apoE or FH were included in the assay ( buffer ) . ( right ) Cell permeability of C3 , fB , fD and fI incubated beads coupled with apoE2 / 488 - Aβ1 - 42 , apoE4 / 488 - Aβ1 - 42 , apoE2 / 488 - Aβ1 - 42 / FH , apoE4 / 488 - Aβ1 - 42 / FH , 488 - Aβ1 - 42 / FH and 488 - Aβ1 - 42 / BSA complexes ( OAβ ) or with MAβ . Also , cells incubated with only buffer were included in the assay ( buffer ) . Cell permeability is calculated by flow cytometry as % of DAPI stained cells in the cell population . The effect of CR3 expression in the phagocytosis was measured using PMA activated U937 cells with ( U937 CR3 cells ) or without ( U937 empty cells ) CR3 expression .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (left) Phagocytosis of complement C3, fB, fD and fI (C) or only buffer (no C) incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH and 488-Aβ1-42/BSA (oligomerized Aβ = OAβ) or with non-oligomerized 488-Aβ1-42 (monomeric Aβ = MAβ). Also, samples without apoE or FH were included in the assay (buffer). (right) Cell permeability of C3, fB, fD and fI incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH and 488-Aβ1-42/BSA complexes (OAβ) or with MAβ. Also, cells incubated with only buffer were included in the assay (buffer). Cell permeability is calculated by flow cytometry as % of DAPI stained cells in the cell population. The effect of CR3 expression in the phagocytosis was measured using PMA activated U937 cells with (U937 CR3 cells) or without (U937 empty cells) CR3 expression.


( A ) 488 - Aβ1 - 42 phagocytosis in the presence of FH and apoE isoform . Flow cytometry data are presented as mean fluorescence intensity of the cell population of the sample .

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

(A) 488-Aβ1-42 phagocytosis in the presence of FH and apoE isoform. Flow cytometry data are presented as mean fluorescence intensity of the cell population of the sample.


( B ) Comparison of mRNA transcripts ( two individual samples obtained from three times repeated experiments ) and MS1 intensities ( from 8 apoE44 / apoE43 and 9 apoE33 / 23 carriers ) between genes or proteins that showed differential expression in vitro ( Fig . 5B ) and in vivo .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of mRNA transcripts (two individual samples obtained from three times repeated experiments) and MS1 intensities (from 8 apoE44/apoE43 and 9 apoE33/23 carriers) between genes or proteins that showed differential expression in vitro (Fig. 5B) and in vivo.


( D ) ( left ) Combined analysis of complement activation markers C3 , C4 , CFH and Clusterin obtained from the detected MS1 spectra between samples from apoE44 ( n = 3 ) , apoE43 ( n = 5 ) , apoE33 ( n = 7 ) and apoE23 ( n = 2 ) carriers ( four markers / patient ) . ( center ) Combined analysis of complement activation markers C3 , C4 , CFH and Clusterin obtained from the detected MS1 spectra between samples that were positive ( n = 10 ) or negative ( n = 7 ) for Aβ pathology ( four markers / patient ) . ( right ) Expression levels of Phosphodiesterase A control , showing similar expression levels in frontal cortex , between samples from apoE44 ( n = 3 ) , apoE43 ( n = 5 ) , apoE33 ( n = 7 ) and apoE23 ( n = 2 ) carriers The data were normalized against neutral apoE33 samples with no apoE association .

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

(D) (left) Combined analysis of complement activation markers C3, C4, CFH and Clusterin obtained from the detected MS1 spectra between samples from apoE44 (n=3), apoE43 (n=5), apoE33 (n=7) and apoE23 (n=2) carriers (four markers/patient). (center) Combined analysis of complement activation markers C3, C4, CFH and Clusterin obtained from the detected MS1 spectra between samples that were positive (n=10) or negative (n=7) for Aβ pathology (four markers/patient). (right) Expression levels of Phosphodiesterase A control, showing similar expression levels in frontal cortex, between samples from apoE44 (n=3), apoE43 (n=5), apoE33 (n=7) and apoE23 (n=2) carriers The data were normalized against neutral apoE33 samples with no apoE association.


( B ) Effect of ND - 011992 on the intracellular ATP level in M . bovis BCG . The bacteria were treated with DMSO ( blue triangle ) , 100 nM Q203 alone ( green triangle ) , 20 μM ND - 011992 alone ( brown square ) , and a dose - range of ND - 011992 in the presence of Q203 ( red squares ) for 15 hours before quantification of intracellular ATP levels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of ND-011992 on the intracellular ATP level in M. bovis BCG. The bacteria were treated with DMSO (blue triangle), 100 nM Q203 alone (green triangle), 20 μM ND-011992 alone (brown square), and a dose-range of ND-011992 in the presence of Q203 (red squares) for 15 hours before quantification of intracellular ATP levels.


( C ) Oxygen consumption assay in M . bovis BCG using methylene blue as an oxygen sensor . ( D ) Oxygen consumption assay in M . bovis BCG using the Seahorse XFe96 Extracellular flux analyser . 12 μM ND - 011992 was injected alone ( green triangles ) , 100 nM Q203 ( blue circles ) , or in combination with Q203 ( red squares ) . For each condition , OCR readings were normalised to the last basal OCR reading before drug injection . OCR : Oxygen Consumption Rate . The dotted lines indicate the timepoints of drug injection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oxygen consumption assay in M. bovis BCG using methylene blue as an oxygen sensor. (D) Oxygen consumption assay in M. bovis BCG using the Seahorse XFe96 Extracellular flux analyser. 12 μM ND-011992 was injected alone (green triangles), 100 nM Q203 (blue circles), or in combination with Q203 (red squares). For each condition, OCR readings were normalised to the last basal OCR reading before drug injection. OCR: Oxygen Consumption Rate. The dotted lines indicate the timepoints of drug injection.


( E ) Dose - dependent inhibition of M . bovis BCG OCR by ND - 011992 ( in the presence of 100 nM Q203 ) measured on a Seahorse XFe96 analyser platform . For each condition , OCR readings were normalised to the last basal OCR reading before drug injection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dose-dependent inhibition of M. bovis BCG OCR by ND-011992 (in the presence of 100 nM Q203) measured on a Seahorse XFe96 analyser platform. For each condition, OCR readings were normalised to the last basal OCR reading before drug injection.


( F ) Effect of ND - 011992 on the OCR of M . smegmatis IMVs using the Oroboros O2k fluorespirometer . IMVs OCR from the parental strain ( green triangles ) , ∆ cydAB knockout ( blue circles ) , and ∆ cydAB complemented with M . tuberculosis CydABDC + ( red squares ) energized with NADH were determined . Q203 was used at 1 μM . 100 % OCR was defined as the OCR of the untreated samples for each strain .

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

(F) Effect of ND-011992 on the OCR of M. smegmatis IMVs using the Oroboros O2k fluorespirometer. IMVs OCR from the parental strain (green triangles), ∆cydAB knockout (blue circles), and ∆cydAB complemented with M. tuberculosis CydABDC+ (red squares) energized with NADH were determined. Q203 was used at 1 μM. 100% OCR was defined as the OCR of the untreated samples for each strain.


( A ) Differential gene expression analysis of H37Rv treated with Q203 , ND - 011992 , or combination ( H37Rv - Combo ) , and ∆ cydAB treated with Q203 compared to untreated controls . The log2 - fold differences in gene expression of various conditions relative to the untreated control is indicated using a sliding scale where higher expression is reflected in red and lower expression is reflected in blue , with a midpoint signifying no difference in white .

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

(A) Differential gene expression analysis of H37Rv treated with Q203, ND-011992, or combination (H37Rv-Combo), and ∆cydAB treated with Q203 compared to untreated controls. The log2-fold differences in gene expression of various conditions relative to the untreated control is indicated using a sliding scale where higher expression is reflected in red and lower expression is reflected in blue, with a midpoint signifying no difference in white.


( C ) Absence of the Cyt - bcc : aa3 sensitized H37Rv to ND - 011992 . M . tuberculosis H37Rv wildtype ( Blue circles ) , ∆ ctaE - qcrCAB ( red squares ) and complemented strain ( green triangles ) were treated with ND - 011992 . Growth inhibitory potency ( MIC50 ) was recorded after 2 to 3 weeks of incubation .

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

(C) Absence of the Cyt-bcc:aa3 sensitized H37Rv to ND-011992. M. tuberculosis H37Rv wildtype (Blue circles), ∆ctaE-qcrCAB (red squares) and complemented strain (green triangles) were treated with ND-011992. Growth inhibitory potency (MIC50) was recorded after 2 to 3 weeks of incubation.


( D ) The reduced minus oxidized difference spectrum of M . smegmatis ΔqcrCAB inverted membrane vesicles ( IMVs ) . The green line represents the spectrum without treatment , while the blue line represents the spectrum of the IMVs in the presence of 10 μM ND - 011992 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The reduced minus oxidized difference spectrum of M. smegmatis ΔqcrCAB inverted membrane vesicles (IMVs). The green line represents the spectrum without treatment, while the blue line represents the spectrum of the IMVs in the presence of 10 μM ND-011992.


( A ) Combined potency of ND - 011992 + Q203 against the drug - resistant clinical isolate M . tuberculosis 123 - 20 - 0047 .

O O O 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) Combined potency of ND-011992 + Q203 against the drug-resistant clinical isolate M. tuberculosis 123-20-0047.


( C ) Growth inhibition assay in M . bovis BCG . 106 bacteria were plated onto 7H10 agar supplemented with DMSO , 25 nM Q203 , 3 µM ND - 011992 , or 25 nM Q203 + 3 µM ND - 011992 .

O O O O O O O O O O O O O O O O O O O O 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) Growth inhibition assay in M. bovis BCG. 106 bacteria were plated onto 7H10 agar supplemented with DMSO, 25 nM Q203, 3 µM ND-011992, or 25 nM Q203 + 3 µM ND-011992.


( D ) Fluctuation analysis in M . tuberculosis H37Rv . M . tuberculosis ΔcydAB was plated on 7H10 containing 100 nM Q203 ( red ) . Parental H37Rv was plated on 7H10 with 100 nM Q203 and 6 µM ND - 011992 or 2 µg / mL rifampicin ( blue ) . The corresponding values of the median frequency of resistance is indicated in the graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluctuation analysis in M. tuberculosis H37Rv. M. tuberculosis ΔcydAB was plated on 7H10 containing 100 nM Q203 (red). Parental H37Rv was plated on 7H10 with 100 nM Q203 and 6 µM ND-011992 or 2 µg/mL rifampicin (blue). The corresponding values of the median frequency of resistance is indicated in the graph.


( A ) Bactericidal activity of ND - 011992 and ND - 011992 - Q203 combination in replicating M . tuberculosis H37Rv . Bedaquiline ( BDQ ) was used at 500 nM , and Q203 at 100 nM . Inoc . : starting inoculum ; DMSO : vehicle control . Bacteria viability was determined by enumerating colony - forming units ( CFU ) on nutrient - agar plates after 15 days of incubation . Dotted line represents 90 % reduction in CFU / mL compared to the initial inoculum at time 0 . * P < 0 . 05 ; * * P < 0 . 01 unpaired Student ' s t - test , one - tailed ; n = 3 ; comparing Q203 alone vs Q203 + ND - 011992 . Exact P - values ( from left to right ) : 0 . 0346 , 0 . 0143 , 0 . 00016 , 0 . 000052 . ( B ) Bactericidal activity of ND - 011992 and ND - 011992 - Q203 combination in nutrient - starved M . tuberculosis H37Rv . Bedaquiline ( BDQ ) was used at 500 nM , isoniazid was used at 1 μM , Q203 was used at 100 nM , and ND - 011992 was used at 3 , 6 , 12 , and 30 μM . Bacteria viability was determined by enumerating colony - forming units ( CFU ) after 15 days of incubation . Dotted line represents 90 % reduction in CFU / mL compared to the initial inoculum at time 0 . * * P < 0 . 01 unpaired Student ' s t - test , one - tailed ; n = 3 ; comparing Q203 alone vs Q203 + ND - 011992 . Exact P - values ( from left to right ) : 0 . 0098 , 0 . 000027 , 0 . 000067 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in replicating M. tuberculosis H37Rv. Bedaquiline (BDQ) was used at 500 nM, and Q203 at 100 nM. Inoc.: starting inoculum; DMSO: vehicle control. Bacteria viability was determined by enumerating colony-forming units (CFU) on nutrient-agar plates after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0. *P < 0.05; **P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0346, 0.0143, 0.00016, 0.000052. (B) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in nutrient-starved M. tuberculosis H37Rv. Bedaquiline (BDQ) was used at 500 nM, isoniazid was used at 1 μM, Q203 was used at 100 nM, and ND-011992 was used at 3, 6, 12, and 30 μM. Bacteria viability was determined by enumerating colony-forming units (CFU) after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0. **P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0098, 0.000027, 0.000067.


( C ) Bactericidal activity of ND - 011992 and ND - 011992 - Q203 combination in M . tuberculosis H37Rv under hypoxia . BDQ was used at 500 nM , isoniazid at 1 μM , metronidazole ( MTZ ) at 200 μM , Q203 at 100 nM , and ND - 011992 at 3 , 6 , 12 , and 30 μM . Bacteria viability was determined by enumerating colony - forming units ( CFU ) after 15 days of incubation . Dotted line represents 90 % reduction in CFU / mL compared to the initial inoculum at time 0 . * * P < 0 . 01 unpaired Student ' s t - test , one - tailed ; n = 3 ; comparing Q203 alone vs Q203 + ND - 011992 . Exact P - values ( from left to right ) : 0 . 0092 , 0 . 00010 , 0 . 00079 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in M. tuberculosis H37Rv under hypoxia. BDQ was used at 500 nM, isoniazid at 1 μM, metronidazole (MTZ) at 200 μM, Q203 at 100 nM, and ND-011992 at 3, 6, 12, and 30 μM. Bacteria viability was determined by enumerating colony-forming units (CFU) after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0.**P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0092, 0.00010, 0.00079.


( D ) Efficacy of ND - 011992 - Q203 combination treatment in a mouse model of acute tuberculosis . Bacterial load ( CFU ) were enumerated in the lungs of mice before treatment , and after 5 days of treatment with either the vehicle control ( brown squares ) , Q203 at 5 mg / kg ( red squares ) , ND - 011992 at 25 mg / kg ( green triangles ) , or ND - 011992 at 25mg / kg with Q203 at 5 mg / kg ( orange diamonds ) . * * P = 0 . 0006 unpaired Student ' s t - test , two - tailed ; n = 4 ; comparing Q203 alone vs Q203 + ND - 011992 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Efficacy of ND-011992-Q203 combination treatment in a mouse model of acute tuberculosis. Bacterial load (CFU) were enumerated in the lungs of mice before treatment, and after 5 days of treatment with either the vehicle control (brown squares), Q203 at 5 mg/kg (red squares), ND-011992 at 25 mg/kg (green triangles), or ND-011992 at 25mg/kg with Q203 at 5 mg/kg (orange diamonds). **P = 0.0006 unpaired Student's t-test, two-tailed; n = 4; comparing Q203 alone vs Q203 + ND-011992.


A , B . Depletion of Ccdc108 markedly affects bead movement by decreasing velocity Fluorescent beads were applied to the dorsal surface of Xenopus embryos . Bead tracks every 500 ms over 2 s are shown ( A ) . Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants , which can be fully rescued by co - injecting with Xenopus ccdc108 mRNA ( B ) . 150 beads from 15 embryos for each condition . Arrowheads indicate beads that showed no movement on the embryo surface through the recording . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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

A, B. Depletion of Ccdc108 markedly affects bead movement by decreasing velocity Fluorescent beads were applied to the dorsal surface of Xenopus embryos. Bead tracks every 500 ms over 2 s are shown (A). Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants, which can be fully rescued by co-injecting with Xenopus ccdc108 mRNA (B). 150 beads from 15 embryos for each condition. Arrowheads indicate beads that showed no movement on the embryo surface through the recording. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


C , D . Defective cilia formation in ccdc108 morphants . Embryos at stage 27 were fixed and stained with the acetylated tubulin ( Ac - tub ) antibody and / or Cep164 antibody . mRNA of a membrane - bound form of GFP ( mGFP ) was co - injected with each morpholino to indicate targeted cells . Representative confocal images ( C ) and 3D - SIM images ( D ) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos . ( C ) 20 images of 20 embryos and ( D ) > 80 MCCs from 6 embryos for each condition . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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

C, D. Defective cilia formation in ccdc108 morphants. Embryos at stage 27 were fixed and stained with the acetylated tubulin (Ac-tub) antibody and/or Cep164 antibody. mRNA of a membrane-bound form of GFP (mGFP) was co-injected with each morpholino to indicate targeted cells. Representative confocal images (C) and 3D-SIM images (D) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos. (C) 20 images of 20 embryos and (D) > 80 MCCs from 6 embryos for each condition. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


F . Defective cilia formation occurs in ccdc108 CRISPR mutants . Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 ( 108sg ) and fixed at stage 27 . Representative 3D - SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants . > 70 MCCs from 6 embryos for each condition . Cell membranes ( mGFP , purple ) , cilia ( Ac - tub , green ) and basal bodies ( Cep164 , yellow ) were labeled with indicated antibodies . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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

F. Defective cilia formation occurs in ccdc108 CRISPR mutants. Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 (108sg) and fixed at stage 27. Representative 3D-SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants. > 70 MCCs from 6 embryos for each condition. Cell membranes (mGFP, purple), cilia (Ac-tub, green) and basal bodies (Cep164, yellow) were labeled with indicated antibodies. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


G . In situ hybridization chain reaction ( HCR ) reveals that ccdc108 mRNA is specially detected in MCCs . Embryos at stage 35 were fixed and subjected to in situ HCR ( red ) . Embryos were then incubated with the Ac - tub antibody ( cyan ) and DAPI ( gray ) .

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

G. In situ hybridization chain reaction (HCR) reveals that ccdc108 mRNA is specially detected in MCCs. Embryos at stage 35 were fixed and subjected to in situ HCR (red). Embryos were then incubated with the Ac-tub antibody (cyan) and DAPI (gray).


C , D . Representative confocal images and plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts ( C ) and olfactory placodes ( D ) . Zebrafish embryos were fixed and stained with the Ac - tub antibody ( green ) , phalloidin ( purple ) and Hoechst ( blue ) . Data information : Quantitative data were from three independent repeats . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * p < 0 . 05 ) . Mean ± s . e . m values are presented .

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

C, D. Representative confocal images and plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts (C) and olfactory placodes (D). Zebrafish embryos were fixed and stained with the Ac-tub antibody (green), phalloidin (purple) and Hoechst (blue). Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). Mean ± s.e.m values are presented.


E . Scanning electron microscope images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes .

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

E. Scanning electron microscope images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes.


H . Representative 3D - SIM images and plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs . mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days and subjected to immunostaining . GFP - CETN1 ( blue ) marked mEPCs infected with lentivirus . Basal bodies ( CEP164 , purple ) and cilia ( Ac - tub , green ) were labeled with indicated antibodies . > 60 MCCs from three independent repeats for each condition were counted . Data information : Quantitative data were from three independent repeats . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * p < 0 . 05 ) . mean ± s . d . values are presented .

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

H. Representative 3D-SIM images and plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs. mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days and subjected to immunostaining. GFP-CETN1 (blue) marked mEPCs infected with lentivirus. Basal bodies (CEP164, purple) and cilia (Ac-tub, green) were labeled with indicated antibodies. > 60 MCCs from three independent repeats for each condition were counted. Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). mean ± s.d. values are presented.


A . Depletion of Ccdc108 causes disorganized basal body distribution in MCCs . Representative 3D - SIM images ( x - y ) and 3D - reconstructions ( x - z ) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells . Cell membranes ( mGFP , blue ) , basal bodies ( Cetn1 , green ) and distal appendages ( Cep164 , purple ) were labeled with indicated antibodies . Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations . Orange arrowheads and numbers mark individual centriole . > 70 MCCs from 6 embryos for each condition were counted . Data information : Quantitative data were from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ) . Mean ± s . d . values are presented .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Depletion of Ccdc108 causes disorganized basal body distribution in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells. Cell membranes (mGFP, blue), basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations. Orange arrowheads and numbers mark individual centriole. > 70 MCCs from 6 embryos for each condition were counted. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.


B . Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs . Orange arrowheads mark centrioles . Percentage of centrioles docked to plasma membrane in each field were scored . At least 7 cells for each sample were counted .

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

B. Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs. Orange arrowheads mark centrioles. Percentage of centrioles docked to plasma membrane in each field were scored. At least 7 cells for each sample were counted.


C . Representative 3D - SIM images reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs . Embryos were fixed and stained with phalloidin ( purple ) . Cilia ( Ac - tub , gray ) were also presented . Apical area of MCCs in each condition were measured ; 80 MCCs from 6 embryos for each condition . D . Representative confocal images show a significant reduction in apical actin following depletion of Ccdc108 . Cell boundaries and apical actin network were marked with mGFP ( green ) and phalloidin ( purple ) and phalloidin intensity levels measured and plotted . > 80 MCCs from 6 embryos for each condition . Data information : Quantitative data were from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ) . Mean ± s . d . values are presented .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3D-SIM images reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Apical area of MCCs in each condition were measured ; 80 MCCs from 6 embryos for each condition. D. Representative confocal images show a significant reduction in apical actin following depletion of Ccdc108. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple) and phalloidin intensity levels measured and plotted. > 80 MCCs from 6 embryos for each condition. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.


A . Live imaging of MCCs shows the distribution of Ccdc108 in MCCs cilia and basal bodies . Embryos expressing GFP - Ccdc108 ( green ) and Cetn4 - RFP ( purple ) were imaged at stage 27 . To better visualize ciliary structures , maximum intensity projections of images stacks for regions containing cilia and basal bodies are shown separately .

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

A. Live imaging of MCCs shows the distribution of Ccdc108 in MCCs cilia and basal bodies. Embryos expressing GFP-Ccdc108 (green) and Cetn4-RFP (purple) were imaged at stage 27. To better visualize ciliary structures, maximum intensity projections of images stacks for regions containing cilia and basal bodies are shown separately.


C . Ccdc108 interacts with IFT proteins . Interactions were further determined by coimmunoprecipitation of endogenous IFT proteins from HEK293T cells with GFP - Trap agarose beads .

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

C. Ccdc108 interacts with IFT proteins. Interactions were further determined by coimmunoprecipitation of endogenous IFT proteins from HEK293T cells with GFP-Trap agarose beads.


D . Live imaging of MCCs shows both Ccdc108 and Ift74 proteins localize to the ciliary axoneme and the basal bodies in MCCs . Embryos expressing mCherry - Ccdc108 ( green ) and GFP - Ift74 ( purple ) were imaged at stage 27 .

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

D. Live imaging of MCCs shows both Ccdc108 and Ift74 proteins localize to the ciliary axoneme and the basal bodies in MCCs. Embryos expressing mCherry-Ccdc108 (green) and GFP-Ift74 (purple) were imaged at stage 27.


A - C . The IFT - interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos : ( A ) impaired multiciliogenesis , ( B ) defective apical migration / docking of centrioles , and ( C ) reduced enrichment of apical F - actin . Embryos at stage 27 were fixed and stained with the acetylated tubulin ( A ; Ac - tub ; green ) , Cep164 ( B ; purple ) and Cetn1 ( B ; green ) antibodies or phalloidin ( purple ) . mRNA of a membrane - bound form of GFP was co - injected with each morpholino to indicate targeted cells ( A , B ) . Typical D - SIM images ( A , B ) and confocal images ( C ) are present . Embryos were treated as described in Figure 1C and expressed mGFP or GFP - Ccdc108 mutants . Plots show results from three independent experiments . ( A ) > 70 MCCs from 6 embryos , and ( C ) > 70 MCCs from 6 embryos for each condition . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are presented .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C. The IFT-interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos: (A) impaired multiciliogenesis, (B) defective apical migration/docking of centrioles, and (C) reduced enrichment of apical F-actin. Embryos at stage 27 were fixed and stained with the acetylated tubulin (A; Ac-tub; green), Cep164 (B; purple) and Cetn1 (B; green) antibodies or phalloidin (purple). mRNA of a membrane-bound form of GFP was co-injected with each morpholino to indicate targeted cells (A,B). Typical D-SIM images (A,B) and confocal images (C) are present. Embryos were treated as described in Figure 1C and expressed mGFP or GFP-Ccdc108 mutants. Plots show results from three independent experiments. (A) > 70 MCCs from 6 embryos, and (C) > 70 MCCs from 6 embryos for each condition. Unpaired two-tailed t-test was performed (*** p<0.001; **p<0.01). Mean ± s.d. values are presented.


A , ift74 CRISPR mutants display defective cilia formation . Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74 . Representative confocal images of the Xenopus epidermis , and plots display defective ciliation . ; 60 MCCs from 6 embryos Cell membranes ( mGFP ) , cilia ( Ac - tub , green ) were labeled with indicated antibodies . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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

A, ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative confocal images of the Xenopus epidermis, and plots display defective ciliation. ;60 MCCs from 6 embryos Cell membranes (mGFP), cilia (Ac-tub, green) were labeled with indicated antibodies. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


B . ift74 CRISPR mutants display defective cilia formation . Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74 . Representative 3D - SIM images of the Xenopus epidermis , and plots display defective ciliation . ; 70 MCCs from 6 embryos for each condition . Cell membranes ( mGFP ) , cilia ( Ac - tub , green ) and basal bodies ( Cep164 ) were labeled with indicated antibodies . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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

B. ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative 3D-SIM images of the Xenopus epidermis, and plots display defective ciliation. ;70 MCCs from 6 embryos for each condition. Cell membranes (mGFP), cilia (Ac-tub, green) and basal bodies (Cep164) were labeled with indicated antibodies. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C . Ift74 depletion affects centriole apical migration in MCCs . Representative 3D - SIM images ( x - y ) and 3D - reconstructions ( x - z ) of MCCs show failure of apical trafficking of basal bodies . Basal bodies ( Cetn1 , green ) and distal appendages ( Cep164 , purple ) were labeled with indicated antibodies . Quantitative plot shows a slight significant reduction in basal bodies by the depletion of Ift74 . > 70 MCCs from 6 embryos for each condition . D . Representative 3D - SIM images and plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion . > 70 MCCs from 6 embryos for each condition . Embryos were fixed and stained with phalloidin ( purple ) . Cilia ( Ac - tub , gray ) were also presented . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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

C. Ift74 depletion affects centriole apical migration in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs show failure of apical trafficking of basal bodies. Basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Quantitative plot shows a slight significant reduction in basal bodies by the depletion of Ift74. >70 MCCs from 6 embryos for each condition. D. Representative 3D-SIM images and plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion. >70 MCCs from 6 embryos for each condition. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


E . Representative confocal images and plot show a significant reduction in apical F - actin enrichment upon Ift74 depletion . > 70 MCCs from 6 embryos for each condition . Cell boundaries and apical actin network were marked with mGFP ( green ) and phalloidin ( purple ) . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images and plot show a significant reduction in apical F-actin enrichment upon Ift74 depletion. >70 MCCs from 6 embryos for each condition. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple). Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C - E . Ccdc108 depletion affects centriolar distribution of IFT - B proteins during centriole migration in MCCs . Embryos were co - injected with GFP - IFTs ( C : Ift74 ; D : Ift80 ; E : Ift88 ; green ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . HA tagged Ccdc108 proteins were co - expressed to test for the rescue of phenotypic defects . Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels . α - tubulin ( α - Tub ) served as loading control . Quantitative plots show that Ccdc108 - depleted MCCs showed a significant decrease of centriolar distribution of IFT - B proteins ( Ift74 , Ift80 and Ift88 ) , and Ccdc108 - IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT - B proteins . > 40 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are also presented .

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

C-E. Ccdc108 depletion affects centriolar distribution of IFT-B proteins during centriole migration in MCCs. Embryos were co-injected with GFP-IFTs (C: Ift74; D: Ift80; E: Ift88; green), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of IFT-B proteins (Ift74, Ift80 and Ift88), and Ccdc108-IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT-B proteins. >40 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are also presented.


A , B . Centriolar distribution of ciliary adhesion complex related proteins Fak and Cp110 are not reduced following Ccdc108 depletion . Embryos were co - injected with GFP - Cp110 ( A ) GFP - Fak ( B ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . > 30 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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

A, B. Centriolar distribution of ciliary adhesion complex related proteins Fak and Cp110 are not reduced following Ccdc108 depletion. Embryos were co-injected with GFP-Cp110 (A) GFP-Fak (B), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. >30 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C , D . Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs . Embryos were co - injected with GFP - Drg1 ( C ) GFP - RBD ( D ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . HA tagged Ccdc108 proteins were co - expressed to test for the rescue of phenotypic defects . Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels . α - tubulin ( α - Tub ) served as loading control . Quantitative plots show that Ccdc108 - depleted MCCs showed a significant decrease of centriolar distribution of Drg1 and RBD , and neither of Ccdc108 - IFT interaction deficient mutants can rescue the centriolar phenotypic defects . > 30 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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

C, D. Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs. Embryos were co-injected with GFP-Drg1 (C) GFP-RBD (D), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of Drg1 and RBD, and neither of Ccdc108-IFT interaction deficient mutants can rescue the centriolar phenotypic defects. >30 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


CSF extract was treated with DMSO , CsA , buffer or His - CnA420 - 508 at the indicated concentrations . Meiotic exit was induced by calcium addition and samples were taken at the indicated time points . Samples were immunoblotted for cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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

CSF extract was treated with DMSO, CsA, buffer or His-CnA420-508 at the indicated concentrations. Meiotic exit was induced by calcium addition and samples were taken at the indicated time points. Samples were immunoblotted for cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was treated with DMSO or CsA . Both reactions were divided and supplemented with the indicated amounts of calcium or H2O . After 20min , samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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

CSF extract was treated with DMSO or CsA. Both reactions were divided and supplemented with the indicated amounts of calcium or H2O. After 20min, samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was supplemented with Myc - XErp1 wt IVT at the indicated dilutions . An empty IVT reaction not expressing Myc - XErp1 served as control . All reactions were divided and treated with DMSO or CsA . Calcium was added to all reactions and samples were taken at the indicated time points , treated with λ - phosphatase and immunoblotted for XErp1 , the Myc - tag , cyclin B2 and α - tubulin . Asterisks indicate unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 wt IVT at the indicated dilutions. An empty IVT reaction not expressing Myc-XErp1 served as control. All reactions were divided and treated with DMSO or CsA. Calcium was added to all reactions and samples were taken at the indicated time points, treated with λ-phosphatase and immunoblotted for XErp1, the Myc-tag, cyclin B2 and α-tubulin. Asterisks indicate unspecific bands.


CSF extract was supplemented with Myc - XErp1 wt IVT . The reaction was divided and treated with DMSO or CsA . Both reactions were treated with calcium , samples were taken at the indicated time points and as indicated incubated with λ - phosphatase . Samples were immunoblotted for XErp1 and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 wt IVT. The reaction was divided and treated with DMSO or CsA. Both reactions were treated with calcium, samples were taken at the indicated time points and as indicated incubated with λ-phosphatase. Samples were immunoblotted for XErp1 and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.


CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT and as indicated treated with DMSO or CsA . Both reactions were treated with calcium and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag , cyclin B2 and α - tubulin . A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A . Arrow marks the meiotic phosphorylation state of Myc - XErp1 . Asterisk indicates unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT and as indicated treated with DMSO or CsA. Both reactions were treated with calcium and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag, cyclin B2 and α-tubulin. A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A. Arrow marks the meiotic phosphorylation state of Myc-XErp1. Asterisk indicates unspecific bands.


CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT that was either wild - type or mutated to alanine at the p90RSK - target sites S335 , T336 and S342 ( Rsk3A ) . Both reactions were divided and treated with DMSO or CsA . Calcium was added and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT that was either wild-type or mutated to alanine at the p90RSK-target sites S335, T336 and S342 (Rsk3A). Both reactions were divided and treated with DMSO or CsA. Calcium was added and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.


CSF extract was treated with Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT . An empty IVT reaction not expressing XErp1 was used as control . The extracts were treated with DMSO or CsA as indicated . Calcium was added , samples were taken at the indicated time points and immunoblotted for cyclin B2 , pSer335 XErp1 and α - tubulin . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisks indicate unspecific bands .

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

CSF extract was treated with Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT. An empty IVT reaction not expressing XErp1 was used as control. The extracts were treated with DMSO or CsA as indicated. Calcium was added, samples were taken at the indicated time points and immunoblotted for cyclin B2, pSer335 XErp1 and α-tubulin. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisks indicate unspecific bands.


Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT was in vitro phosphorylated by recombinant PKA and isolated by α - Myc immunoprecipitation . The immunoprecipitate was supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1 . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisk indicates the IgG heavy chain .

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

Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT was in vitro phosphorylated by recombinant PKA and isolated by α-Myc immunoprecipitation. The immunoprecipitate was supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisk indicates the IgG heavy chain.


CSF extract was supplemented with mRNA encoding Flag - Cdc20 that was either wild - type or mutated to serine at Thr64 , Thr68 and Thr79 ( 3TS ) . After expression , calcium was added , samples were taken at the indicated time points and resolved by either conventional or Phos - tagTM SDS - PAGE . Samples were immunoblotted for the Flag - tag , Cdc27 and cyclin B2 .

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

CSF extract was supplemented with mRNA encoding Flag-Cdc20 that was either wild-type or mutated to serine at Thr64, Thr68 and Thr79 (3TS). After expression, calcium was added, samples were taken at the indicated time points and resolved by either conventional or Phos-tagTM SDS-PAGE. Samples were immunoblotted for the Flag-tag, Cdc27 and cyclin B2.


CSF extract was supplemented with Myc - XErp1 wt IVT and treated with DMSO or CsA . Calcium was added , samples were taken at the indicated time points , resolved by either conventional or Phos - tagTM SDS - PAGE and immunoblotted for Cdc20 , Cdc27 and cyclin B2 . A control sample was treated with λ - phosphatase . A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig . EV4A . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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

CSF extract was supplemented with Myc-XErp1 wt IVT and treated with DMSO or CsA. Calcium was added, samples were taken at the indicated time points, resolved by either conventional or Phos-tagTM SDS-PAGE and immunoblotted for Cdc20, Cdc27 and cyclin B2. A control sample was treated with λ-phosphatase. A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig. EV4A. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was supplemented with Flag - Cdc20 wt IVT coupled to α - Flag beads . Calcium was added and Flag - Cdc20 was isolated by α - Flag immunoprecipitation at the indicated time points . Input and pellet ( IP : Flag ) samples were immunoblotted for the Flag - tag , pThr68 Cdc20 and p150 ( Glued ) . The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2 . Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain . Asterisks in other immunoblots indicate unspecific bands .

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

CSF extract was supplemented with Flag-Cdc20 wt IVT coupled to α-Flag beads. Calcium was added and Flag-Cdc20 was isolated by α-Flag immunoprecipitation at the indicated time points. Input and pellet (IP: Flag) samples were immunoblotted for the Flag-tag, pThr68 Cdc20 and p150(Glued). The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2. Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain. Asterisks in other immunoblots indicate unspecific bands.


CSF extract was supplemented with Flag - Cdc20 wt IVT . Flag - Cdc20 was re - isolated by α - Flag immunoprecipitation , supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the Flag - tag and pThr68 Cdc20 . The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA . Asterisk indicates the IgG heavy chain .

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

CSF extract was supplemented with Flag-Cdc20 wt IVT. Flag-Cdc20 was re-isolated by α-Flag immunoprecipitation, supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the Flag-tag and pThr68 Cdc20. The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA. Asterisk indicates the IgG heavy chain.


A Affinity measurements by biolayer interferometry ( BLI ) of bipNbs NM1267 and NM1268 . NM1267 and NM1268 were applied with concentrations ranging from 5 - 0 . 625 nM and 20 ‑ 2 . 5 nM , respectively ( illustrated with gradually lighter shades ) on immobilized RBD derived from SARS - CoV - 2 B . 1 ( RBDB . 1 ) . Global 1 : 1 fits are illustrated as dashed lines . Global 1 : 1 fits are illustrated as dashed lines and binding affinity ( KD ) , association ( kon ) and dissociation constant ( koff ) determined for the individual bipNbs are summarized .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Affinity measurements by biolayer interferometry (BLI) of bipNbs NM1267 and NM1268. NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD derived from SARS-CoV-2 B.1 (RBDB.1). Global 1:1 fits are illustrated as dashed lines. Global 1:1 fits are illustrated as dashed lines and binding affinity (KD), association (kon) and dissociation constant (koff) determined for the individual bipNbs are summarized.


A - P Affinity measurements by BLI of bipNb NM1267 and NM1268 on recently identified RBD variants B . 1 . 1 . 7 ( Alpha ) ( A , I ) , B . 1 . 351 ( Beta ) ( B , J ) , P1 ( Gamma ) ( C , K ) , B . 1 . 617 . 2 ( Delta ) ( D , L ) , B . 1 . 429 ( Epsilon ) ( E , M ) , P3 ( Theta ) ( F , N ) , B . 1 . 617 . 1 ( Kappa ) ( G , O ) and A . 23 . 1 ( H , P ) . NM1267 and NM1268 were applied with concentrations ranging from 5 - 0 . 625 nM and 20 ‑ 2 . 5 nM , respectively ( illustrated with gradually lighter shades ) on immobilized RBD variants . Global 1 : 1 fits are illustrated as dashed lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-P Affinity measurements by BLI of bipNb NM1267 and NM1268 on recently identified RBD variants B.1.1.7 (Alpha) (A, I), B.1.351 (Beta) (B, J), P1 (Gamma) (C, K), B.1.617.2 (Delta) (D, L), B.1.429 (Epsilon) (E, M), P3 (Theta) (F, N), B.1.617.1 (Kappa) (G, O) and A.23.1 (H, P). NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD variants. Global 1:1 fits are illustrated as dashed lines.


A - F Neutralization potency of NM1267 and NM1268 were analyzed in Caco ‑ 2 cells using the SARS - CoV - 2 B . 1 ( A , D ) SARS - CoV - 2 B . 1 . 351 ( Beta ) ( B , E ) and SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) ( C , F ) . Infection normalized to virus - only infection control is illustrated as percent of infection ( infection [ % ] ) . Data are presented as mean ± SEM of three ( n = 3 ) biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Neutralization potency of NM1267 and NM1268 were analyzed in Caco‑2 cells using the SARS-CoV-2 B.1 (A, D) SARS-CoV-2 B.1.351 (Beta) (B, E) and SARS-CoV-2 B.1.617.2 (Delta) (C, F). Infection normalized to virus-only infection control is illustrated as percent of infection (infection [%]). Data are presented as mean ± SEM of three (n = 3) biological replicates.


C Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 15 ) or NM1267 ( n = 12 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . survival were monitored for 14 days . , * * * * P < 0 . 0001 , by log - rank

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 15) or NM1267 (n = 12) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1. survival were monitored for 14 days. , ****P < 0.0001, by log-rank test


D Nasal swabs were collected from six mice per group ( n = 6 ) at the indicated time points and viral load was determined by plaque - assay . Bars represent mean ± SEM . * * P < 0 . 01 , by unpaired t

O O O O O O O O O O O O O O O O O O O O O O O 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 Nasal swabs were collected from six mice per group (n = 6) at the indicated time points and viral load was determined by plaque-assay. Bars represent mean ± SEM. **P < 0.01, by unpaired t test


Histopathological analysis of mice , which were intranasally treated with bipNb NM1267 or control Nb NM1251 and subsequently infected with 3 * 103 PFU SARS - CoV - 2 B . 1 . A - B Serial tissue sections revealed severe inflammation ( H & E ) and numerous widespread SARS - CoV - 2 RNA positive alveolar epithelia cells and macrophages ( in situ hybridization ( ISH ) ) in lungs of infected , control - treated mice . In infected and NM1267 - treated animals no inflammation or only small focal areas with inflammation and a few SARS - CoV - 2 RNA positive cells were observed . Scale bars represent 1 mm and 200 µm , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Histopathological analysis of mice, which were intranasally treated with bipNb NM1267 or control Nb NM1251 and subsequently infected with 3*103 PFU SARS-CoV-2 B.1. A-B Serial tissue sections revealed severe inflammation (H&E) and numerous widespread SARS-CoV-2 RNA positive alveolar epithelia cells and macrophages (in situ hybridization (ISH)) in lungs of infected, control-treated mice. In infected and NM1267-treated animals no inflammation or only small focal areas with inflammation and a few SARS-CoV-2 RNA positive cells were observed. Scale bars represent 1 mm and 200 µm, respectively.


B , Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 8 ) , NM1267 ( n = 9 ) or NM1268 ( n = 9 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) . Weight loss were monitored for 14 days . Dashed line indicates humane endpoint and symbols represent mean ± SEM * * * * P < 0 . 0001 in orange between NM1251 and NM1267 and in purple between NM1251 and NM1268 , * P < 0 . 1 in black between NM1267 and NM1268 , by two - way ANOVA with Sidak ' s multiple comparison

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 8), NM1267 (n = 9) or NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2 (Delta). Weight loss were monitored for 14 days. Dashed line indicates humane endpoint and symbols represent mean ± SEM ****P < 0.0001 in orange between NM1251 and NM1267 and in purple between NM1251 and NM1268, *P < 0.1 in black between NM1267 and NM1268, by two-way ANOVA with Sidak's multiple comparison test