A . Blood - stage growth rate of concavin ( - ) parasites in comparison to wild type . Data points represent parasites growing in individual mice , n indicates total number . P - value is calculated using the Mann Whitney test . Shown is the mean ± SEM .

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A. Blood-stage growth rate of concavin(-) parasites in comparison to wild type. Data points represent parasites growing in individual mice, n indicates total number. P-value is calculated using the Mann Whitney test. Shown is the mean ± SEM.


B . Gametocyte to ookinete conversion of 3 biological replicates . P - value calculated using the Mann Whitney test . Shown is the mean ± SEM . C . Average speed of moving ookinetes . Data points represent individual ookinetes , n indicates total number of cells from 3 biological replicates . P - values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison . D . Ookinete images of wild type and concavin ( - ) parasites revealing similar cell shapes . Scale bar 5 µm .

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

B. Gametocyte to ookinete conversion of 3 biological replicates. P-value calculated using the Mann Whitney test. Shown is the mean ± SEM. C. Average speed of moving ookinetes. Data points represent individual ookinetes, n indicates total number of cells from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison. D. Ookinete images of wild type and concavin(-) parasites revealing similar cell shapes. Scale bar 5 µm.


E . Oocyst development of concavin ( - ) parasites compared to wild type . Data points represent individual midguts observed between d12 - 17 post infection from 3 independent cage feeds . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison .

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E. Oocyst development of concavin(-) parasites compared to wild type. Data points represent individual midguts observed between d12-17 post infection from 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison.


F . Mosquito infections resulted in deformed sporozoites in the salivary gland . Shown are example images of different sporozoites arranged to illustrate their rounding up over time . G . Quantification of sporozoite rounding up over time in the midgut ( MG ) the haemolymph ( HL ) and salivary gland ( SG ) at the indicated days post infection from three biological replicates . Sporozoites were classified as either normal , deformed or round as illustrated in F .

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F. Mosquito infections resulted in deformed sporozoites in the salivary gland. Shown are example images of different sporozoites arranged to illustrate their rounding up over time. G. Quantification of sporozoite rounding up over time in the midgut (MG) the haemolymph (HL) and salivary gland (SG) at the indicated days post infection from three biological replicates. Sporozoites were classified as either normal, deformed or round as illustrated in F.


H . Average speed of salivary gland sporozoites . Data points represent individual sporozoites , n indicates total number from 3 biological replicates . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test .

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

H. Average speed of salivary gland sporozoites. Data points represent individual sporozoites, n indicates total number from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


I . Selected trajectories of manually tracked sporozoites .

I. Selected trajectories of manually tracked sporozoites.


A . Oocyst development of concavin ( - ) parasites complemented with either the P . berghei gene or the P . falciparum orthologue fused to GFP . Data points represent individual midguts observed between d12 - 17 post infection of 3 independent cage feeds . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test .

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

A. Oocyst development of concavin(-) parasites complemented with either the P. berghei gene or the P. falciparum orthologue fused to GFP. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


B . Average speed of salivary gland sporozoites . Data points represent individual sporozoites of 3 independent biological replicates . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test .

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

B. Average speed of salivary gland sporozoites. Data points represent individual sporozoites of 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


C . Sum table of mosquito infections of wild type , concavin ( - ) and complemented lines expressing either P . berghei ( Pb ) or P . falciparum ( Pf ) concavin - GFP . Numbers determined on d17 post mosquito infection . Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections . Note the difference of normally shaped and total sporozoites for concavin ( - ) parasites . Images show infection of salivary glands . Scale bar : 100

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C. Sum table of mosquito infections of wild type, concavin(-) and complemented lines expressing either P. berghei (Pb) or P. falciparum (Pf) concavin-GFP. Numbers determined on d17 post mosquito infection. Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections. Note the difference of normally shaped and total sporozoites for concavin(-) parasites. Images show infection of salivary glands. Scale bar: 100 µm


D . Pb concavin - GFP and Pf concavin - GFP localization in blood and mosquito stages . Nuclei ( blue ) stained with Hoechst . Scale bar : 5 µm .

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D. Pb concavin-GFP and Pf concavin-GFP localization in blood and mosquito stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.


E . Localization of P . berghei concavin - GFP in liver stages . Nuclei ( blue ) stained with Hoechst . Scale bar : 5 µm .

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E. Localization of P. berghei concavin-GFP in liver stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.


F . Immunofluorescence images of permeabilized and unpermeabilized concavin - GFP expressing salivary gland sporozoites stained with an anti - GFP antibody . Note that a GFP signal could only be detected after permeabilization , excluding concavin localization on the parasite surface as illustrated in the model . Scale bar : 5 µm .

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

F. Immunofluorescence images of permeabilized and unpermeabilized concavin-GFP expressing salivary gland sporozoites stained with an anti-GFP antibody. Note that a GFP signal could only be detected after permeabilization, excluding concavin localization on the parasite surface as illustrated in the model. Scale bar: 5 µm.


A , B . Time series of concavin - GFP ( A ) PhiL1 - GFP ( B ) sporozoites before and after bleaching and quantification of the fluorescence signal over time of 3 technical replicates . * indicates time of bleaching . Shown is the mean ± SD .

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A, B. Time series of concavin-GFP (A) PhiL1-GFP (B) sporozoites before and after bleaching and quantification of the fluorescence signal over time of 3 technical replicates. * indicates time of bleaching. Shown is the mean ± SD.


C . Super resolution ( STED ) imaging of PhiL1 - GFP and CSP as well as concavin - GFP and CSP . Cells were stained with an anti - GFP antibody in combination with Atto - 594 ( green ) in addition to an anti - CSP staining in combination with Atto - 647 . Images were deconvolved using the Richardson - Lucy algorithm . The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji . Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test . Scale bar : 1 µm .

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

C. Super resolution (STED) imaging of PhiL1-GFP and CSP as well as concavin-GFP and CSP. Cells were stained with an anti-GFP antibody in combination with Atto-594 (green) in addition to an anti-CSP staining in combination with Atto-647. Images were deconvolved using the Richardson-Lucy algorithm. The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji. Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. Scale bar: 1 µm.


D . Localization of PhiL1 - GFP ( green ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . E . Localization of SiR - Tubulin ( red ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . F . Localization of CSP ( red ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . Data information : Asterisks in D - F indicate apical part of the sporozoite .

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D. Localization of PhiL1-GFP (green) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. E. Localization of SiR-Tubulin (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. F. Localization of CSP (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. Data information: Asterisks in D-F indicate apical part of the sporozoite.


B . Expression and localization of concavinC7A - GFP in blood stage parasites . Scale bar 5 µm .

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B. Expression and localization of concavinC7A-GFP in blood stage parasites. Scale bar 5 µm.


C . Oocysts number in infected mosquitoes . Data points represent individual midguts observed between d12 - 17 post infection of 3 independent cage feeds . Shown is the mean ± SEM . P - value calculated using the Mann Whitney test .

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C. Oocysts number in infected mosquitoes. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-value calculated using the Mann Whitney test.


D . Quantification of concavinC7A - GFP cell shapes at the indicated days from midgut ( MG ) or salivary gland ( SG ) derived sporozoites . Numbers above bars indicate investigated sporozoites .

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D. Quantification of concavinC7A-GFP cell shapes at the indicated days from midgut (MG) or salivary gland (SG) derived sporozoites. Numbers above bars indicate investigated sporozoites.


E . Average speed of salivary gland sporozoites . Data points represent individual sporozoites from 3 independent biological replicates . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test .

E. Average speed of salivary gland sporozoites. Data points represent individual sporozoites from 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


F . Localization of concavinC7A - GFP in normal and deformed sporozoites ( arrowheads point to deformations ) . Scale bar : 5 µm .

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F. Localization of concavinC7A-GFP in normal and deformed sporozoites (arrowheads point to deformations). Scale bar: 5 µm.


A . Growth curve of blood stage parasites in C57BL / 6 mice infected by the bite of 10 mosquitoes ( left ) or 10 . 000 sporozoites intra venously ( right ) at two different times post mosquito infection as indicated . Shown is the mean ± SEM . Mice infected with Concavin ( - ) parasites by bite represent 2 independent biological replicates with 3 mice each . Wild type bite back and Concavin ( - ) intra venous injections represent one biological replicate using 3 or 4 mice respectively .

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

A. Growth curve of blood stage parasites in C57BL/6 mice infected by the bite of 10 mosquitoes (left) or 10.000 sporozoites intra venously (right) at two different times post mosquito infection as indicated. Shown is the mean ± SEM. Mice infected with Concavin(-) parasites by bite represent 2 independent biological replicates with 3 mice each. Wild type bite back and Concavin(-) intra venous injections represent one biological replicate using 3 or 4 mice respectively.


C . Liver cell invasion assay of concavin - gfp and concavin ( - ) parasites . Parasites are positively stained for CSP in case they remain extracellular . Both , normal and deformed parasites were detected intracellularly . Graph shows quantification of CSP positive ( red , hence extracellular ) and negative ( grey , hence intracellular ) sporozoites . Small graph shows the percentage of deformed and normally shaped intracellular concavin ( - ) sporozoites . Data points represent the 4 individual biological replicates with n indicating the numbers of sporozoites observed . Shown is the mean ± SEM . P - values are calculated using the Mann Whitney test . Scale bar : 5 µm .

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

C. Liver cell invasion assay of concavin-gfp and concavin(-) parasites. Parasites are positively stained for CSP in case they remain extracellular. Both, normal and deformed parasites were detected intracellularly. Graph shows quantification of CSP positive (red, hence extracellular) and negative (grey, hence intracellular) sporozoites. Small graph shows the percentage of deformed and normally shaped intracellular concavin(-) sporozoites. Data points represent the 4 individual biological replicates with n indicating the numbers of sporozoites observed. Shown is the mean ± SEM. P-values are calculated using the Mann Whitney test. Scale bar: 5 µm.


D . Liver - stage development of concavin ( - ) parasites compared to wildtype , both expressing cytoplasmic GFP ( white ) . Parasite size was measured 24h and 48h post infection . Data points represent individual parasites from 4 independent biological replicates . Shown is the mean ± SEM . P - values calculated using the Mann Whitney test . Scale bar : 5 µm .

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

D. Liver-stage development of concavin(-) parasites compared to wildtype, both expressing cytoplasmic GFP (white). Parasite size was measured 24h and 48h post infection. Data points represent individual parasites from 4 independent biological replicates. Shown is the mean ± SEM. P-values calculated using the Mann Whitney test. Scale bar: 5 µm.


E . Sporozoite ejection of immobilized concavin ( - ) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes . * indicates individual sporozoites in the ejected saliva . Scale bar : 10 µm .

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

E. Sporozoite ejection of immobilized concavin(-) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes. * indicates individual sporozoites in the ejected saliva. Scale bar: 10 µm.


F , G . Only normally shaped concavin ( - ) sporozoites released by salivary glands move on helical paths ( arrows ) through polyacrylamide gels that mimic the skin ( F ) . Quantification of two individual experiments Scale bar 10 µm . ( G ) : only normal shaped sporozoites were able to migrate through the gel .

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F, G. Only normally shaped concavin(-) sporozoites released by salivary glands move on helical paths (arrows) through polyacrylamide gels that mimic the skin (F). Quantification of two individual experiments Scale bar 10 µm. (G): only normal shaped sporozoites were able to migrate through the gel.


A . Percentage of mosquitoes depositing WT or concavin ( - ) sporozoites in the skin of a mouse during a bite ( left ) and sporozoites deposited during a mosquito bite ( right ) . P - values are calculated using the Fishers exact test ( left ) and the Mann Whitney test ( right ) . Bite transmission experiments were repeated over 3 independent mosquito infections for the KO and 2 independent mosquito infections for the WT . For each mosquito infection 2 - 3 mice were imaged , for a total of 3 - 5 ears .

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

A. Percentage of mosquitoes depositing WT or concavin(-) sporozoites in the skin of a mouse during a bite (left) and sporozoites deposited during a mosquito bite (right). P-values are calculated using the Fishers exact test (left) and the Mann Whitney test (right). Bite transmission experiments were repeated over 3 independent mosquito infections for the KO and 2 independent mosquito infections for the WT. For each mosquito infection 2-3 mice were imaged, for a total of 3-5 ears.


B . Maximum fluorescence intensity projections encoded by color for time from movies showing migrating sporozoites after mosquito - bite transmission . Graphs and camembert diagrams below show fraction of motile and immotile sporozoites after 10 , 20 and 30 minutes of recording , numbers analysed as well as sporozoite speed . Pooled data from 4 - 5 WT or 5 - 8 concavin ( - ) bite sites per time point . Scale bar : 50 µm . P - values are calculated using the Mann Whitney test .

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

B. Maximum fluorescence intensity projections encoded by color for time from movies showing migrating sporozoites after mosquito-bite transmission. Graphs and camembert diagrams below show fraction of motile and immotile sporozoites after 10, 20 and 30 minutes of recording, numbers analysed as well as sporozoite speed. Pooled data from 4-5 WT or 5-8 concavin (-) bite sites per time point. Scale bar: 50 µm. P-values are calculated using the Mann Whitney test.


C . Motile WT or concavin ( - ) sporozoites entering blood vessels during the first hour following micro - injection . Number above bars shows numbers of sporozoites observed . Pooled data from four WT or five concavin ( - ) experiments . P - values are calculated using the Fishers exact test .

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

C. Motile WT or concavin(-) sporozoites entering blood vessels during the first hour following micro-injection. Number above bars shows numbers of sporozoites observed. Pooled data from four WT or five concavin(-) experiments. P-values are calculated using the Fishers exact test.


D , E ( D ) Deformation and ( E ) disintegration of concavin ( - ) sporozoites migrating in the skin . Individual images corresponding to the frames shown on the right are indicated in distinct colours in the maximum projection ( left ) of 13 - min movies . Arrowheads indicate constrictions of the parasites . Graphs below time - lapse show that deformation and disintegration are preceded by a decrease in speed . Color of dots correspond to the time - points displayed in the time - lapse images . Scale bars : 10 µm .

O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E (D) Deformation and (E) disintegration of concavin(-) sporozoites migrating in the skin. Individual images corresponding to the frames shown on the right are indicated in distinct colours in the maximum projection (left) of 13-min movies. Arrowheads indicate constrictions of the parasites. Graphs below time-lapse show that deformation and disintegration are preceded by a decrease in speed. Color of dots correspond to the time-points displayed in the time-lapse images. Scale bars: 10 µm.


F . Percentage of deformation and disintegration events observed in 54 WT and 57 concavin ( - ) sporozoites that were tracked for at least 10 min . Pooled data from 5 WT or 10 concavin ( - ) bite sites .

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F. Percentage of deformation and disintegration events observed in 54 WT and 57 concavin(-) sporozoites that were tracked for at least 10 min. Pooled data from 5 WT or 10 concavin(-) bite sites.


B , Same as in A , with the exception that cells expressing flag - praja2rm or praja2 deletion mutants ( praja21 - 530 , praja21 - 630 and praja2Δ530 - 630 ) were included in the analysis .

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B, Same as in A, with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21-530, praja21-630 and praja2Δ530-630) were included in the analysis.


B . MST signal ( normalized fluorescence ) of P1 ( red curve ) , P2 ( green curve ) and P3 ( cyan curve ) plotted against TBC1D31 , at increasing concentrations of peptides . The threading modelled structure of the overlapping binding segment of praja2 ( praja2550 - 570 ) is shown .

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B. MST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550-570) is shown.


F . MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides : wild - type ( red curve ) , R948A - R951A ( violet curve ) and R957 - R959D - H960A ( orange curve ) .

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F. MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild-type (red curve), R948A-R951A (violet curve) and R957-R959D-H960A (orange curve).


D . Total lysates from mouse brain were immunoprecipitated with anti - TBC1D31 antibodies or with control IgG . The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies .

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D. Total lysates from mouse brain were immunoprecipitated with anti-TBC1D31 antibodies or with control IgG. The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies.


E . Same as in C , with the exception that cells were co - transfected with flag - OFD1 vector and with control siRNA ( siCNT ) or siRNA targeting praja2 . F . Quantitative analysis of the experiments shown in E . A mean value ± SD of three independent experiments is shown . Student ' s t test , * p < 0 , 05 . G

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E. Same as in C, with the exception that cells were co-transfected with flag-OFD1 vector and with control siRNA (siCNT) or siRNA targeting praja2. F. Quantitative analysis of the experiments shown in E. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05. G


C . HEK293 cells transfected with siRNAs were serum deprived , pre - treated with cycloheximide and stimulated with FSK at different time point . Lysates were immunoblotted for endogenous praja2 and OFD1 . Hsp90 was used as loading control . D . Quantitative analysis of the experiments shown in C . A mean value ± SD of three independent experiments is shown . Student ' s t test * p < 0 , 05 ; * * < 0 , 01 versus sipraja2 ( + FSK ) and basal values ( 0 ) . E

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C. HEK293 cells transfected with siRNAs were serum deprived, pre-treated with cycloheximide and stimulated with FSK at different time point. Lysates were immunoblotted for endogenous praja2 and OFD1. Hsp90 was used as loading control. D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test * p<0,05; **<0,01 versus sipraja2 (+FSK) and basal values (0). E


H . Immunoblot analysis of flag - OFD1 or flag - E97G mutant in HEK293 cells stimulated with FSK as in C . I . Quantitative analysis of the experiments shown in H . A mean value ± SD of three independent experiments is shown . Student ' s t test , * p < 0 , 05 versus E97G mutant ( + FSK ) and basal values ( 0 ) .

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H. Immunoblot analysis of flag-OFD1 or flag-E97G mutant in HEK293 cells stimulated with FSK as in C. I. Quantitative analysis of the experiments shown in H. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05 versus E97G mutant (+FSK) and basal values (0).


A . siRNA transfected HEK293 cells were serum deprived and stained for acetylated - tubulin , TBC1D31 and DRAQ5 . B . Quantitative analysis of the experiments shown in A . A mean value ± SD of four independent experiments is shown . Student ' s t test * p < 0 , 05 . C

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A. siRNA transfected HEK293 cells were serum deprived and stained for acetylated-tubulin, TBC1D31 and DRAQ5. B. Quantitative analysis of the experiments shown in A. A mean value ± SD of four independent experiments is shown. Student's t test *p< 0,05. C


C . HEK293 cells expressing flag - OFD1 or flag - S735A were serum deprived and subjected to staining analysis for acetylated α - tubulin , flag and DRAQ5 . Images were collected by super - resolution microscopy ( upper panels ) . Where indicated , flag - OFD1 or flag - S735A was co - transfected with cilia - APEX - GFP vector ( middle panels ) . NIH3T3 expressing flag - OFD1 or flag - S735A were stained with anti - ARL13B and anti - flag antibodies . D , E . Statistical analysis of the experiments shown in D . A mean value ± SD of three independent experiments is shown . For each experimental group , a minimum of 40 cilia were analyzed . Student ' s t test p * * * < 0 , 001 . F

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C. HEK293 cells expressing flag-OFD1 or flag-S735A were serum deprived and subjected to staining analysis for acetylated α-tubulin, flag and DRAQ5. Images were collected by super-resolution microscopy (upper panels). Where indicated, flag-OFD1 or flag-S735A was co-transfected with cilia-APEX-GFP vector (middle panels). NIH3T3 expressing flag-OFD1 or flag-S735A were stained with anti-ARL13B and anti-flag antibodies. D, E. Statistical analysis of the experiments shown in D. A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analyzed. Student's t test p***<0,001. F


F . HEK293 cells transiently transfected with flag - OFD1 or flag - S735A were serum deprived , treated with FSK ( 6 hours ) and stained for flag , acetylated - tubulin and DRAQ5 . G . Statistical analysis of the experiments shown in F . A mean value ± SD of three independent experiments is shown . Cells analysed for each experiment : 300 for NT , 80 for flag - OFD1 and 80 for flag - S735A . Student ' s t test , p * < 0 , 05 , p * * < 0 , 01 . H

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F. HEK293 cells transiently transfected with flag-OFD1 or flag-S735A were serum deprived, treated with FSK (6 hours) and stained for flag, acetylated-tubulin and DRAQ5. G. Statistical analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for flag-OFD1 and 80 for flag-S735A. Student's t test, p*<0,05, p**<0,01. H


B . Confocal images of cilia of the neural tube cells in the wild - type , Ol - TBC1D31 KD , Ol - TBC1D31 KD + hTBC1D31 , wild - type hOFD1 , Ol - TBC1D31 KD + wild - type hOFD1 , hOFD1S735A , Ol - TBC1D31 KD + hOFD1S735A , Ol - TBC1D31 KD + hOFD1S735D and Ol - TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti - acetylated α - tubulin antibody ( green ) and DAPI ( blue ) .

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B. Confocal images of cilia of the neural tube cells in the wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, wild-type hOFD1, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735D and Ol-TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti-acetylated α-tubulin antibody (green) and DAPI (blue).


A - E Echocardiographic analysis of ( A ) posterior wall thickness , ( B ) septum thickness , ( C ) left ventricle end diastolic diameter , ( D ) fractional area change and ( E ) ejection fraction . n = 8 per genotype .

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A-E Echocardiographic analysis of (A) posterior wall thickness, (B) septum thickness, (C) left ventricle end diastolic diameter, (D) fractional area change and (E) ejection fraction. n= 8 per genotype.


F Comparison of TAZ gene expression , normalized to S12 , in heart , liver , and kidney , analyzed in triplicates by quantitative PCR . n = 5 per genotype . G TAZ gene expression in percent . Wild - type is set to 100 % .

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F Comparison of TAZ gene expression, normalized to S12, in heart, liver, and kidney, analyzed in triplicates by quantitative PCR. n= 5 per genotype.G TAZ gene expression in percent. Wild-type is set to 100%.


H , I Lipid species profiles of cardiolipin ( H ) and monolysocardiolipin ( I ) in isolated cardiac mitochondria analyzed by mass spectrometry . n = 4 per genotype .

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H, I Lipid species profiles of cardiolipin (H) and monolysocardiolipin (I) in isolated cardiac mitochondria analyzed by mass spectrometry. n= 4 per genotype.


J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues .

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J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues.


A Periodic measurement of the oxygen consumption rate ( OCR ) of mitochondria , isolated from 5 mice per genotype , before and after the administration of the indicated compounds ( left ) . Quantification of OCR in independent experiments , n = 8 per genotype ( right ) .

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A Periodic measurement of the oxygen consumption rate (OCR) of mitochondria, isolated from 5 mice per genotype, before and after the administration of the indicated compounds (left). Quantification of OCR in independent experiments, n= 8 per genotype (right).


B OCR rates of cardiac mitochondria after administration of pyruvate , malate and ADP , n = 7 per genotype .

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B OCR rates of cardiac mitochondria after administration of pyruvate, malate and ADP, n= 7 per genotype.


A Mitochondrial membranes , isolated from cardiac tissue , were solubilized in 1 % digitonin and analyzed by BN - PAGE and Western - blotting with the indicated antibodies .

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A Mitochondrial membranes, isolated from cardiac tissue, were solubilized in 1% digitonin and analyzed by BN-PAGE and Western- blotting with the indicated antibodies.


B Cardiac mitochondria were prepared as in ( A ) and analyzed on a large pore BN - gel for better resolution of supercomplexes .

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B Cardiac mitochondria were prepared as in (A) and analyzed on a large pore BN-gel for better resolution of supercomplexes.


C Cardiac mitochondria were solubilized in 0 . 4 % DDM and complexes were separated by BN - PAGE and analyzed by Western - blotting with the indicated antibodies .

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C Cardiac mitochondria were solubilized in 0.4% DDM and complexes were separated by BN-PAGE and analyzed by Western- blotting with the indicated antibodies.


D Mitochondria were isolated from kidney and liver and analyzed as in ( A ) .

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D Mitochondria were isolated from kidney and liver and analyzed as in (A).


E Quantification of ROS production over time in cardiac mitochondria using the ROS - specific fluorescence - based sensor H2DCFDA . One representative measurement is shown . Analyses were performed in triplicates and confirmed for three animals per genotype . Significance was calculated using Student ' s T - TEST . sh , shTAZ .

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E Quantification of ROS production over time in cardiac mitochondria using the ROS-specific fluorescence-based sensor H2DCFDA. One representative measurement is shown. Analyses were performed in triplicates and confirmed for three animals per genotype. Significance was calculated using Student's T-TEST. sh, shTAZ.


A Enzymatic activity of ubiquinone - cytochrome c oxidase in mitochondria isolated from cardiac tissue . The ubiquinone analogue decylubiquinone was used in this assay . Data were standardized to malate - dehydrogenase activity and presented as percent of wild - type , n = 4 per genotype .

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A Enzymatic activity of ubiquinone-cytochrome c oxidase in mitochondria isolated from cardiac tissue. The ubiquinone analogue decylubiquinone was used in this assay. Data were standardized to malate-dehydrogenase activity and presented as percent of wild-type, n= 4 per genotype.


B Cytochrome c reductase activity , standardized to malate - dehydrogenase activity in cardiac mitochondria , were plotted as % of wild - type , n = 3 per genotype .

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B Cytochrome c reductase activity, standardized to malate-dehydrogenase activity in cardiac mitochondria, were plotted as % of wild-type, n= 3 per genotype.


C Protein levels of Cox1 and Rieske compared to VDAC3 in three different dilutions of isolated mitochondria , separated on SDS gel and visualized by Western - blotting .

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C Protein levels of Cox1 and Rieske compared to VDAC3 in three different dilutions of isolated mitochondria, separated on SDS gel and visualized by Western-blotting.


D Respiratory chain complexes in cardiac mitochondria were separated on BN - PAGE and stained for activity of complex I , II , IV and F1FO - ATPase . sh , shTAZ ; Q , ubiquinone .

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D Respiratory chain complexes in cardiac mitochondria were separated on BN-PAGE and stained for activity of complex I, II, IV and F1FO- ATPase. sh, shTAZ; Q, ubiquinone.


A Enzymatic activity of the succinate dehydrogenase in isolated cardiac mitochondria from 3 animals per genotype .

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A Enzymatic activity of the succinate dehydrogenase in isolated cardiac mitochondria from 3 animals per genotype.


B Mitochondrial membranes were solubilized in 1 % digitonin and analyzed by BN - PAGE and Western - blotting using antibodies against SDH components SDHA and SDHB ( upper panel ) . For a loading control , samples were separated on SDS gel and analyzed by Western - blotting ( lower panel ) .

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B Mitochondrial membranes were solubilized in 1% digitonin and analyzed by BN-PAGE and Western-blotting using antibodies against SDH components SDHA and SDHB (upper panel). For a loading control, samples were separated on SDS gel and analyzed by Western-blotting (lower panel).


C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western - blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm . Lower panel : quantification of signal ratio SDHA / VDAC3 in shTAZ and control ( set to 100 % ) .

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C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western- blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm. Lower panel: quantification of signal ratio SDHA/VDAC3 in shTAZ and control (set to 100%).


D Analysis of gene expression in cardiac tissue analyzed in ( C ) by qPCR using primers against indicated mRNA . Data of one representative measurement , normalized to b - actin with WT set to 100 % are shown . Identical results were obtained in further experiments with two different pairs of animals .

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D Analysis of gene expression in cardiac tissue analyzed in (C) by qPCR using primers against indicated mRNA. Data of one representative measurement, normalized to b-actin with WT set to 100% are shown. Identical results were obtained in further experiments with two different pairs of animals.


E Upper panel : SDH complex of cardiac , liver and kidney were analyzed by BN - PAGE as in ( B ) ; lower panel : the same samples were analyzed by SDS gel and Western - blotting .

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E Upper panel: SDH complex of cardiac, liver and kidney were analyzed by BN-PAGE as in (B); lower panel: the same samples were analyzed by SDS gel and Western-blotting.


F , G Analysis of enzymatic activity of the succinate dehydrogenase related to the malate dehydrogenase activity in mitochondria isolated from kidney ( F ) and liver ( G ) . Values of 3 animals per genotype are plotted as percent of wild - type . sh , shTAZ .

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F, G Analysis of enzymatic activity of the succinate dehydrogenase related to the malate dehydrogenase activity in mitochondria isolated from kidney (F) and liver (G). Values of 3 animals per genotype are plotted as percent of wild-type. sh, shTAZ.


A Flow cytometry analysis of cardiac troponinT ( cTNT ) expression was performed at day 60 post cardiac differentiation . 98 % of cTNT + cardiomyocytes ( CM ) were obtained after metabolic selection .

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A Flow cytometry analysis of cardiac troponinT (cTNT) expression was performed at day 60 post cardiac differentiation. 98% of cTNT+ cardiomyocytes (CM) were obtained after metabolic selection.


B RT - PCR showing the expression of indicated genes in undifferentiated iPSCs and differentiated iPSC - CMs of BTHS patient TAZ10 and a healthy control .

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B RT-PCR showing the expression of indicated genes in undifferentiated iPSCs and differentiated iPSC-CMs of BTHS patient TAZ10 and a healthy control.


C Immunostaining of control ( left ) and BTHS iPSC - CMs at day 60 post differentiation for cardiac troponin T ( cTNT ) and Connexin 43 ( Cx43 ) . Scale bars : 50µm .

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C Immunostaining of control (left) and BTHS iPSC-CMs at day 60 post differentiation for cardiac troponin T (cTNT) and Connexin 43 (Cx43). Scale bars: 50µm.


D Quantitation of sarcomere organization . Left panel : Immunostaining of cTNT , MLC2a ( myosin light chain ) and a - actinin at day 60 post cardiac differentiation . Right panel : The bar graphs at the right panel represent the sarcomere regularity from 3 independent experiments . Error bars represent standard deviation for the comparison between control and BTHS patient TAZ10 . Significance was analyzed using Student ' s T - TEST * * P = 0 . 002 and * * * P = 0 . 0006 . Scale bars : 50 µm .

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D Quantitation of sarcomere organization. Left panel: Immunostaining of cTNT, MLC2a (myosin light chain) and a-actinin at day 60 post cardiac differentiation. Right panel: The bar graphs at the right panel represent the sarcomere regularity from 3 independent experiments. Error bars represent standard deviation for the comparison between control and BTHS patient TAZ10. Significance was analyzed using Student's T-TEST **P = 0.002 and ***P = 0.0006. Scale bars: 50 µm.


E Measurement of cell surface areas . Cell surface area was measured in single cardiomyocytes ( n = at least 30 measurements each group from N = 3 independent experiments each group ) at day 60 post cardiac differentiation after immunostaining of sarcomeric a - actinin . Scale bars : 50 µm .

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E Measurement of cell surface areas. Cell surface area was measured in single cardiomyocytes (n = at least 30 measurements each group from N = 3 independent experiments each group) at day 60 post cardiac differentiation after immunostaining of sarcomeric a-actinin. Scale bars: 50 µm.


A Oxygen consumption rate ( OCR ) of cardiomyocytes from patient TAZ10 and control at basal conditions and after the administration of the indicated compounds .

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A Oxygen consumption rate (OCR) of cardiomyocytes from patient TAZ10 and control at basal conditions and after the administration of the indicated compounds.


B Mitochondrial membranes analyzed by BN - PAGE and Western - blotting using indicated antibodies . For a loading control samples were separated on SDS gel and analyzed by Western - blotting ( lower panel ) .

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B Mitochondrial membranes analyzed by BN-PAGE and Western-blotting using indicated antibodies. For a loading control samples were separated on SDS gel and analyzed by Western-blotting (lower panel).


C Western blot analysis of SDHA protein levels compared to VDAC3 . Quantification of SDHA relative to VDAC3 in percent with control set to 100 % . Error bars indicate range of values of two independent experiments .

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C Western blot analysis of SDHA protein levels compared to VDAC3. Quantification of SDHA relative to VDAC3 in percent with control set to 100%. Error bars indicate range of values of two independent experiments.


D Analysis of SDHA gene expression in triplicates by qPCR in cardiomyocytes , analyzed in ( C ) .

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D Analysis of SDHA gene expression in triplicates by qPCR in cardiomyocytes, analyzed in (C).


D . Representative immunofluorescence images of MEFs 48h after transgene induction stained for the indicated reprogramming factors . Scale bar represents 50 μm ( n = 3 biological replicates ) .

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D. Representative immunofluorescence images of MEFs 48h after transgene induction stained for the indicated reprogramming factors. Scale bar represents 50 μm (n = 3 biological replicates).


E . Log fluorophore expression superimposed on Uniform Manifold Approximation and Projection ( UMAP ) embedding of the dataset . Shown are fluorophore expression levels ( gene exp . ) on a logarithmic ( ln ) scale . F . Log transgene expression superimposed on a UMAP embedding of the dataset . Shown are transgene expression levels ( gene exp . ) on an a logarithmic ( ln ) scale .

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

E. Log fluorophore expression superimposed on Uniform Manifold Approximation and Projection (UMAP) embedding of the dataset. Shown are fluorophore expression levels (gene exp.) on a logarithmic (ln) scale. F. Log transgene expression superimposed on a UMAP embedding of the dataset. Shown are transgene expression levels (gene exp.) on an a logarithmic (ln) scale.


G . UMAP embedding of scRNA - seq dataset colored by technical replicate . Orange : Replicate 1 , Blue : Replicate

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

G. UMAP embedding of scRNA-seq dataset colored by technical replicate. Orange: Replicate 1, Blue: Replicate 2


H . Cell cycle score superimposed on UMAP embedding of the dataset . See unsupervised analysis of single - cell RNA - seq section in Methods for further details .

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H. Cell cycle score superimposed on UMAP embedding of the dataset. See unsupervised analysis of single-cell RNA-seq section in Methods for further details.


D . Log - normalized expression z - score of genes up - ( top panels ) and downregulated ( bottom panels ) by Ascl1 ( left , blue violins ) and MyoD1 ( right , yellow violins ) versus their scaled expression levels ( x - axis ) binned into 10 % intervals . For each box , the centerline defines the median , the height of the box is given by the interquartile range ( IQR ) , the whiskers are given by 1 . 5 * IQR and outliers are given as points beyond the minimum or maximum whisker .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Log-normalized expression z-score of genes up- (top panels) and downregulated (bottom panels) by Ascl1 (left, blue violins) and MyoD1 (right, yellow violins) versus their scaled expression levels (x-axis) binned into 10% intervals. For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker.


E . Fate titration plot of Ascl1 and MyoD1 double positive cells Shown are decision boundaries for indicated fates according to transcription factor levels . MyoD1 and Ascl1 expression shown on the x - and y - axis are log - normalized expression values scaled into the dynamic range of the single - positive condition Cells are colored according to their lineage endpoint as determined by CellRank .

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

E. Fate titration plot of Ascl1 and MyoD1 double positive cells Shown are decision boundaries for indicated fates according to transcription factor levels. MyoD1 and Ascl1 expression shown on the x- and y-axis are log-normalized expression values scaled into the dynamic range of the single-positive condition Cells are colored according to their lineage endpoint as determined by CellRank.


F . Representative immunofluorescence images of cells 3 days after transgene induction . Cells were stained for the fluorescent reporter present on the PiggyBac construct of the reprogramming factor . Arrows indicate multinucleated cells . Scale bar represents 50 µm . mutAscl1 = mutant Ascl1 . G . Quantification of the percentage of cells with more than one nucleus among all transfected cells within a given condition . Data points represent biological replicates ( n = 4 ) . For each box , the centerline defines the median , the height of the box is given by the interquartile range ( IQR ) , the whiskers are given by 1 . 5 * IQR and outliers are given as points beyond the minimum or maximum whisker . Pairwise comparisons performed with Mann - Whitney U test , correction for multiple testing performed with Benjamini - Hochberg correction . * : p < 0 . 05 ( MyoD1 vs Ascl1 : p = 0 . 03 , MyoD1 vs mutAscl1 : p = 0 . 03 , MyoD1 vs Ascl1 & MyoD1 : p = 0 . 03 , MyoD1 vs mutAscl1 & MyoD1 : p = 0 . 03 , Ascl1 vs Ascl1 & MyoD1 : p = 0 . 03 , Ascl1 vs mutAscl1 & MyoD1 : p = 0 . 03 , mutAscl1 vs Ascl1 & MyoD1 : p = 0 . 03 , mutAscl1 vs mutAscl1 & MyoD1 : p = 0 .

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

F. Representative immunofluorescence images of cells 3 days after transgene induction. Cells were stained for the fluorescent reporter present on the PiggyBac construct of the reprogramming factor. Arrows indicate multinucleated cells. Scale bar represents 50 µm. mutAscl1 = mutant Ascl1. G. Quantification of the percentage of cells with more than one nucleus among all transfected cells within a given condition. Data points represent biological replicates (n = 4). For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker. Pairwise comparisons performed with Mann-Whitney U test, correction for multiple testing performed with Benjamini-Hochberg correction. *: p < 0.05 (MyoD1 vs Ascl1: p = 0.03, MyoD1 vs mutAscl1: p = 0.03, MyoD1 vs Ascl1 & MyoD1: p = 0.03, MyoD1 vs mutAscl1 & MyoD1: p = 0.03, Ascl1 vs Ascl1 & MyoD1: p = 0.03, Ascl1 vs mutAscl1 & MyoD1: p = 0.03, mutAscl1 vs Ascl1 & MyoD1: p = 0.03, mutAscl1 vs mutAscl1 & MyoD1: p = 0.03


( A ) in vitro kinase assays were performed using purified recombinant kinases ( ULK1 and ULK2 ) and substrate ( ATG16L1 ) in the presence of radiolabelled ATP . ULK and ATG16L1 inputs were examined by western blot ( WB ) and substrate phosphorylation was analyzed by autoradiography ( AR ) . Data information : Unless otherwise indicated experiments were performed three times .

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

(A) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB) and substrate phosphorylation was analyzed by autoradiography (AR). Data information: Unless otherwise indicated experiments were performed three times.


( B ) Full - length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay . ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography . Data information : Unless otherwise indicated experiments were performed three times .

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

(B) Full-length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay. ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography. Data information: Unless otherwise indicated experiments were performed three times.


( D ) Full - length or mutated HA - ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay . Inputs were analysed by WB and target phosphorylation by AR . Data information : Unless otherwise indicated experiments were performed three times .

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

(D) Full-length or mutated HA-ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay. Inputs were analysed by WB and target phosphorylation by AR. Data information: Unless otherwise indicated experiments were performed three times.


( E ) HEK293A cells were transfected with wild - type or phospho - dead ATG16L1 in the presence of wild - type or kinase - dead ULK1 . Phosphorylation of ATG16L1 ( S278 or S287 ) and inputs were examined by WB . Data information : Unless otherwise indicated experiments were performed three times .

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

(E) HEK293A cells were transfected with wild-type or phospho-dead ATG16L1 in the presence of wild-type or kinase-dead ULK1. Phosphorylation of ATG16L1 (S278 or S287) and inputs were examined by WB. Data information: Unless otherwise indicated experiments were performed three times.


( A ) Wild - type , ULK1 / 2 double knockout ( dKO ) , or IKKα KO mouse embryonic fibroblasts ( MEFs ) were incubated with either complete medium , amino acid - deficient DMEM , or HBSS for 1 hour . Samples were immunoblotted using the indicated antibodies . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

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

(A) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( B ) Wild - type , ULK1 / 2 dKO , or IKKα KO MEFs were infected with log phase Salmonella for 2 hours ; bacteria - containing media was then removed and cells were incubated with gentamycin ( 50 µg / mL ) - containing DMEM for 2 hours . Samples were immunoblotted using the indicated antibodies . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

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

(B) Wild-type, ULK1/2 dKO, or IKKα KO MEFs were infected with log phase Salmonella for 2 hours; bacteria-containing media was then removed and cells were incubated with gentamycin (50 µg/mL)-containing DMEM for 2 hours. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( C ) Wild - type , ULK1 / 2 dKO , or IKKα KO MEFs cells were infected with Salmonella for 1 hours . Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B . Representative images are shown ( scale bars , 10 μm and 3 μm ) . Quantification was generated from 8 fields of view from a representative experiment . The experiments were repeated twice . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type, ULK1/2 dKO, or IKKα KO MEFs cells were infected with Salmonella for 1 hours. Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 μm and 3 μm). Quantification was generated from 8 fields of view from a representative experiment. The experiments were repeated twice. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( D ) Wild - type , ULK1 / 2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour . Xenophagy rates were examined through Colony Forming Unit ( CFU ) assays . Quantification of infection rates by immunofluorescence is demonstrated in the right panel . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

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

(D) Wild-type, ULK1/2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour. Xenophagy rates were examined through Colony Forming Unit (CFU) assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( A ) HEK293A cells were transfected with either flag - tagged WT ATG16L1 or T300A ATG16L1 . ULK1 was co - transfected in increasing amounts where indicated . Cleavage of ATG16L1 was analyzed by WB of whole cell lysates . Levels of ATG16L1 cleavage were quantified from 3 biological repeats ( right panel ) . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with either flag-tagged WT ATG16L1 or T300A ATG16L1. ULK1 was co-transfected in increasing amounts where indicated. Cleavage of ATG16L1 was analyzed by WB of whole cell lysates. Levels of ATG16L1 cleavage were quantified from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( B ) HEK293A cells were transfected with either tagged wild - type , T300A , or S278 / T300A ATG16L1 in the presence or absence of ULK1 . Cleavage of ATG16L1 was analyzed by WB . Levels of ATG16L1 cleavage were measured from 3 biological repeats ( right panel ) . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with either tagged wild-type, T300A, or S278/T300A ATG16L1 in the presence or absence of ULK1. Cleavage of ATG16L1 was analyzed by WB. Levels of ATG16L1 cleavage were measured from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( C ) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan - caspase inhibitor Z - VAD - FMK ( 15 µM ) for 4 hours . Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

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

(C) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan-caspase inhibitor Z-VAD-FMK (15 µM) for 4 hours. Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( D ) Wild - type or T300A - expressing HEK293A were treated with Salmonella in the presence or absence of ULK1 / 2 inhibitor ( 16 μM ) for the indicated time points . Expression of ATG16L1 was analysed by WB . The experiments were performed twice .

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

(D) Wild-type or T300A-expressing HEK293A were treated with Salmonella in the presence or absence of ULK1/2 inhibitor (16 μM) for the indicated time points. Expression of ATG16L1 was analysed by WB. The experiments were performed twice.


( E ) ATG16L1 knock - out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour . Xenophagy rates were examined through CFU assays . Quantification of infection rates by immunofluorescence is demonstrated in the right panel . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

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

(E) ATG16L1 knock-out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour. Xenophagy rates were examined through CFU assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( A ) Wild - type MEF cells were infected with Salmonella for 25 minutes . Phospho - ATG16L1 , total ATG16L1 , and LPS were stained and analysed by immunofluorescence . Representative immunofluorescent images are shown ( scale bars , 10 μm and 1 μm ) . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

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

(A) Wild-type MEF cells were infected with Salmonella for 25 minutes. Phospho-ATG16L1, total ATG16L1, and LPS were stained and analysed by immunofluorescence. Representative immunofluorescent images are shown (scale bars, 10 μm and 1 μm). Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( B ) Wild - type and ULK1 / 2 dKO were infected with Salmonella for 25 minutes . Immunofluorescence was performed using antibodies against LPS and ATG16L1 . Representative immunofluorescent images are shown on the left panel ( scale bars , 10 μm and 2 μm ) . Quantification of ATG16L1 - positive bacteria from 7 fields of view from a representative experiment is shown in the right panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

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

(B) Wild-type and ULK1/2 dKO were infected with Salmonella for 25 minutes. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Representative immunofluorescent images are shown on the left panel (scale bars, 10 μm and 2 μm). Quantification of ATG16L1-positive bacteria from 7 fields of view from a representative experiment is shown in the right panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( C ) ATG16L1 knock - out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour . Bacteria were stained using anti - LPS antibodies to analyze localization in addition to ATG16L1 . Representative immunofluorescent images of ATG16L1 and LPS are shown ( scale bars , 5μm and 1 μm ) . Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

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

(C) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to ATG16L1. Representative immunofluorescent images of ATG16L1 and LPS are shown (scale bars, 5μm and 1 μm). Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( D ) ATG16L1 knock - out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour . Bacteria were stained using anti - LPS antibodies to analyze localization in addition to the autophagy marker LC3B . Representative immunofluorescent images of LC3B and LPS are shown ( scale bars , 5μm and 1 μm ) . Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

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

(D) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to the autophagy marker LC3B. Representative immunofluorescent images of LC3B and LPS are shown (scale bars, 5μm and 1 μm). Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( B ) HEK293T cells transiently expressing FLAG and FLAG - tagged TEX264 were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of the lysates ) and immunoprecipitates ( from 80 % of the lysates ) were analyzed by immunoblotting using the indicated antibodies .

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(B) HEK293T cells transiently expressing FLAG and FLAG-tagged TEX264 were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies.


( C ) HEK293T cells transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody and detected with the indicated antibodies .

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(C) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.


( D and E ) MEFs stably expressing TEX264 - GFP or its mutant were cultured in starvation media and immunostained with an anti - LC3 antibody . Bars : 10 µm and 1 µm ( insets ) ( D ) . Quantification of the number of TEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 48 cells for each cell type ( E ) .

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(D and E) MEFs stably expressing TEX264-GFP or its mutant were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 1 µm (insets) (D). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 48 cells for each cell type (E).


( F and G ) WT and TEX264 - KO HeLa cells ( with or without the indicated TEX264 mutants with a C - terminal FLAG tag ) stably expressing the ER - phagy reporter ( ss - RFP - GFP - KDEL ) were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , cells were cultured in starvation medium lacking amino acids and serum for 9 h ( F ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± standard error of the mean ( SEM ) of three independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( G ) .

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

(F and G) WT and TEX264-KO HeLa cells (with or without the indicated TEX264 mutants with a C-terminal FLAG tag) stably expressing the ER-phagy reporter (ss-RFP-GFP-KDEL) were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured in starvation medium lacking amino acids and serum for 9 h (F). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (G).


( A and B ) MEFs stably expressing TEX264 - GFP were cultured in starvation media with or without bafilomycin A1 ( Baf A1 ) and immunostained with anti - pTEX264 and anti - LC3 antibodies . Bars : 10 µm and 1 µm ( insets ) ( A ) . Quantification of the number of pTEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 35 cells for each cell type ( B ) .

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(A and B) MEFs stably expressing TEX264-GFP were cultured in starvation media with or without bafilomycin A1 (Baf A1) and immunostained with anti-pTEX264 and anti-LC3 antibodies. Bars: 10 µm and 1 µm (insets) (A). Quantification of the number of pTEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 35 cells for each cell type (B).


( C ) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h , and cell homogenates were subjected to an OptiPrep membrane flotation analysis .

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(C) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h, and cell homogenates were subjected to an OptiPrep membrane flotation analysis.


( D , E and F ) WT , ATG3 - KO , and FIP200 - KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h . Cell lysates were analyzed by immunoblotting using the indicated antibodies ( D ) . Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown ( E and F ) . Data represent the mean ± SEM of three independent experiments .

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

(D, E and F) WT, ATG3-KO, and FIP200-KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies (D). Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown (E and F). Data represent the mean ± SEM of three independent experiments.


( A ) HeLa cells were cultured with 20 nM CK1 inhibitor ( D4476 ) or CK2 inhibitor ( CX4945 ) for 3 h , followed by starvation ( with the inhibitors ) for 3 h . Cell lysates were analyzed by immunoblotting using the indicated antibodies .

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(A) HeLa cells were cultured with 20 nM CK1 inhibitor (D4476) or CK2 inhibitor (CX4945) for 3 h, followed by starvation (with the inhibitors) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies.


( B ) Recombinant TEX264 - FLAG was incubated with or without CK2 and the CK2 inhibitor ( CX4945 ) in the presence of 200 µM ATP for 1 h .

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(B) Recombinant TEX264-FLAG was incubated with or without CK2 and the CK2 inhibitor (CX4945) in the presence of 200 µM ATP for 1 h.


( C ) HEK293T cells transiently expressing TEX264 - FLAG and one of the three Halo - CK2A isoforms or Halo - CK2B were subjected to immunoprecipitation with an anti - FLAG antibody and detected with anti - Halo and anti - FLAG antibodies .

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(C) HEK293T cells transiently expressing TEX264-FLAG and one of the three Halo-CK2A isoforms or Halo-CK2B were subjected to immunoprecipitation with an anti-FLAG antibody and detected with anti-Halo and anti-FLAG antibodies.


( D and E ) HEK293T cells transiently expressing WT or TEX264 - FLAG mutants were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of the lysates ) and immunoprecipitates ( from 80 % of the lysates ) were analyzed by immunoblotting using the indicated antibodies ( D ) . The band intensities in the IP fractions were quantified , and the phospho - serine : FLAG ratio was calculated . Data represent the mean ± SEM of five independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( E ) .

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

(D and E) HEK293T cells transiently expressing WT or TEX264-FLAG mutants were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies (D). The band intensities in the IP fractions were quantified, and the phospho-serine:FLAG ratio was calculated. Data represent the mean ± SEM of five independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (E).


( F and G ) MEFs stably expressing TEX264 - GFP were cultured with the CK2 inhibitor for 3 h followed by starvation ( with the CK2 inhibitor ) and immunostained with an anti - LC3 antibody . Scale bars represent 10 μm and 1 μm ( insets ) ( F ) . Quantification of the number of TEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by an unpaired two - tailed Student ' s t - test . Data were collected from 85 cells for each cell type ( G ) .

O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and G) MEFs stably expressing TEX264-GFP were cultured with the CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) and immunostained with an anti-LC3 antibody. Scale bars represent 10 μm and 1 μm (insets) (F). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by an unpaired two-tailed Student's t-test. Data were collected from 85 cells for each cell type (G).


( H and I ) WT HeLa cells stably expressing the ER - phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation ( with the CK2 inhibitor ) for 6 h ( H ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± SEM of six independent experiments . Differences were statistically analyzed by an unpaired two - tailed Student ' s t - test ( I ) .

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

(H and I) WT HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) for 6 h (H). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of six independent experiments. Differences were statistically analyzed by an unpaired two-tailed Student's t-test (I).


( B ) HEK293T cells transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody and detected with the indicated antibodies .

(B) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.


( C and D ) MEFs stably expressing TEX264 - GFP or its mutants were cultured in starvation media and immunostained with an anti - LC3 antibody . Bars : 10 µm and 5 µm ( insets ) ( C ) . The intensity of the GFP signal under starvation conditions was quantified at TEX264 - GFP puncta and the ER . The puncta : ER signal ratio was calculated . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 70 puncta for each cell type ( D ) .

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(C and D) MEFs stably expressing TEX264-GFP or its mutants were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 5 µm (insets) (C). The intensity of the GFP signal under starvation conditions was quantified at TEX264-GFP puncta and the ER. The puncta:ER signal ratio was calculated. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 70 puncta for each cell type (D).


( E and F ) WT and TEX264 - KO ( expressing WT or TEX264 - FLAG mutants ) HeLa cells stably expressing the ER - phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , the cells were cultured in starvation medium lacking amino acids and serum for 9 h ( E ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± SEM of four independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( F ) .

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

(E and F) WT and TEX264-KO (expressing WT or TEX264-FLAG mutants) HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, the cells were cultured in starvation medium lacking amino acids and serum for 9 h (E). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (F).


( b ) Density gradient fractions of NRK cells starved for 8 h were analysed by transmission electron microscopy ( upper panel ) and immunoblotted for LC3 or LAMP1 ( lower panel ) . Proteins in fraction 1 were identified by mass spectrometry analysis . Three replicates were carried out from three independent purifications . Scale bar , 2 μm .

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

(b) Density gradient fractions of NRK cells starved for 8 h were analysed by transmission electron microscopy (upper panel) and immunoblotted for LC3 or LAMP1 (lower panel). Proteins in fraction 1 were identified by mass spectrometry analysis. Three replicates were carried out from three independent purifications. Scale bar, 2 μm.


( e ) NRK cells grown on the SAMCell were transfected with FAM - RNAi by reverse transfection and the transfection efficiency was monitored by microscopy . Left panel , FAM - RNAi - positive cells ; right panel , differential interference contrast ( DIC ) image of the cells in the left panel .

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(e) NRK cells grown on the SAMCell were transfected with FAM-RNAi by reverse transfection and the transfection efficiency was monitored by microscopy. Left panel, FAM-RNAi-positive cells; right panel, differential interference contrast (DIC) image of the cells in the left panel.


( a ) NRK cells stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or Cltc ( clathrin heavy chain ) - RNAi and starved for 12 h . Scale bar , 5 μm . ( b ) Cells from a were assessed for enlarged autolysosomes after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d .

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(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 12 h. Scale bar, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative TEM micrographs of nonspecific - or Cltc - RNAi - transfected NRK cells after 12 h of serum starvation . Scale bar , 5 μm .

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(c) Representative TEM micrographs of nonspecific- or Cltc-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm.