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

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

(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 B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-ORGANISM O O O B-DISEASE O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

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

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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 .

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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 .

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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 .

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

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

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 .

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

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

A 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 B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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 .

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


C 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 ) . survival were monitored for 14 days . * * * P < 0 . 001 in orange between NM1251 and NM1267 and * * * * P < 0 . 0001 in purple between NM1251 and NM1268 , by log - rank

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

C 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). survival were monitored for 14 days. ***P < 0.001 in orange between NM1251 and NM1267 and ****P < 0.0001 in purple between NM1251 and NM1268, by log-rank test


( a ) Knockdown of dRagA and dRagC decreased dS6K phosphorylation ( Thr 398 ) . Drosophila S2 cells untreated ( lane 2 ) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies .

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

(a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (Thr 398). Drosophila S2 cells untreated (lane 2) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies.


( b ) dRagA and dRagC are not required for dAkt phosphorylation . Drosophila S2 cells untreated ( lanes 1 - 3 ) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min . Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated . NC , negative control RNAi .

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

(b) dRagA and dRagC are not required for dAkt phosphorylation. Drosophila S2 cells untreated (lanes 1-3) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min. Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated. NC, negative control RNAi.


( c ) dRagA and dRagC function in parallel with TSC2 and PTEN . dRagA and / or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi , as indicated . dTSC2 or dPTEN RNAi treatment increased pdS6K ( Thr 398 ) and this increase was compromised by dRagA or / and dRagC RNAi . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

(c) dRagA and dRagC function in parallel with TSC2 and PTEN. dRagA and/or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi, as indicated. dTSC2 or dPTEN RNAi treatment increased pdS6K (Thr 398) and this increase was compromised by dRagA or/and dRagC RNAi. Full scans of blots are provided in Supplementary Information, Fig. S6.


( a ) Constitutively active RagA and RagB stimulate S6K phosphorylation . 200 ng of each mammalian RagA , RagB , RagC or RagD construct was co - transfected with HA - S6K ( 20 ng ) into HEK293 cells . Their corresponding dominant - negative mutants ( RagAT21N , RagBT54N , RagCS75N , RagDS76N ) and constitutively active mutants ( RagAQ66L , RagBQ99L , RagCQ120L , RagDQ121L ) were also tested . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated .

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

(a) Constitutively active RagA and RagB stimulate S6K phosphorylation. 200 ng of each mammalian RagA, RagB, RagC or RagD construct was co-transfected with HA-S6K (20 ng) into HEK293 cells. Their corresponding dominant-negative mutants (RagAT21N, RagBT54N, RagCS75N, RagDS76N) and constitutively active mutants (RagAQ66L, RagBQ99L, RagCQ120L, RagDQ121L) were also tested. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.


( b ) RagA has a dominant role over RagC in regulating S6K phosphorylation . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) . The different Rag mutants used in the transfection are indicated at the top of each lane .

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

(b) RagA has a dominant role over RagC in regulating S6K phosphorylation. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng). The different Rag mutants used in the transfection are indicated at the top of each lane.


( c ) Rag regulates TORC1 but not TORC2 activity . Each indicated Rag construct ( 200 ng ) was co - transfected with either HA - S6K ( 20 ng ) , Myc - 4EBP1 ( 20 ng ) or of GST - Akt ( 100 ng ) . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

(c) Rag regulates TORC1 but not TORC2 activity. Each indicated Rag construct (200 ng) was co-transfected with either HA-S6K (20 ng), Myc-4EBP1 (20 ng) or of GST-Akt (100 ng). Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


( a ) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) into HEK293 cells . Cells were starved of amino acids for 1 h before collection . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated .

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

(a) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng) into HEK293 cells. Cells were starved of amino acids for 1 h before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.


( b ) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid ( AA ) stimulation . pcDNA3 ( 200 ng , lanes 1 and 2 ) or each indicated Rag construct was co - transfected with HA - S6K into HEK293 cells . Cells were starved of amino acids for 1 h ( - AA ) and either remained in the starvation medium or were stimulated with amino - acid - containing - medium ( + AA ) for 30 min before collection . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated .

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

(b) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid (AA) stimulation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K into HEK293 cells. Cells were starved of amino acids for 1 h (-AA) and either remained in the starvation medium or were stimulated with amino-acid-containing-medium (+AA) for 30 min before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.


( c ) RagAT21N and RagCQ120L suppress insulin - induced stimulation of S6K phosphorylation . pcDNA3 ( 200 ng , lanes 1 and 2 ) or each indicated Rag construct was co - transfected with HA - S6K ( 20 ng ) or GST - Akt ( 100 ng ) into HeLa cells . Cells were serum - starved overnight and stimulated with insulin ( 400 nM ) for 30 min . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

(c) RagAT21N and RagCQ120L suppress insulin-induced stimulation of S6K phosphorylation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K (20 ng) or GST-Akt (100 ng) into HeLa cells. Cells were serum-starved overnight and stimulated with insulin (400 nM) for 30 min. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


( a ) dRagA positively regulates wing compartment size . Wild - type or mutant dRagA transgenes were expressed in posterior compartments with the en - GAL4 driver . The ratios of representative posterior to anterior compartment areas are shown . Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression . Data are mean ± s . d . , * P = 7 . 32 × 10 − 4 ( n = 7 ) , * * P = 6 . 18 × 10 − 4 ( n = 12 ) , Student ' s two - tailed t - test , ( n is the number of adult wings analysed ) .

O O O B-GENEPROD O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) dRagA positively regulates wing compartment size. Wild-type or mutant dRagA transgenes were expressed in posterior compartments with the en-GAL4 driver. The ratios of representative posterior to anterior compartment areas are shown. Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression. Data are mean ± s.d., *P = 7.32 × 10−4 (n = 7), **P = 6.18 × 10−4 (n = 12), Student's two-tailed t-test, (n is the number of adult wings analysed).


( b ) dRagA positively regulates wing cell size . The average area of posterior compartment cells from en - GAL4 UAS - dRagA adult wings is shown . Cells expressing dRagAQ61L are significantly larger and dRagAT61N - expressing cells are smaller than controls . Data are mean ± s . d . , * P = 1 . 15 × 10 − 3 ( n = 7 ) , * * P = 0 . 025 ( n = 12 ) , Student ' s 2 - tailed t - test ( n represents number of adult wings analysed ) .

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

(b) dRagA positively regulates wing cell size. The average area of posterior compartment cells from en-GAL4 UAS-dRagA adult wings is shown. Cells expressing dRagAQ61L are significantly larger and dRagAT61N-expressing cells are smaller than controls. Data are mean ± s.d., *P = 1.15 × 10−3 (n = 7), **P = 0.025 (n = 12), Student's 2-tailed t-test (n represents number of adult wings analysed).


( c , d ) dRag GTPases positively regulate larval fat body cell size . ( c ) Cell area of clonally - induced dRagA - expressing cells or dRagC homozygous mutant cells relative to neighbouring wild - type control cells is shown . Cell area was determined from phalloidin - stained fixed fat body samples from fed or 48 h starved larvae . Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions . Cells expressing dRagAT61N and dRagC loss - of - function cells were significantly smaller than control cells only under nutrient replete conditions . Data are means ± s . d . , * P = 2 . 04 × 10 − 3 ( n = 5 ) , * * P = 2 . 94 × 10 − 6 ( n = 14 ) , * * * P = 3 . 79 × 10 − 7 ( n = 14 ) , * * * * P = 1 . 36 × 10 − 5 ( n = 30 ) , Student ' s two - tailed t - test . ( d ) Representative examples of fat body cells with altered dRagA activity . Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels , and dRagC homozygous mutant cells are marked by absence of GFP in the right panel . Scale bar represents 50 μm .

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

(c, d) dRag GTPases positively regulate larval fat body cell size. (c) Cell area of clonally-induced dRagA-expressing cells or dRagC homozygous mutant cells relative to neighbouring wild-type control cells is shown. Cell area was determined from phalloidin-stained fixed fat body samples from fed or 48 h starved larvae. Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions. Cells expressing dRagAT61N and dRagC loss-of-function cells were significantly smaller than control cells only under nutrient replete conditions. Data are means ± s.d., *P = 2.04 × 10−3 (n = 5), **P = 2.94 × 10−6 (n = 14), ***P = 3.79 × 10−7 (n = 14), ****P = 1.36 × 10−5 (n = 30), Student's two-tailed t-test. (d) Representative examples of fat body cells with altered dRagA activity. Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels, and dRagC homozygous mutant cells are marked by absence of GFP in the right panel. Scale bar represents 50 μm.


( a ) Rag acts through TORC1 to regulate S6K phosphorylation . HEK293 cells were transfected with constructs as indicated . Co - expression of of mTORKD construct ( 600 ng ) or rapamycin treatment ( rapa , 20 nM , 30 min ) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation . The protein level of mTORKD was determined by immunoblotting with anti - mTOR antibody .

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

(a) Rag acts through TORC1 to regulate S6K phosphorylation. HEK293 cells were transfected with constructs as indicated. Co-expression of of mTORKD construct (600 ng) or rapamycin treatment (rapa, 20 nM, 30 min) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation. The protein level of mTORKD was determined by immunoblotting with anti-mTOR antibody.


( b ) RagA / RagC and TSC1 / TSC2 independently regulate S6K phosphorylation . HEK293 cells were transfected with 200 ng of each Rag and / or ′ constructs as indicated . Amino acid starvation for 1 h ( - AA ) is indicated . Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies , as indicated .

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

(b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or ′ constructs as indicated. Amino acid starvation for 1 h (-AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.


( c ) TSC2 and RagA / B independently affect S6K phosphorylation . HA - S6K ( 20 ng ) was transfected into HeLa cells with or without RNAi against human TSC2 , RagA and RagB as indicated .

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

(c) TSC2 and RagA/B independently affect S6K phosphorylation. HA-S6K (20 ng) was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated.


( d ) RagAT21N and RagCQ120L do not block Rheb - induced S6K phosphorylation . RagAT21N and RagCQ120L ( 200 ng each ) were transfected into HEK293 cells with or without Rheb construct ( 20 ng ) . S6K was included in the co - transfection . Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

(d) RagAT21N and RagCQ120L do not block Rheb-induced S6K phosphorylation. RagAT21N and RagCQ120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


( a , b ) dRagC is not required for Rheb - induced cell growth . ( a ) The area of Rheb - overexpressing cells in control or dRagC mutant ( dRagC − / − ) backgrounds under fed conditions , relative to that of neighbouring control cells which were assigned a value of 1 . Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds . Data are mean ± s . d . , * P = 0 . 034 ( n = 5 ) , * * P = 6 . 1 × 10 − 3 ( n = 5 ) , Student ' s two - tailed t - test ( n represents number of experimental samples ) . ( b ) A representative example of a clone of Rheb - overexpressing cells in a dRagC − / − animal . Rheb transgene - expressing cells are marked by co - expression of GFP . Cell boundaries are labelled by phalloidin staining in red ; nuclei are labelled by DAPI in blue . Scale bar represents 50 μm .

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

(a, b) dRagC is not required for Rheb-induced cell growth. (a) The area of Rheb-overexpressing cells in control or dRagC mutant (dRagC−/−) backgrounds under fed conditions, relative to that of neighbouring control cells which were assigned a value of 1. Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds. Data are mean ± s.d., *P = 0.034 (n = 5), **P = 6.1 × 10−3 (n = 5), Student's two-tailed t-test (n represents number of experimental samples). (b) A representative example of a clone of Rheb-overexpressing cells in a dRagC−/− animal. Rheb transgene-expressing cells are marked by co-expression of GFP. Cell boundaries are labelled by phalloidin staining in red; nuclei are labelled by DAPI in blue. Scale bar represents 50 μm.


( c , d ) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells . ( c ) Relative area of clonally - induced Rheb26 . 2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body . Clonally induced Rheb26 . 2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild - type and in dRagAQ61L expressing backgrounds . Data are mean ± s . d . , * P = 2 . 91 × 10 − 4 ( n = 7 ) , * * P = 2 . 59 × 10 − 8 ( n = 5 ) , Student ' s two - tailed t - test ( fed conditions where n represents number of experimental samples ) . ( d ) A representative example of Rheb homozygous mutant cells ( marked by lack of GFP , arrows ) in fat body ubiquitously expressing UAS - dRagAQ61L . GFP - positive control cells in this experiment are a mixture of Rheb + / − and Rheb + / + . Scale bar represents 50 μm .

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

(c, d) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells. (c) Relative area of clonally-induced Rheb26.2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body. Clonally induced Rheb26.2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild-type and in dRagAQ61L expressing backgrounds. Data are mean ±s.d., *P = 2.91 × 10−4 (n = 7), **P = 2.59 × 10−8 (n = 5), Student's two-tailed t-test (fed conditions where n represents number of experimental samples). (d) A representative example of Rheb homozygous mutant cells (marked by lack of GFP, arrows) in fat body ubiquitously expressing UAS-dRagAQ61L. GFP-positive control cells in this experiment are a mixture of Rheb+/− and Rheb+/+. Scale bar represents 50 μm.


( a - d ) dRagAQ61L suppresses autophagy . ( a ) Drosophila fat body cells clonally expressing dRagAQ61L ( marked in green by GFP expression ) failed to accumulate autolysosomes ( shown in surrounding control cells by punctate Lysotracker Red staining ) in response to starvation for 4 h . Nuclei are marked in blue by DAPI . ( b - d ) Induction of autophagosomes in response to 4 - h starvation is shown by the punctate pattern of GFP - Atg8a expression in control fat body cells ( b ) , but not in cells expressing dRagAQ61L ( c ) . The average number of GFP - Atg8a - marked autophagosomes per cell in control and dRagAQ61L - expressing clones is shown ( d ) . Data are mean ± s . d . , * P = 2 . 91 × 10 − 6 , Students two - tailed t - test ( n = 33 fat body samples imaged per genotype ) . Scale bars represent 25 μm in each panel .

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

(a-d) dRagAQ61L suppresses autophagy. (a) Drosophila fat body cells clonally expressing dRagAQ61L (marked in green by GFP expression) failed to accumulate autolysosomes (shown in surrounding control cells by punctate Lysotracker Red staining) in response to starvation for 4 h. Nuclei are marked in blue by DAPI. (b-d) Induction of autophagosomes in response to 4-h starvation is shown by the punctate pattern of GFP-Atg8a expression in control fat body cells (b), but not in cells expressing dRagAQ61L (c). The average number of GFP-Atg8a-marked autophagosomes per cell in control and dRagAQ61L-expressing clones is shown (d). Data are mean ± s.d., *P = 2.91 × 10−6, Students two-tailed t-test (n = 33 fat body samples imaged per genotype). Scale bars represent 25 μm in each panel.


( e ) RagA regulates LC3 conversion in mammalian cells . Myc - LC3 was co - transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated . One day after transfection , cells were cultured in amino - acid - sufficienct medium ( + AA ) or amino - acid - depleted medium ( − AA ) for 4 h before collection . Western blotting for Myc - LC3 and HA - Rag were performed . Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant - negative RagA . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

(e) RagA regulates LC3 conversion in mammalian cells. Myc-LC3 was co-transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated. One day after transfection, cells were cultured in amino-acid-sufficienct medium (+AA) or amino-acid-depleted medium (−AA) for 4 h before collection. Western blotting for Myc-LC3 and HA-Rag were performed. Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant-negative RagA. Full scans of blots are provided in Supplementary Information, Fig. S6.