HeLa were released from single thymidine block , after 7 hr , BAY 1816032 or DMSO was added for 5 hr . were then fixed subjected to immunofluorescence Example of anaphase are shown ( D ) . Scale bars , 10 μm .

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HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining Example images of anaphase cells are shown (D). Scale bars, 10 μm.


E HeLa were released from single thymidine block , after 7 hr , BAY 1816032 or DMSO was added for 5 hr . were then fixed subjected to immunofluorescence of anaphases with PICH was determined ( E ) .

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E HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining The percentage of anaphases with PICH was determined (E).


HeLa were arrested in a prometaphase - like state with nocodazole treatment for 5hr , then BAY 1816032 or DMSO was added for 1 hr in continued presence of nocodazole . Then , nocodazole was removed by washing into fresh medium containing BAY 1816032 or DMSO . After 1 hr , were fixed subjected to immunofluorescence . Example are shown ( F ) . of anaphases with PICH was determined ( G ) . Arrows point to UFBs . Scale bars , 10 μm .

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

HeLa cells were arrested in a prometaphase-like state with nocodazole treatment for 5hr, and then BAY 1816032 or DMSO was added for 1 hr in the continued presence of nocodazole. Then, nocodazole was removed by washing into fresh medium containing BAY 1816032 or DMSO. After 1 hr, cells were fixed and subjected to immunofluorescence staining. Example images are shown (F). The percentage of anaphases with PICH was determined (G). Arrows point to the UFBs. Scale bars, 10 μm.


HeLa Sgo1 - K492A transfected with indicated siRNA were synchronized treated with BAY 1816032 or DMSO then immunostained with indicated antibodies . Example of anaphase are shown ( H ) . of anaphases with PICH was determined in at least 256 in each condition . Means S . D . s from three independent experiments are shown ( I ) . Example of prometaphase immunostained with indicated antibodies are shown ( J ) . Data information : Means error bars representing S . D . are shown unpaired t ) . Scale bars , 10 μm .

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HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO and then immunostained with the indicated antibodies. Example images of anaphase cells are shown (H). The percentage of anaphases with PICH was determined in at least 256 cells in each condition. Means and S.D.s from three independent experiments are shown (I). Example images of prometaphase cells immunostained with the indicated antibodies are shown (J). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


HeLa Sgo1 - K492A transfected with indicated siRNA were synchronized treated with BAY 1816032 or DMSO Lysates of nocodazole - arrested mitotic were immunoblotted ( K ) .

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HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO Lysates of nocodazole-arrested mitotic cells were immunoblotted (K).


A HeLa were transfected with plasmids encoding CB - GFP or CB - TOP2A - GFP . Lysates of asynchronously growing were immunoblotted with indicated antibodies .

O O O 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 HeLa cells were transfected with plasmids encoding CB-GFP or CB-TOP2A-GFP. Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies.


HeLa transfected with siRNA plasmids encoding indicated were released from single thymidine block . At 12 hr post - release , were fixed subjected to immunofluorescence with DAPI antibodies for PICH , GFP CENP - C . Example of anaphase are shown ( B ) . of anaphases with PICH was determined in at least 152 in each condition . Means S . D . s from three independent experiments are shown ( C ) . Arrows point to UFBs . Data information : Means error bars representing S . D . are shown unpaired t ) . Scale bars , 10 μm .

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

HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells were fixed and subjected to immunofluorescence staining with DAPI and the antibodies for PICH, GFP and CENP-C. Example images of anaphase cells are shown (B). The percentage of anaphases with PICH was determined in at least 152 cells in each condition. Means and S.D.s from three independent experiments are shown (C). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


HeLa transfected with siRNA plasmids encoding indicated were released from single thymidine block . At 12 hr post - release , Lysates of nocodazole - arrested mitotic were immunoblotted ( D ) . * represents non - specific bands .

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

HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells Lysates of nocodazole-arrested mitotic cells were immunoblotted (D). * represents non-specific bands.


E , F HeLa transiently expressing CB - GFP or CB - TOP2A - GFP were released from single thymidine block , after 7 hr , BAY 1816032 or DMSO was added for 5 hr . were then fixed subjected to immunofluorescence as in ( B ) . Example of anaphase are shown ( E ) . of anaphases with PICH was determined in at least 142 in each condition . Means S . D . s from three independent experiments are shown ( F ) . Arrows point to UFBs . Data information : Means error bars representing S . D . are shown ; unpaired t ) . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F HeLa cells transiently expressing CB-GFP or CB-TOP2A-GFP were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining as in (B). Example images of anaphase cells are shown (E). The percentage of anaphases with PICH was determined in at least 142 cells in each condition. Means and S.D.s from three independent experiments are shown (F). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.


HeLa transfected with siRNA plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized then subjected to immunofluorescence with DAPI , ACA antibodies for PICH GFP . Example of anaphase are shown ( G ) . Arrows point to UFBs . Scale bars , 10 μm .

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

HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. Example images of anaphase cells are shown (G). Arrows point to the UFBs. Scale bars, 10 μm.


HeLa transfected with siRNA plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized then subjected to immunofluorescence with DAPI , ACA antibodies for PICH GFP . of anaphases with PICH was determined in at least 174 in each condition . Means S . D . s from three independent experiments are shown ( H ) . Data information : Means error bars representing S . D . are shown unpaired t ) .

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

HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. The percentage of anaphases with PICH was determined in at least 174 cells in each condition. Means and S.D.s from three independent experiments are shown (H). Data information: Means and error bars representing S.D. are shown unpaired t test).


HeLa transfected with siRNA plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized then Lysates of asynchronous were immunoblotted ( I ) .

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

HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then Lysates of asynchronous cells were immunoblotted (I).


( A ) H1299 were transfected with Myc - β - catenin , CK1α MDMX mutants . Phosphorylation of β - catenin S45 was determined by western blot . Co - expression of MDMX blocked increase of pS45 level . MDMXSG ( W200S / W201G ) MDMX - 1 - 300 were defective for CK1α binding .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1299 cells were transfected with Myc-β-catenin, CK1α and MDMX mutants. Phosphorylation of β-catenin S45 was determined by western blot. Co-expression of MDMX blocked the increase of pS45 level. MDMXSG (W200S/W201G) and MDMX-1-300 were defective for CK1α binding.


( B ) GST - β - catenin - 1 - 200 was incubated with purified FLAG - CK1α , FLAG - MDMX ATP at indicated ratios . Phosphorylation of β - catenin S45 was determined by western blot . MDMXS289A contains mutation of CK1α phosphorylation site .

O O O O O O O O O O O O O O O O O O O O O O O O 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) GST-β-catenin-1-200 was incubated with purified FLAG-CK1α, FLAG-MDMX and ATP at indicated ratios. Phosphorylation of β-catenin S45 was determined by western blot. MDMXS289A contains mutation of CK1α phosphorylation site.


( C ) Kinetic characterization of CK1α inhibition by MDMX . Reaction velocity as a function of β - catenin peptide substrate increasing MDMX concentrations at 100 µM ATP 2 nM CK1α . Data were fitted to Michaelis - Menten equation . resulting parameter values are shown in table .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kinetic characterization of CK1α inhibition by MDMX. Reaction velocity as a function of β-catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation. The resulting parameter values are shown in the table.


( D ) Reaction velocity as a function of ATP increasing MDMX concentrations at 800 µM peptide substrate 2 nM CK1α . Data were fitted to Michaelis - Menten equation .

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(D) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation.


( A , B ) H1299 human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX analyzed by western blot .

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

(A, B) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot.


( C ) H1299 were transiently transfected with CK1α plasmid analyzed for β - catenin phosphorylation by western blot .

(C) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β-catenin phosphorylation by western blot.


( D ) fractions of H1299 stably expressing MDMX were analyzed for indicated markers .

(D) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers.


( E ) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β - catenin S45 kinase activity in a titration .

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) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β-catenin S45 kinase activity in a titration assay.


( F ) Extracts of H1299 separately transfected with MDMX or CK1α were mixed in presence of CK1α inhibitors . In vitro formation of MDMX - CK1α was detected by MDMX IP followed by CK1α western blot .

O O O O O O O O O O O O O O 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) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors. In vitro formation of MDMX-CK1α complex was detected by MDMX IP followed by CK1α western blot.


( A , B , C , D ) were treated with MDMX siRNA for 48 hrs analyzed for β - catenin pS45 level by western blot .

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(A, B, C, D) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β-catenin pS45 level by western blot.


( E ) H1299 293T were treated with indicated siRNA for 48 hrs . activity of β - catenin S45 kinase in extracts was determined using an ELISA . results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 05 ( Student ' s t ) .

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

(E) H1299 and 293T cells were treated with indicated siRNA for 48 hrs. The activity of β-catenin S45 kinase in cell extracts was determined using an ELISA assay. The results are average of 3 experiments (mean +/- SD). *p<0.05 (Student's t test).


( B GST - β - catenin - 1 - 200 was incubated with ATP , CK1α GST - MDMX mutants at indicated ratios . level of pS45 was determined by western blot .

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(B GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.


C , D ) GST - β - catenin - 1 - 200 was incubated with ATP , CK1α GST - MDMX mutants at indicated ratios . level of pS45 was determined by western blot .

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C, D) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.


( B ) Purified FLAG - CK1α was incubated with ATP , GST - MDMX , GST - β - catenin - 1 - 200 , pDI peptide ( LTFEHYWAQLTS , 5 µM ) , pDI3A peptide ( LTAEHYAAQATS , 5 µM ) . β - catenin pS45 level was determined by western blot .

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(B) Purified FLAG-CK1α was incubated with ATP, GST-MDMX, GST-β-catenin-1-200, pDI peptide (LTFEHYWAQLTS, 5 µM), and pDI3A peptide (LTAEHYAAQATS, 5 µM). β-catenin pS45 level was determined by western blot.


( C ) H1299 were cotransfected with MDMX , CK1α p53 mutants . MDMX - CK1α binding was determined by IP - western blot .

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(C) H1299 cells were cotransfected with MDMX, CK1α and p53 mutants. MDMX-CK1α binding was determined by IP-western blot.


( D ) Endogenous p53 in A549 ( wt ) Panc1 ( R273H ) were depleted by CRISPR / Cas9 knockout . β - catenin pS45 level was determined by western blot .

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

(D) Endogenous p53 in A549 (wt) and Panc1 (R273H) cells were depleted by CRISPR/Cas9 knockout. β-catenin pS45 level was determined by western blot.


( E ) β - catenin S45 kinase activity in A549 Panc1 was

(E) The β-catenin S45 kinase activity in A549 and Panc1 cells was determined


( A ) H1299 were transfected with MDMX CK1α mutants . MDMX - CK1α binding was determined by IP - western blot .

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

(A) H1299 cells were transfected with MDMX and CK1α mutants. MDMX-CK1α binding was determined by IP-western blot.


( B ) GST - β - catenin - 1 - 200 was incubated with ATP , CK1α GST - MDMX . level of pS45 was determined by western blot .

O O O O O O O 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) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX. The level of pS45 was determined by western blot.


( C ) GST - β - catenin - 1 - 200 was incubated with identical amounts of purified FLAG - CK1α mutants ATP . level of pS45 was determined by western blot .

(C) GST-β-catenin-1-200 was incubated with identical amounts of purified FLAG-CK1α mutants and ATP. The level of pS45 was determined by western blot.


( D ) . ELISA plates coated with GST - β - catenin - 1 - 200 was incubated with purified FLAG - CK1α mutants , ATP , MDMX . level of pS45 was determined by ELISA . results are average of 3 experiments ( mean + / - SD ) .

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

(D). ELISA plates coated with GST-β-catenin-1-200 was incubated with purified FLAG-CK1α mutants, ATP, and MDMX. The level of pS45 was determined by ELISA assay. The results are average of 3 experiments (mean +/- SD).


( A ) MEF lines derived from 3 p53 - / - ; MDMXSG / SG 3 p53 - / - ; MDMXwt / wt were analyzed for β - catenin pS45 level by western blot .

O O O O O O O O O O O O O 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) MEF cell lines derived from 3 p53-/-;MDMXSG/SG and 3 p53-/-;MDMXwt/wt embryos were analyzed for β-catenin pS45 level by western blot.


( B ) CK1α activity in MEF extracts was determined using ELISA kinase activity . results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 01 ( Student ' s t ) .

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

(B) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay. The results are average of 3 experiments (mean +/- SD). *p<0.01 (Student's t test).


( C ) Volcano plot of differential gene expression between p53 - / - ; MDMXSG / SG p53 - / - ; MDMXwt / wt MEF lines .

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

(C) Volcano plot of differential gene expression between p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt MEF lines.


( D ) Multiple Wnt signaling genes were down regulated in p53 - / - ; MDMXSG / SG MEF lines .

(D) Multiple Wnt signaling genes were down regulated in p53-/-;MDMXSG/SG MEF cell lines.


( E ) RT - PCR confirmation of Wnt pathway genes down - regulated in p53 - / - ; MDMXSG / SG MEF ( average of 3 lines , mean + / - SD ) thymus from 1 month old mice ( average of 3 mice , mean + / - SD ) . * * p < 0 . 01 , * p < 0 . 05 ( Student ' s t ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-PCR confirmation of Wnt pathway genes down-regulated in p53-/-;MDMXSG/SG MEF (average of 3 cell lines, mean +/- SD) and thymus from 1 month old mice (average of 3 mice, mean +/- SD). **p<0.01, *p<0.05 (Student's t test).


( F ) p53 - / - ; MDMXSG / SG p53 - / - ; MDMXwt / wt were treated with conditioned medium containing Wnt3a . Axin2 mRNA level was determined by RT - PCR ( average of 3 experiments , mean + / - SD ) . * p < 0 . 05 ( Student ' s t ) .

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

(F) p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt cells were treated with conditioned medium containing Wnt3a. Axin2 mRNA level was determined by RT-PCR (average of 3 experiments, mean +/- SD). *p<0.05 (Student's t test).


( A ) Identical starting number of from MEF lines were serially passaged following identical schedules . differences in number were determined after 3 5 passages . results are average of 3 experiments ( mean + / - SD ) .

O O O O O O O O O O O O O O O O O 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) Identical starting number of cells from the MEF lines were serially passaged following identical schedules. The differences in cell number were determined after 3 and 5 passages. The results are average of 3 experiments (mean +/- SD).


( B ) Identical number of from MEF lines were plated at low density cultured for 14 days . Colony formation efficiency was determined by crystal violate .

(B) Identical number of cells from the MEF lines were plated at low density and cultured for 14 days. Colony formation efficiency was determined by crystal violate staining.


( C ) Survival curves of p53 - / - ; MDMXSG / SG p53 - / - ; MDMXwt / wt mice . Thymic lymphomas were major cause of mortality in both genotypes .

O O O O O O O O O O O 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) Survival curves of p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt mice. Thymic lymphomas were the major cause of mortality in both genotypes.


Time - lapse microscopy of sporozoites , 10 min after intradermal inoculation into mice with CD31 - labeled vascular endothelia . Maximum projection over 240 sec shows trajectories of moving sporozoites ( green ) blood vessels ( magenta ) . green structures in first two panels are autofluorescent hair follicles . Scale bar , 50 μm .

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

Time-lapse microscopy of sporozoites, 10 min after intradermal inoculation into mice with CD31-labeled vascular endothelia. Maximum intensity projection over 240 sec shows trajectories of moving sporozoites (green) and blood vessels (magenta). The light green structures in the first two panels are autofluorescent hair follicles. Scale bar, 50 μm.


( A ) P . berghei sporozoite trajectories obtained with manual ( left ) automated ( right ) tracking methods . ( B ) Image processing automated tracking method . Shown are illustrating each step of automated tracking : raw data image shows first frame of time - lapse microscopy of P . berghei sporozoites ( red ) after intradermal inoculation , followed by same image after background subtraction thresholding using Fiji software . Spot detection was run using ICY software final image shows overlay with tracks generated using spot tracking plugin of ICY software . Scale bars , 50 μm . ( C ) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual automated tracking methods . Data is displayed in box whisker plots with whiskers showing 10 to 90 percentiles values below above whiskers shown individually . Horizontal indicates median speed . No statistically significant differences between corresponding timepoints of manual automated tracking methods were found ( Kruskal - Wallis ) . ( D ) Displacement of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual automated tracking methods . To normalize between tracks of different duration , displacement is displayed as final displacement per 30 seconds interval . Data is displayed in box whisker plots with whiskers showing 10 to 90 percentiles values below above whiskers shown individually . Horizontal indicates median . only statistically significant difference in sporozoite displacement between manual automated tracking methods was at 120 minutes ( Kruskal - Wallis , * * * * p < 0 . 0001 ) . For panels C & D , a varying number of videos were processed for each time point : C : 5 min ( 3 videos / 37 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 112 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 95 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 63 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 77 manual tracks / 162 automated tracks ) , 120 min ( 6 videos / 44 manual tracks / 151 automated tracks ) . D : 5 min ( 3 videos / 61 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 74 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 47 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 53 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 65 manual tracks / 162 automated tracks ) , 120 min ( 6 videos / 48 manual tracks / 151 automated tracks ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. berghei sporozoite trajectories obtained with manual (left) and automated (right) tracking methods. (B) Image processing and automated tracking method. Shown are images illustrating each step of automated tracking: The raw data image shows the first frame of time-lapse microscopy of P. berghei sporozoites (red) after intradermal inoculation, followed by the same image after background subtraction and thresholding using Fiji software. Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software. Scale bars, 50 μm. (C) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median speed. No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found (Kruskal-Wallis test). (D) Displacement of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. To normalize between tracks of different duration, displacement is displayed as final displacement per 30 seconds interval. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median. The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes (Kruskal-Wallis test, ****p<0.0001). For panels C&D, a varying number of videos were processed for each time point: C: 5 min (3 videos/37 manual tracks/203 automated tracks), 10 min (6 videos/112 manual tracks/292 automated tracks), 20 min (4 videos/95 manual tracks/184 automated tracks), 30 min (6 videos/63 manual tracks/235 automated tracks), 60 min (7 videos/77 manual tracks/162 automated tracks), and 120 min (6 videos/44 manual tracks/151 automated tracks). D: 5 min (3 videos/61 manual tracks/203 automated tracks), 10 min (6 videos/74 manual tracks/292 automated tracks), 20 min (4 videos/47 manual tracks/184 automated tracks), 30 min (6 videos/53 manual tracks/235 automated tracks), 60 min (7 videos/65 manual tracks/162 automated tracks), and 120 min (6 videos/48 manual tracks/151 automated tracks).


( A - C ) Tracks generated through automated tracking were plotted to a common origin to visualize dispersal P . berghei ( A ) , P . yoelii ( B ) P . falciparum ( C ) at 5 , 10 , 30 60 min after intradermal inoculation . For each timepoint , tracks are representative of average displacement of entire sporozoite population . Track numbers used for each timepoint were : 5 min , 40 tracks ; 10 min , 32 tracks ; 30 min , 25 tracks ; 60 min , 21 tracks . ( D - F ) Displacement of P . berghei ( D ) , P . yoelii ( E ) P . falciparum ( F ) sporozoites 5 - 120 min after inoculation in box whisker plots with whiskers displaying 10 - 90 percentiles values below above whiskers shown individually . Horizontal lines show median displacement at each time point . values in boxes indicate fraction of tracks displacing over 75 micrometers at each time point . Within a given species , difference in sporozoite displacement between 5 min 120 min is statistically significant ( Kruskal - Wallis : P . berghei p < 0 . 0001 , P . yoelii p < 0 . 0001 , P . falciparum p < 0 . 01 ) . Comparisons between species showed no statistically significant difference in displacements for any given time point , with exception of 10 min time point in which there was a significant difference between P . berghei P . falciparum ( Kruskal - Wallis , * p < 0 . 05 ) . For panels ( D - F ) , a varying number of videos were processed for each time point after inoculation : ( D ) Same dataset as used for Figure 2D : 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) 120 min ( 6 videos / 151 tracks ) . ( E ) 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , 120 min ( 4 videos / 180 tracks ) . ( F ) 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , 120 min ( 2 videos / 103 tracks ) .

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

(A-C) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P. berghei (A), P. yoelii (B) and P. falciparum (C) at 5, 10, 30 and 60 min after intradermal inoculation. For each timepoint, the tracks are representative of the average displacement of the entire sporozoite population. Track numbers used for each timepoint were: 5 min, 40 tracks; 10 min, 32 tracks; 30 min, 25 tracks; 60 min, 21 tracks. (D-F) Displacement of P. berghei (D), P. yoelii (E) and P. falciparum (F) sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show median displacement at each time point. Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point. Within a given species, the difference in sporozoite displacement between 5 min and 120 min is statistically significant (Kruskal-Wallis test: P. berghei p<0.0001, P. yoelii p<0.0001, P. falciparum p<0.01). Comparisons between species showed no statistically significant difference in displacements for any given time point, with the exception of the 10 min time point in which there was a significant difference between P. berghei and P. falciparum (Kruskal-Wallis test, *p<0.05). For panels (D-F), a varying number of videos were processed for each time point after inoculation: (D) Same dataset as used for Figure 2D: 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks) and 120 min (6 videos/151 tracks). (E) 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). (F) 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).


( A ) Speed of P . berghei , P . yoelii P . falciparum sporozoites 5 - 120 min after inoculation in box whisker plots with whiskers displaying 10 - 90 percentiles values below above whiskers shown individually . Horizontal lines show median speed at each time point . Comparisons among species showed statistically significant difference in sporozoite speeds at 20 min time point ( Kruskal - Wallis , * * * * p < 0 . 0001 for both comparisons ) 120 min time point ( Kruskal - Wallis , * * p < 0 . 006 for both comparisons ) . ( B ) Linear regression of apparent speed shows a more rapid decrease for P . berghei ( slope = - 0 . 005 ) than for P . yoelii P . falciparum ( slopes = - 0 . 002 ) . Symbols indicate mean values + / - standard deviation . A varying number of videos were processed for each time point after inoculation , using same data set used for Figure 3 : P . berghei ; 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) , 120 min ( 6 videos / 151 tracks ) . P . yoelii ; 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , 120 min ( 4 videos / 180 tracks ) . P . falciparum ; 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , 120 min ( 2 videos / 103 tracks ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Speed of P. berghei, P. yoelii and P. falciparum sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. Comparisons among the species showed statistically significant difference in sporozoite speeds at the 20 min time point (Kruskal-Wallis test, ****p<0.0001 for both comparisons) and the 120 min time point (Kruskal-Wallis test, **p<0.006 for both comparisons). (B) Linear regression of apparent speed shows a more rapid decrease for P. berghei (slope = -0.005) than for P. yoelii and P. falciparum (slopes = -0.002). Symbols indicate mean values +/- standard deviation. A varying number of videos were processed for each time point after inoculation, using the same data set used for Figure 3: P. berghei; 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks), and 120 min (6 videos/151 tracks). P. yoelii; 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). P. falciparum; 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).


( A ) number of motile non - motile sporozoites moving in dermis of mice was manually counted in time courses spanning 120 minutes is displayed as a of total sporozoites observed 5 min after inoculation , for P . berghei ( left panel ) , P . yoelii ( center panel ) , P . falciparum ( right panel ) . ( B ) Fold - change in number of motile sporozoites at 60 120 min post - inoculation compared to number of motile sporozoites at 5 min . data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites . ( P . berghei : n = 2 , P . yoelii n = 4 , P . falciparum n = 2 ) .

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

(A) The number of motile and non-motile sporozoites moving in the dermis of mice was manually counted in time courses spanning 120 minutes and is displayed as a percentage of total sporozoites observed 5 min after inoculation, for P. berghei (left panel), P. yoelii (center panel), and P. falciparum (right panel). (B) Fold-change in the number of motile sporozoites at 60 and 120 min post-inoculation compared to the number of motile sporozoites at 5 min. The data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites. (P. berghei: n=2, P. yoelii n=4, P. falciparum n=2).


( A ) Maximum projection shows trajectories of moving P . berghei , P . yoelii P . falciparum sporozoites ( green ) , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bars , 25 μm . See movies EV4 - EV6 . ( B ) Lymphatic blood vessel invasion of P . berghei , P . yoelii P . falciparum sporozoites moving in mouse dermis . Combined invasion events observed in 4 min videos recorded at 5 , 10 20 min after intradermal injection of sporozoites ( left panel ) 60 120 min after intradermal injection ( right panel ) . n = number of videos scored for each Plasmodium species . To view a blood vessel entry event by P . yoelii or P . falciparum , see movies EV7 EV8 . There were no statistically significant differences in lymphatic or blood vessel entry among three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P . falciparum sporozoites compared to P . berghei P . yoelii sporozoites at 60 to 120 min after inoculation ( Kruskal - Wallis , p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximum intensity projection shows trajectories of moving P. berghei, P. yoelii and P. falciparum sporozoites (green), engaging with CD31-labeled vascular endothelia (magenta). Scale bars, 25 μm. See movies EV4 - EV6. (B) Lymphatic and blood vessel invasion of P. berghei, P. yoelii and P. falciparum sporozoites moving in mouse dermis. Combined invasion events observed in 4 min videos recorded at 5, 10 and 20 min after intradermal injection of sporozoites (left panel) and 60 and 120 min after intradermal injection (right panel). n = number of videos scored for each Plasmodium species. To view a blood vessel entry event by P. yoelii or P. falciparum, see movies EV7 and EV8. There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P. falciparum sporozoites compared to P. berghei and P. yoelii sporozoites at 60 to 120 min after inoculation (Kruskal-Wallis test, p<0.05).


( A ) Maximum projection of video acquired 10 min after inoculation of P . falciparum sporozoites visualizes trajectories of , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bar , 50 μm . See movie EV9 . ( B ) Displacement of sporozoites 5 to 60 min after inoculation . Data is displayed in box whisker plots with whiskers showing 10 to 90 percentiles values below above whiskers shown individually . Horizontal lines show median at each time point . values indicate fraction of tracks displacing over 75 microns . ( C ) Apparent speed of sporozoites 5 to 60 min after inoculation . Data is displayed in box whisker plots with whiskers showing 10 to 90 percentiles values below above whiskers shown individually . Horizontal lines show median speed at each time point . For panels B - C , data shown originates from of 3 - 4 videos per time point a varying number of tracks were processed for each time point after inoculation : 5 min ( 157 tracks ) , 10 min ( 568 tracks ) , 20 min ( 286 tracks ) , 30 min ( 171 tracks ) , 60 min ( 101 tracks ) . No motile sporozoites were observed 120 min after inoculation . ( D ) Proportion of motile non - motile sporozoites was manually counted is displayed as of sporozoites observed 5 min after inoculation . data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximum intensity projection of video acquired 10 min after inoculation of P. falciparum sporozoites visualizes trajectories of parasites, engaging with CD31-labeled vascular endothelia (magenta). Scale bar, 50 μm. See movie EV9. (B) Displacement of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median at each time point. Percentage values indicate the fraction of tracks displacing over 75 microns. (C) Apparent speed of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. For panels B-C, data shown originates from the analysis of 3-4 videos per time point and a varying number of tracks were processed for each time point after inoculation: 5 min (157 tracks), 10 min (568 tracks), 20 min (286 tracks), 30 min (171 tracks), 60 min (101 tracks). No motile sporozoites were observed 120 min after inoculation. (D) Proportion of motile and non-motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation. The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites.


( E ) Sporozoite track straightness , of displacement to track of P . falciparum sporozoites moving in mouse dermis , human skin ex vivo , human skin graft , at 5 - 10 min post intradermal injection . Data is displayed in box whisker plots with horizontal showing median whiskers extending to minimum maximum values . Comparisons showed statistically significant differences in track straightness between human skin graft both mouse skin ( * * * * p = 0 . 0001 ) human skin ex vivo ( * * * * p < 0 . 0001 ) using Tukey ' s multiple comparisons . A varying number of tracks were processed for each environment : mouse skin : 17 videos , 822 tracks ; human skin ex vivo : 12 videos , 761 tracks ; human skin graft : 9 videos , 718 tracks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sporozoite track straightness, the ratio of displacement to track length of P. falciparum sporozoites moving in mouse dermis, human skin ex vivo, and human skin graft, at 5-10 min post intradermal injection. Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values. Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin (****p=0.0001) and human skin ex vivo (****p<0.0001) using Tukey's multiple comparisons test. A varying number of tracks were processed for each environment: mouse skin: 17 videos, 822 tracks; human skin ex vivo: 12 videos, 761 tracks; human skin graft: 9 videos, 718 tracks.


( F ) P . falciparum sporozoites engage with enter blood vessels in human skin graft . Maximum projection shows trajectories of moving P . falciparum sporozoites ( green ) , engaging with human CD31 - labeled vascular endothelia ( magenta ) . Scale bar , 25 μm . See movie EV10 . Graph shows lymphatic blood vessel entry events of P . falciparum sporozoites in human skin graft . Combined invasion events observed at 5 - 20 min after intradermal injection in n number of videos . To view a blood vessel invasion event P . falciparum in human skin graft ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) P. falciparum sporozoites engage with and enter blood vessels in human skin graft. Maximum intensity projection shows trajectories of moving P. falciparum sporozoites (green), engaging with human CD31-labeled vascular endothelia (magenta). Scale bar, 25 μm. See movie EV10. Graph shows lymphatic and blood vessel entry events of P. falciparum sporozoites in human skin graft. Combined invasion events observed at 5-20 min after intradermal injection in n number of videos. To view a blood vessel invasion event P. falciparum in human skin graft,


( A ) Activity of mAb 2A10 on sporozoite speed displacement : Data are displayed in box whisker plots with whiskers displaying 10 to 90 percentiles values below above whiskers shown individually . Horizontal lines show median . For displacement data , values indicate fraction of tracks displacing over 75 microns . Statistical : Mann - Whitney , p = 0 . 0001 ( speed data ) p = 0 . 0098 ( displacement data ) . Data shown originates from of 5 videos obtained at 5 separate imaging sessions , which generated a total number of 217 ( naïve ) 136 ( 2A10 ) sporozoite tracks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activity of mAb 2A10 on sporozoite speed and displacement: Data are displayed in box and whisker plots with whiskers displaying the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median. For the displacement data, percentage values indicate the fraction of tracks displacing over 75 microns. Statistical analysis: Mann-Whitney, p=0.0001 (speed data) and p=0.0098 (displacement data). Data shown originates from the analysis of 5 videos obtained at 5 separate imaging sessions, which generated a total number of 217 (naïve) and 136 (2A10) sporozoite tracks.


( B ) Activity of mAb 2A10 on proportion of motile sporozoites ( left panel ) blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with mAb 2A10 naïve controls were manually counted at 10 min post P . falciparum sporozoite inoculation . 5 videos for each condition , from 5 independent imaging sessions were scored for motility . 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry . Statistical : motile , Mann - Whitney , p = 0 . 0317 ; Blood vessel entry , Fisher ' s exact , p = 0 . 0163

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activity of mAb 2A10 on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P. falciparum sporozoite inoculation. 5 videos for each condition, from 5 independent imaging sessions were scored for motility. 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry. Statistical analysis: Percent motile, Mann-Whitney, p=0.0317; Blood vessel entry, Fisher's exact test, p=0.0163


( B ) Activity of mAb 2A10 on proportion of motile sporozoites ( left panel ) blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with mAb 2A10 naïve controls were manually counted at 10 min post P . falciparum sporozoite inoculation . 5 videos for each condition , from 5 independent imaging sessions were scored for motility . 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry . Statistical : motile , Mann - Whitney , p = 0 . 0317 ; Blood vessel entry , Fisher ' s exact , p = 0 .


( C ) Activity of human mAbs 10 CIS43 , specific for P . falciparum CSP , VRC01 , an isotype control human mAb specific for HIV - 1 , on proportion of motile sporozoites ( left panel ) blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with indicated mAbs were taken 10 min after sporozoite inoculation manually counted . Data are pooled from 7 biological replicates , naïve VRC01 ( n = 2 ) , naïve mAb 10 ( n = 2 ) , VRC01 CIS43 ( n = 3 ) . No comparisons were statistically significant ( Mann - Whitney for motile Fisher ' s exact for blood vessel entry ) .

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(C) Activity of human mAbs 10 and CIS43, specific for P. falciparum CSP, and VRC01, an isotype control human mAb specific for HIV-1, on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with the indicated mAbs were taken 10 min after sporozoite inoculation and manually counted. Data are pooled from 7 biological replicates, naïve and VRC01 (n=2), naïve and mAb 10 (n=2), VRC01 and CIS43 (n=3). No comparisons were statistically significant (Mann-Whitney for percent motile and Fisher's exact test for blood vessel entry).


( A ) of single - transcriptomics dataset for mouse visual cortex ( Tasic et al , 2016 ) revealed that expression of metabolism - associated gene Cox6a2 is highly restricted to PV + interneurons . Cox6a2 is expressed in subtypes of PV + interneurons ( marked in blue color ) but rarely detected in other neuronal subtypes from mouse visual cortex . Cox6a1 , which encodes other isoform of COX6A , is expressed in subtypes .

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(A) Analysis of the single-cell transcriptomics dataset for the mouse visual cortex (Tasic et al, 2016) revealed that the expression of the metabolism-associated gene Cox6a2 is highly restricted to PV+ interneurons. Cox6a2 is expressed in all subtypes of PV+ interneurons (marked in blue color) but rarely detected in other neuronal subtypes from the mouse visual cortex. Cox6a1, which encodes the other protein isoform of COX6A, is expressed in all subtypes.


( B RT - PCR showing expression of Cox6a2 in brain heart .

(B RT-PCR showing expression of Cox6a2 in the brain and heart.


C ) Western - blot showing expression of Cox6a2 in brain heart .

C) Western-blot showing expression of Cox6a2 in the brain and heart.


( D ) Western - blot showed that COX6A2 is first detected in brain at postnatal day 0 ( P0 ) . Then , COX6A2 gradually increase until P60 .

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(D) Western-blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 (P0). Then, COX6A2 protein levels gradually increase until P60.


COX6A2 immunoreactivity is detected in PV + interneurons in different brain areas of adult mice , including cortex ( general overview in E , single PV + interneuron magnification in F ) arrowheads indicate PV / Cox6a2 double positive .

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COX6A2 immunoreactivity is detected in PV+ interneurons in different brain areas of adult mice, including the cortex (general overview in E, and single PV+ interneuron magnification in F) White arrowheads indicate PV/Cox6a2 double positive cells.


COX6A2 immunoreactivity is detected in PV + interneurons in hippocampus ( G ) striatum ( H ) . arrowheads indicate PV / Cox6a2 double positive .

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COX6A2 immunoreactivity is detected in PV+ interneurons in hippocampus (G) and striatum (H). White arrowheads indicate PV/Cox6a2 double positive cells.


( I , J ) Quantifications showing almost 100 % of co - localization of COX6A2 + PV + in somatosensory ( SS ) motor ( MT ) cortices , hippocampus ( HI ) of adult mice ( n = 4 , 2 months old , n sections = 24 SS , 16 MT , 8 HI ) .

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(I,J) Quantifications showing almost 100% of co-localization of COX6A2+ cells and PV+ cells in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult mice (n animals=4, 2 months old, n sections=24 SS, 16 MT, 8 HI).


( K ) COX6A2 immunoreactivity in cortex at P9 ( n = 2 , n sections = 12 SS , 8 MT , 4 HI ) . arrowheads indicate Cox6a2 positive .

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(K) COX6A2 immunoreactivity in the cortex at P9 (n animals=2, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate Cox6a2 positive cells.


( L - N ) As soon as parvalbumin immunoreactivity becomes visible at P13 , almost 100 % of PV + are co - labelled with COX6A2 immunoreactivity in SS MT cortices , HI of P13 mice ( n = 2 ) .

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(L-N) As soon as parvalbumin immunoreactivity becomes visible at P13, almost 100% of PV+ cells are co-labelled with COX6A2 immunoreactivity in the SS and MT cortices, and the HI of P13 mice (n=2).


( A , B ) COX6A2 immunoreactivity is virtually restricted to PV + interneurons in somatosensory ( SS ) motor ( MT ) cortices , hippocampus ( HI ) of adult rat ( n = 1 , 2 . 5 months old , n sections = 12 SS , 8 MT , 4 HI ) . arrowheads indicate PV / Cox6a2 double positive .

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(A,B) COX6A2 immunoreactivity is virtually restricted to PV+ interneurons in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult rat (n animals=1, 2.5 months old, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate PV/Cox6a2 double positive cells.


( C , D ) In SS of adult rhesus monkey , COX6A2 immunoreactivity is present exclusively in PV + ( n = 1 , 1 - year - old , n sections = 6 ) . arrowheads indicate PV / Cox6a2 double positive .

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(C,D) In the SS of adult rhesus monkey, COX6A2 immunoreactivity is present exclusively in PV+ cells (n animals=1, 1-year-old, n sections=6). White arrowheads indicate PV/Cox6a2 double positive cells.


( E , F ) In adult humans , cortical PV + interneurons from Brodmann areas 9 10 ( BA9 BA10 ) are co - labelled with COX6A2 immunoreactivity . However , PV + interneurons only represent 60 % of in which COX6A2 is detected ( n human subjects = 2 , 59 - 69 - year - old , n sections = 6 BA9 , 6 BA10 ) . arrowheads indicate PV / Cox6a2 double positive .

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(E,F) In adult humans, all cortical PV+ interneurons from Brodmann areas 9 and 10 (BA9 and BA10) are co-labelled with COX6A2 immunoreactivity. However, PV+ interneurons only represent 60% of the cells in which COX6A2 is detected (n human subjects=2, 59- and 69-year-old, n sections=6 BA9, 6 BA10). White arrowheads indicate PV/Cox6a2 double positive cells.


( A ) showing absence of COX6A2 immunoreactivity in cortex of Cox6a2 - / - mice .

(A) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2-/- mice.


( B , C ) Parvalbumin immunoreactivity in PV + interneurons was significantly decreased in somatosensory cortex of Cox6a2 - / - mice compared to wild - types ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . C : two - unpaired Student ' s t - .

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(B,C) Parvalbumin immunoreactivity in PV+ interneurons was significantly decreased in the somatosensory cortex of Cox6a2-/- mice compared to wild-types (n=4 WT, 4 Cox6a2-/-; 2-month-old). C: two-tailed unpaired Student's t-test.


( D ) In Cox6a2 - / - mice , cortical PV + interneurons showed a fewer number of synapses innervating principal neurons ( n = 3 WT , 3 Cox6a2 - / - ; 2 - month - old ) . Two - unpaired Student ' s t - .

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(D) In Cox6a2-/- mice, cortical PV+ interneurons showed a fewer number of synapses innervating principal neurons (n=3 WT, 3 Cox6a2-/-; 2-month-old). Two-tailed unpaired Student's t-test.


( E ) To analyze changes in oxidative stress in PV + interneurons , somatosensory motor cortices of WT Cox6a2 - / - mice were stained with an antibody against 8 - OHdG , a marker of oxidative damage . ( F ) 8 - OHdG immunoreactivity was significantly higher in PV + null in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - unpaired Mann - Whitney .

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(E) To analyze changes in oxidative stress in PV+ interneurons, the somatosensory and motor cortices of WT and Cox6a2-/- mice were stained with an antibody against 8-OHdG, a marker of oxidative DNA damage. (F) 8-OHdG immunoreactivity was significantly higher in PV+ null in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( G ) density of PNNs around PV + interneurons residing in somatosensory motor cortices was analyzed by measuring immunoreactivity of WFA that stains PNNs . ( H ) WFA immunoreactivity around PV + was lower in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - unpaired Mann - Whitney .

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(G) The density of PNNs around PV+ interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs. (H) WFA immunoreactivity around PV+ cells was lower in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( A ) PV + interneurons were sorted from cortices of PV - EGFP WT Cox6a2 - / - mice at P15 processed for bulk sequencing ( n = 4 WT , 6 Cox6a2 - / - ; P15 ) . shows expression for top down - ( red ) upregulated ( blue ) genes in Cox6a2 - / - PV + interneurons relative to their WT counterparts .

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(A) PV+ interneurons were sorted from cortices of PV-EGFP WT and Cox6a2-/- mice at P15 and processed for bulk RNA sequencing (n= 4 WT, 6 Cox6a2-/-; P15). Heatmap shows expression for top down- (red) and upregulated (blue) genes in Cox6a2-/- PV+ interneurons relative to their WT counterparts.


( B ) Cox6a2 expression was effectively abolished in Cox6a2 - / - PV + interneurons . In addition , Cox6a2 knockout led to a decreased expression of Gad1 Gad2 genes ( n = 4 WT , 6 Cox6a2 - / - ) . Two - unpaired Mann - Whitney .

(B) Cox6a2 expression was effectively abolished in Cox6a2-/- PV+ interneurons. In addition, Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes (n= 4 WT, 6 Cox6a2-/-). Two-tailed unpaired Mann-Whitney tests.


( C ) Changes in expression of oxidative stress - related genes ( green ) , genes coding for AMPA receptors ( orange ) , sodium channels ( red ) , potassium channels ( magenta ) , internal calcium signaling ( blue ) . Color scale bars show normalized read .

(C) Changes in expression of oxidative stress-related genes (green), genes coding for AMPA receptors (orange), sodium channels (red), potassium channels (magenta), and internal calcium signaling proteins (blue). Color scale bars show normalized read counts.


( D ) Representative of PV + interneurons from PV - CRE PV - CRE ; Cox6a2fl / fl mice showing PercevalHR after excitation at 930 nm 840 nm . arrowheads indicate PercevalHR expressing . Scale bar : 20 μm .

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(D) Representative images of PV+ interneurons from PV-CRE and PV-CRE;Cox6a2fl/fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm. White arrowheads indicate PercevalHR expressing cells. Scale bar: 20 μm.


( E ) PV + interneurons from PV - CRE ; Cox6a2fl / fl mice exhibited significantly lower ATP - to - ADP ratios than PV + interneurons from PV - CRE mice throughout entire duration of recordings ( 23 from 4 PV - CRE mice ; 15 from 6 PV - CRE ; Cox6a2 - / - mice ) .

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(E) PV+ interneurons from PV-CRE;Cox6a2fl/fl mice exhibited significantly lower ATP-to-ADP ratios than PV+ interneurons from PV-CRE mice throughout the entire duration of the recordings (23 cells from 4 PV-CRE mice; 15 cells from 6 PV-CRE;Cox6a2-/- mice).


( A , B ) resting potential amplitude of action potentials in WT Cox6a2 - / - PV + interneurons , respectively . Two - unpaired Student ' s t - .

(A,B) The resting membrane potential and amplitude of action potentials in WT and Cox6a2-/- PV+ interneurons, respectively. Two-tailed unpaired Student's t-tests.


( C , D ) threshold for action potentials increased over maturation in WT but not in Cox6a2 - / - PV + interneurons . As a result , threshold for action potentials was significantly lower in P34 - 45 Cox6a2 - / - PV + interneurons when compared to their WT counterparts . D : two - unpaired Student ' s t - .

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(C,D) The threshold for action potentials increased over maturation in WT but not in Cox6a2-/- PV+ interneurons. As a result, the threshold for action potentials was significantly lower in P34-45 Cox6a2-/- PV+ interneurons when compared to their WT counterparts. D: two-tailed unpaired Student's t-tests.


( E , F ) Trains of action potentials were evoked by 2 sec depolarizing current pulses . amplitude difference ( difference between mean amplitude of 1st - 10th action potentials 190th - 200th action potentials ) was significantly higher in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . In addition , amplitude difference decreased with maturation in WT but not in Cox6a2 - / - PV + interneurons . F : two - unpaired Mann - Whitney .

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(E,F) Trains of action potentials were evoked by 2 sec depolarizing current pulses. The amplitude difference (difference between the mean amplitude of 1st-10th action potentials and 190th-200th action potentials) was significantly higher in P34-45 Cox6a2-/- compared to WT PV+ interneurons. In addition, the amplitude difference decreased with maturation in WT but not in Cox6a2-/- PV+ interneurons. F: two-tailed unpaired Mann-Whitney tests.


( G ) While in WT PV + interneurons maximal firing frequency increased during maturation , such increase was abolished by Cox6a2 knockout . Thus , maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . Two - unpaired Student ' s t - .

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(G) While in WT PV+ interneurons the maximal firing frequency increased during maturation, such increase was abolished by Cox6a2 knockout. Thus, maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34-45 Cox6a2-/- compared to WT PV+ interneurons. Two-tailed unpaired Student's t-tests.


( H ) Recorded PV + interneurons were filled with biocytin their dendritic was reconstructed using Imaris software . Scale bars : 30 μm .

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(H) Recorded PV+ interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software. Scale bars: 30 μm.


( I , J ) Cox6a2 - / - PV + interneurons showed a negative correlation between dendritic between full branch depth ( maximal number of branches between soma a terminal dendritic point ) , which contrasted with expected positive correlation between these parameters in WT PV + interneurons . Linear regression of Pearson correlation .

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(I,J) Cox6a2-/- PV+ interneurons showed a negative correlation between age and dendritic length and between age and full branch depth (maximal number of branches between the soma and a terminal dendritic point), which contrasted with the expected positive correlation between these parameters in WT PV+ interneurons. Linear regression of Pearson correlation.


( K , L ) WT Cox6a2 - / - PV + interneurons showed similar values for dendritic full branch depth at P22 - P33 . However , at P34 - P45 , Cox6a2 - / - PV + interneurons showed lower dendritic full branch depth than WT PV + interneurons . Two - way ANOVA followed by Tukey ' s multiple comparisons .

(K,L) WT and Cox6a2-/- PV+ interneurons showed similar values for dendritic length and full branch depth at P22-P33. However, at P34-P45, Cox6a2-/- PV+ interneurons showed lower dendritic length and full branch depth than WT PV+ interneurons. Two-way ANOVA followed by Tukey's multiple comparisons tests.


( M , N ) While younger WT Cox6a2 - / - PV + interneurons have similar dendritic tree complexities ( M ) , at later stage of maturation Cox6a2 - / - PV + interneurons show a reduction in dendritic tree complexity in comparison to WT PV + interneurons ( N ) . Multiple t - , two - stage step - up method of Benjamini , Krieger Yekutieli .

(M,N) While younger WT and Cox6a2-/- PV+ interneurons have similar dendritic tree complexities (M), at the later stage of maturation Cox6a2-/- PV+ interneurons show a reduction in dendritic tree complexity in comparison to WT PV+ interneurons (N). Multiple t-tests, two-stage step-up method of Benjamini, Krieger and Yekutieli.


( B ) In OFT , Cox6a2 - / - mice travelled a longer moved faster than WT mice . Cox6a2 - / - mice also moved more often from outer zone to inner zone of arena . Two - unpaired Student ' s t - .

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(B) In the OFT, Cox6a2-/- mice travelled a longer distance and moved faster than WT mice. Cox6a2-/- mice also moved more often from the outer zone to the inner zone of the arena. Two-tailed unpaired Student's t-tests.


( D ) In SIT , Cox6a2 - / - mice also travelled a longer with higher mean velocity than WT mice . Additionally , both Cox6a2 - / - WT mice spent more time exploring container with stranger mouse than empty container . Total moved , mean velocity , f [ number of crossings ] outer to inner zone : two - unpaired Student ' s t - . Social interaction : two - way ANOVA followed by Tukey ' s multiple comparisons .

(D) In the SIT, Cox6a2-/- mice also travelled a longer distance with higher mean velocity than WT mice. Additionally, both Cox6a2-/- and WT mice spent more time exploring the container with the stranger mouse than the empty container. Total distance moved, mean velocity, and f [number of crossings] outer to inner zone: two-tailed unpaired Student's t-test. Social interaction: two-way ANOVA followed by Tukey's multiple comparisons test.


( F ) startle responses were significantly lower in Cox6a2 - / - mice when loud stimulus ( 120 dB ) was presented alone first 5 times . Two - way ANOVA followed by Tukey ' s multiple comparisons .

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(F) The startle responses were significantly lower in Cox6a2-/- mice when the loud stimulus (120 dB) was presented alone the first 5 times. Two-way ANOVA followed by Tukey's multiple comparisons tests.


( G ) PPI of acoustic startle response was similar at pre - pulses for Cox6a2 - / - WT mice . Two - way ANOVA followed by Sidak ' s multiple comparisons .

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(G) The PPI of the acoustic startle response was similar at all pre-pulses for Cox6a2-/- and WT mice. Two-way ANOVA followed by Sidak's multiple comparisons tests.


was extracted from young fertile adults fed ad libitum or fasted for 6 h . ddCts ( delta delta cycle thresholds ) were calculated normalizing to ama - 1 efficiency of primer sets as previously described . Means + s . e . m . are depicted . lipl - 1 to lipl - 5 , n = 6 independent experiments . lipl - 6 to lipl - 8 , n = 3independent experiments . Significant t - derived P - values are indicated . WT , wild type .

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RNA was extracted from young fertile adults fed ad libitum or fasted for 6 h. ddCts (delta delta cycle thresholds) were calculated normalizing to ama-1 and the efficiency of the primer sets as previously described. Means+s.e.m. are depicted. lipl-1 to lipl-5, n = 6 independent experiments. lipl-6 to lipl-8, n = 3independent experiments. Significant t-test derived P-values are indicated. WT, wild type.


( a ) Representative confocal of LIPL - 1 : : GFP LIPL - 3 : : TagRFP localization relative to lysosomal marker PGP - 2 show that LIPL - 1 LIPL - 3 are localized to lysosomal - related organelle .

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(a) Representative confocal images of LIPL-1::GFP and LIPL-3::TagRFP localization relative to the lysosomal marker PGP-2 show that LIPL-1 and LIPL-3 are localized to the lysosomal-related organelle.


( b ) Acid lipase activity , measured in 1 - day adult whole lysates , shows that lipl - 1lipl - 3 double mutant have reduced acidic lipolytic capacity . Mean ± s . d . are presented relative to wild type ( WT ) ; significant differences are indicated ; n = 4 independent experiments .

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(b) Acid lipase activity, measured in 1-day adult whole lysates, shows that lipl-1lipl-3 double mutant animals have reduced acidic lipolytic capacity. Mean±s.d. are presented relative to wild type (WT); significant differences are indicated; n = 4 independent experiments.


( c ) Oil Red O ( ORO ) fatty acid methyl ester ( FAME ) analyses of wild - type lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant L3 larvae show that lysosomal lipases regulate fat stores ( see Fig . 2d for adult measurements ) . Means ± s . e . m . are presented relative to wild type ; significant differences are indicated ; n = 3 independent experiments . ( d ) ORO of wild - type lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant young adults fasted for 4 h shows that LIPL - 1 LIPL - 3 contribute to fat mobilization following fasting . ( Mean ± s . e . m . relative to fed wild type ) . Wild - type fasted worms show 20 % less ORO signal than fed ad libitum ( P ≤ 0 . 001 ) , whereas lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant worms show 7 % reduction in ORO signal ( a difference that is not significant to well - fed fat at P ≤ 0 . 01 but it is significant at P ≤ 0 . 05 ) . n = 3 independent experiments .

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(c) Oil Red O (ORO) staining and fatty acid methyl ester (FAME) analyses of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant L3 larvae show that lysosomal lipases regulate cytosolic fat stores (see Fig. 2d for adult measurements). Means±s.e.m. are presented relative to wild type; significant differences are indicated; n = 3 independent experiments. (d) ORO quantification of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant young adults fasted for 4 h shows that LIPL-1 and LIPL-3 contribute to fat mobilization following fasting. (Mean percentage ± s.e.m. relative to fed wild type). Wild-type fasted worms show 20% less ORO signal than animals fed ad libitum (P≤0.001), whereas lipl-1(tm1954) lipl-3(tm4498) double mutant worms show 7% reduction in ORO signal (a difference that is not significant to well-fed fat levels at P≤0.01 but it is significant at P≤0.05). n = 3 independent experiments.


( e ) Representative transmission electron microscopy of well - fed 6 h - fasted wild - type lipl - 1lipl - 3 mutant ( 11 , 500 × ) show that lipl mutants have more lipid - droplet stores in ad libitum fed fasted conditions . of vesicle number is depicted as mean + s . e . m . , n = 4 independent experiments .

(e) Representative transmission electron microscopy images of well-fed and 6 h-fasted wild-type and lipl-1lipl-3 mutant animals (11,500×) show that the lipl mutants have more lipid-droplet stores in ad libitum fed and fasted conditions. Quantification of vesicle number and size is depicted as mean+s.e.m., n = 4 independent experiments.


( f ) Representative of well - fed transgenic expressing LIPL - 3 : : TagRFP ATGL : : GFP shows that LIPL - 3 does not localize to lipid droplets .

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(f) Representative images of well-fed transgenic animals expressing LIPL-3::TagRFP and ATGL::GFP shows that LIPL-3 does not localize to lipid droplets.


( g ) ORO of wild - type lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant young adults treated post - developmentally ( from L4 ) with RNAi against essential autophagy genes lgg - 1 lgg - 2 or vector control show that lipl - 1 lipl - 3 autophagy genes lgg - 1 lgg - 2 are in same fat regulatory pathway . ( Mean + s . e . m . relative to wild type on vector control is depicted . ) n = 4 independent experiments .

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(g) ORO staining and quantification of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant young adults treated post-developmentally (from L4) with RNAi against the essential autophagy genes lgg-1 and lgg-2 or vector control show that lipl-1 lipl-3 and the autophagy genes lgg-1 lgg-2 are in the same fat regulatory pathway. (Mean percentage+s.e.m. relative to wild type on vector control is depicted.) n = 4 independent experiments.


( a ) Expression of lipl genes in well - fed mxl - 3 ( ok1947 ) mxl - 3 ( tm2580 ) young adults normalized to same well - fed wild - type ( WT ) worms shows that mxl - 3 loss of function is sufficient to induce lipl - 1 to 3 lipl - 5 . Mean ddCts + s . e . m . are depicted . n = 6 independent experiments for lipl - 1 to lipl - 5 , n = 4 independent experiments for lipl - 6 to lipl - 8 .

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(a) Expression of lipl genes in well-fed mxl-3(ok1947) and mxl-3(tm2580) young adults normalized to same age well-fed wild-type (WT) worms shows that mxl-3 loss of function is sufficient to induce lipl-1 to 3 and lipl-5. Mean ddCts+s.e.m. are depicted. n = 6 independent experiments for lipl-1 to lipl-5, and n = 4 independent experiments for lipl-6 to lipl-8.


( b ) L4 larvae were fasted was extracted at indicated times . Mean ddCts + s . e . m . show that response mxl - 3 orchestrates is transient . Time 0 , n = 6 independent experiments ; time 3 - 12 h , n = 4 independent experiments ; time 18 - 24 h , n = 2 independent experiments .

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(b) L4 larvae were fasted and RNA was extracted at the indicated times. Mean ddCts+s.e.m. show that the response mxl-3 orchestrates is transient. Time 0, n = 6 independent experiments; time 3-12 h, n = 4 independent experiments; time 18-24 h, n = 2 independent experiments.


( c ) Immunostainings of well - fed or 6 or 12 h fasted MXL - 3 : : GFP young adults are presented . of GFP - positive nuclei relative to total intestinal nuclei ( 4 , 6 - diamidino - 2 - phenylindole , DAPI ) of two independent experiments shows that MXL - 3 transiently delocalizes from intestinal nuclei during fasting .

(c) Immunostainings of well-fed or 6 or 12 h fasted MXL-3::GFP young adults are presented. Quantification of GFP-positive nuclei relative to total intestinal nuclei (4,6-diamidino-2-phenylindole, DAPI) of two independent experiments shows that MXL-3 transiently delocalizes from the intestinal nuclei during fasting.


( d ) ChIP - quantitative PCR ( ChIP - qPCR ) of well - fed 6 h fasted mixed - stage worms expressing MXL - 3 : : GFP presented as Ct in αGFP immunoprecipitated normalized to input relative to a mock promoter region ( CACTAT site − 88 of ama - 1 gene ) shows that MXL - 3 vacates lipl promoters during early fasting . Three sets of primers surrounding CACGTG target sites found up to 500 bp of ATG of lipl - 1 lipl - 3 genes were used . A representative experiment is presented ; see raw data of two independent experiments in Supplementary Table S9 .

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(d) ChIP-quantitative PCR (ChIP-qPCR) analysis of well-fed and 6 h fasted mixed-stage worms expressing MXL-3::GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region (CACTAT site −88 of ama-1 gene) shows that MXL-3 vacates the lipl promoters during early fasting. Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl-1 and lipl-3 genes were used. A representative experiment is presented; see raw data of two independent experiments in Supplementary Table S9.


( e ) Wild - type or lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant grown on control RNAi plates were transferred as L4 larvae to control or mxl - 3 RNAi plates , incubated for 12 h at 20 ° C , processed for total FAMEs ; as a reference , an aliquot of wild - type young adults on control RNAi was fasted for 6 h . FAME ( mean of fed wild type ± s . e . m . ) shows that acute mxl - 3 inactivation , as fasting , reduces fat stores in a lipl - dependent manner . n = 3 independent experiments . NSD , P - value > 0 . 05 .

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(e) Wild-type or lipl-1(tm1954) lipl-3(tm4498) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl-3 RNAi plates, incubated for 12 h at 20 °C, and processed for total FAMEs; as a reference, an aliquot of wild-type young adults on control RNAi bacteria was fasted for 6 h. FAME quantification (mean percentage of fed wild type ±s.e.m.) shows that acute mxl-3 inactivation, as fasting, reduces fat stores in a lipl-dependent manner. n = 3 independent experiments. NSD, P-value>0.05.


( a ) Transcriptional of hlh - 30 mxl - 3 - dependent lipl genes measured in 5 h fasted wild - type ( WT ) or hlh - 30 ( tm1978 ) young - adult worms depicted as mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms show that hlh - 30 is induced following fasting HLH - 30 induces lysosomal lipolysis . N = 4 independent experiments for lipl data , n = 2 independent experiments for hlh - 30 mRNA . HLH - 30 deficiency fully abrogates lipl - 2 , 3 5 ( P0 . 0001 ) , impairs lipl - 1 ( P0 . 01 ) transcriptional activation following fasting .

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(a) Transcriptional levels of hlh-30 and the mxl-3-dependent lipl genes measured in 5 h fasted wild-type (WT) or hlh-30(tm1978) young-adult worms depicted as mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms show that hlh-30 is induced following fasting and HLH-30 induces lysosomal lipolysis. N = 4 independent experiments for lipl data, n = 2 independent experiments for hlh-30 mRNA. HLH-30 deficiency fully abrogates lipl-2, 3 and 5 (P0.0001), and impairs lipl-1 (P0.01) transcriptional activation following fasting.


( b ) treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults . Mean + s . e . m . of three independent experiments shows that inhibition of TOR is sufficient to induce hlh - 30 transcription .

(b) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults. Mean+s.e.m. of three independent experiments shows that inhibition of TOR is sufficient to induce hlh-30 transcription.


( c ) L3 carrying rescuing construct hlh - 30P : : HLH - 30 : : mGFP : : hlh - 30 3 ′ UTR ( mGFP , monomeric GFP , UTR , untranslated region ) , well fed or fasted for 8 h show that HLH - 30 is enriched in intestinal nuclei of fasted worms ( exposure time : well fed , 500 ms ; fasted , 100 ms ) .

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(c) L3 animals carrying the rescuing construct hlh-30P::HLH-30::mGFP::hlh-30 3′UTR (mGFP, monomeric GFP, and UTR, untranslated region), well fed or fasted for 8 h show that HLH-30 is enriched in intestinal nuclei of fasted worms (exposure time: well fed, 500 ms; fasted, 100 ms).


( d ) ORO of wild - type or hlh - 30 ( tm1978 ) young adults fasted for 8 h reveals that hlh - 30 is required for optimal lipid mobilization on fasting . Mean percentages + s . e . m . are shown below relative to well - fed worms treated in parallel , n = 3 independent experiments .

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(d) ORO staining of wild-type or hlh-30(tm1978) young adults fasted for 8 h reveals that hlh-30 is required for optimal lipid mobilization on fasting. Mean percentages+s.e.m. are shown below relative to well-fed worms treated in parallel, n = 3 independent experiments.


( e ) mxl - 3 hlh - 30 transcriptional in wild - type , hlh - 30 ( tm1978 ) mxl - 3 ( ok1947 ) mutants in basal fasted states show that mxl - 3 transcription is hlh - 30 independent , hlh - 30 transcription is mxl - 3 independent . Expression is presented relative to wild - type fed as mean ddCt + s . e . m . No significant differences relative to wild type in same feeding state were observed , n = 3 independent experiments .

(e) mxl-3 and hlh-30 transcriptional levels in wild-type, hlh-30(tm1978) and mxl-3(ok1947) mutants in the basal and fasted states show that mxl-3 transcription is hlh-30 independent, and hlh-30 transcription is mxl-3 independent. Expression is presented relative to wild-type fed animals as mean ddCt+s.e.m. No significant differences relative to wild type in the same feeding state were observed, n = 3 independent experiments.


( f ) Representative ChIP - qPCR of well - fed 6 h fasted mixed stage worms expressing HLH - 30 : : GFP presented as in Fig . 3d shows that HLH - 30 occupies lipl promoters during early fasting . See raw data of two independent experiments in Supplementary Table S9 .

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(f) Representative ChIP-qPCR analysis of well-fed and 6 h fasted mixed stage worms expressing HLH-30::GFP presented as in Fig. 3d shows that HLH-30 occupies the lipl promoters during early fasting. See raw data of two independent experiments in Supplementary Table S9.


( g ) Transcriptional of mxl - 3 - dependent lipl genes in wild - type , mxl - 3 ( ok1947 ) , hlh - 30 ( tm1978 ) or mxl - 3 ( ok1947 ) ; hlh - 30 ( tm1978 ) double mutant young - adult worms show that hlh - 30 suppresses constitutive - induction - of - - lipl - genes phenotype of mxl - 3 mutant ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent experiments .

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(g) Transcriptional levels of the mxl-3-dependent lipl genes in wild-type, mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant young-adult worms show that hlh-30 suppresses the constitutive-induction-of-the-lipl-genes phenotype of mxl-3 mutant animals (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments.


( a ) LRO ( lysosome - related organelle ) live Nile red - stained of hlh - 30 ( tm1978 ) wild - type ( WT ) L3 larvae starved for 60 h show that hlh - 30 is required for expansion of lysosomal compartment following fasting . as mean ± s . e . m . relative to fasted wild - type worms is also presented ; n = 3 independent experiments .

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(a) LRO (lysosome-related organelle) live Nile red-stained images of hlh-30(tm1978) and wild-type (WT) L3 larvae starved for 60 h show that hlh-30 is required for the expansion of the lysosomal compartment following fasting. Quantification as mean±s.e.m. relative to fasted wild-type worms is also presented; n = 3 independent experiments.


( b ) Transcriptional of autophagy genes lgg - 1 , lgg - 2 atg - 16 . 2 in fasted wild - type hlh - 30 ( tm1978 ) young - adult worms show that transcriptional activation of autophagy following fasting is hlh - 30 dependent ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent

(b) Transcriptional levels of the autophagy genes lgg-1, lgg-2 and atg-16.2 in fasted wild-type and hlh-30(tm1978) young-adult worms show that transcriptional activation of autophagy following fasting is hlh-30 dependent (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments


( c ) mean ( + s . d . ) of L1 larvae alive after 48 h fasting in minimum media shows that hlh - 30 ( tm1978 ) mutant are sensitive to starvation . n = 3 independent experiments . For L4 survival see Supplementary Fig . S6g .

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(c) The mean percentage (+ s.d.) of L1 larvae alive after 48 h fasting in minimum media shows that hlh-30 (tm1978) mutant animals are sensitive to starvation. n = 3 independent experiments. For L4 survival see Supplementary Fig. S6g.


( a ) Expression analyses of liver of C57BL / 6J 9 - week fasted overnight ( ON ) for 10 h relative to siblings feeding ad libitum show that LipA ( mouse lysosomal acid lipase ) , Map1lc3a ( mammalian lgg - 1 / lgg - 2 ) Tfeb ( mammalian hlh - 30 ) but not Max ( mammalian mxl - 3 ) are transcriptionally linked to nutrients in mouse liver ( median + s . e . m . fold change ; ddCt ) . of expression were normalized to ActB Cog2 as internal controls ; but Max differences are significant ( P0 . 05 ) , n = 3 independent experiments . Cyp4a14 is a positive control .

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(a) Expression analyses of the liver of C57BL/6J 9-week females fasted overnight (ON) for 10 h relative to siblings feeding ad libitum show that LipA (mouse lysosomal acid lipase), Map1lc3a (mammalian lgg-1/lgg-2) and Tfeb (mammalian hlh-30) but not Max (mammalian mxl-3) are transcriptionally linked to nutrients in the mouse liver (median+s.e.m. fold change; ddCt). Levels of expression were normalized to ActB and Cog2 as internal controls; all but Max differences are significant (P0.05), n = 3 independent experiments. Cyp4a14 is a positive control.


( b ) Expression analyses of control or hTFEB siRNA treated HepG2 incubated in EBSS minimal medium for 2 , 4 or 8 h , compared with expression of control or hTFEB siRNA treated hepatocytes in complete media , show that LAL ( human lysosomal acid lipase ) , MAP1LC3A TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes , that LAL MAP1LC3A induction under nutrient deprivation are TFEB dependent ( median dCt + s . e . m . ) . of expression were normalized to ACTB as internal control ; butMAX differences are significant ( P0 . 05 ) , n = 3 independent experiments . IGFBP is a positive control . EBSS , Earles ' s balanced salt solution .

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(b) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2, 4 or 8 h, compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media, show that LAL (human lysosomal acid lipase), MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes, and that LAL and MAP1LC3A induction under nutrient deprivation are TFEB dependent (median dCt+s.e.m.). Levels of expression were normalized to ACTB as internal control; all butMAX differences are significant (P0.05), n = 3 independent experiments. IGFBP is a positive control. EBSS, Earles's balanced salt solution.


( a ) Wild - type ( WT ) , mxl - 3 ( ok1947 ) , hlh - 30 ( tm1978 ) or mxl - 3 ( ok1947 ) ; hlh - 30 ( tm1978 ) double mutant were used in longevity epistasis analyses , revealing that hlh - 30 suppresses mxl - 3 extended lifespan phenotype . were incubated at 20 ° C transferred every other day until cessation of reproduction . cumulative survival curve is depicted . Kaplan - Meier statistics , calculated using SSPS 17 , are also shown .

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(a) Wild-type (WT), mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant were used in longevity epistasis analyses, revealing that hlh-30 suppresses the mxl-3 extended lifespan phenotype. Animals were incubated at 20°C and transferred every other day until cessation of reproduction. The cumulative survival curve is depicted. Kaplan-Meier statistics, calculated using SSPS 17, are also shown.


( b ) Live Nile red of wild - type lipl - 1lipl - 3 double mutant worms shows that lipl - 1 lipl - 3 contribute to clearance of lipids from endocytic pathway . Mean + s . e . m . signal is shown as relative to wild type ; significant differences are indicated ; n = 4 independent experiments .

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(b) Live Nile red quantification of wild-type and lipl-1lipl-3 double mutant worms shows that lipl-1 and lipl-3 contribute to the clearance of lipids from the endocytic pathway. Mean+s.e.m. signal intensity is shown as percentage relative to wild type; significant differences are indicated; n = 4 independent experiments.


( c , d ) overexpressing LIPL - 1 or LIPL - 3 were incubated at 25 ° C transferred every other day until cessation of reproduction . Data presented as in Fig . 7a show that activated lysosomal lipolysis extends C . elegans lifespan . a Survival presented as mean lifespan ( LS ) ± s . e . m . b Number of uncensored ( that crawled off plate , bagged , exploded or became contaminated were censored ) .

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(c,d) Animals overexpressing LIPL-1 or LIPL-3 were incubated at 25 °C and transferred every other day until cessation of reproduction. Data presented as in Fig. 7a show that activated lysosomal lipolysis extends C. elegans lifespan. a Survival presented as mean lifespan (LS) ± s.e.m. b Number of uncensored animals (animals that crawled off the plate, bagged, exploded or became contaminated were censored).


( A ) EM of cortical synapses derived from WT MPS - IIIA mice at different ages . of synaptic vesicles was quantified from 400 - 500 vesicles ( taken from 5 mice for each genotype at each time point ) expressed as average of vesicle diameter ( nm ) . number of synaptic vesicles per synapse was quantified from 20 different ( taken from 5 mice for each genotype at each time point ) , normalized by of synaptic cleft expressed as % of WT . synaptic density was measured from 20 different ( taken from 5 mice for each genotype at each time point ) as number of synapses / ( # / 500μm2 ) expressed as % of WT . Arrows indicate synaptic cleft , while asterisks indicate abnormal

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(A) EM analysis of cortical synapses derived from WT and MPS-IIIA mice at different ages. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 mice for each genotype at each time point) and expressed as the average of vesicle diameter (nm). The number of synaptic vesicles per synapse was quantified from 20 different images (taken from 5 mice for each genotype at each time point), normalized by the length of synaptic cleft and expressed as % of WT. The synaptic density was measured from 20 different images (taken from 5 mice for each genotype at each time point) as the number of synapses/area(#/500μm2) and expressed as % of WT.Arrows indicate the synaptic cleft, while asterisks indicate abnormal vacuoles


( B , C ) of lysosomal compartment was evaluated by WB ( B ) in brain of MPS - IIIA mice at indicated ages . Quantitation of WB by densitometry ( ImageJ ) was shown ( B ) .

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(B, C) The size of the lysosomal compartment was evaluated by WB (B) analysis in the brain of MPS-IIIA mice at indicated ages. Quantitation of WB by densitometry analysis (ImageJ) was shown (B).


( B , C ) of lysosomal compartment was evaluated by IF ( C ) in brain of MPS - IIIA mice at indicated ages .

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(B, C) The size of the lysosomal compartment was evaluated by IF (C) analysis in the brain of MPS-IIIA mice at indicated ages.