( B - C ) Western blot showing expression of indicated in adaptive HCC with or without ATP5B silencing .

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(B-C) Western blot showing the expression of indicated proteins in adaptive HCC cells with or without ATP5B silencing.


( D - E ) Transwell assays showing migration invasion of adaptive HCC with or without silence of ATP5B ( 5 × 104 adaptive Huh7 , 1 × 105 adaptive PLC / PRF / 5 ) , Scale bars , 100 μm . ( n = 3 biological replicates , Two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(D-E) Transwell assays showing the migration and invasion of adaptive HCC cells with or without the silence of ATP5B (5×104 adaptive Huh7 cells, 1×105 adaptive PLC/PRF/5 cells), Scale bars, 100 μm. (n=3 biological replicates, Two-way ANOVA). Data information: Data are presented as means ± S.D.


( G ) Western blot showing expression of EMT markers in with ( shTOM ) or without ( shNC ) knockdown of TOMM34 or overexpression of ATP5B .

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(G) Western blot showing the expression of EMT markers in cells with (shTOM) or without (shNC) knockdown of TOMM34 or overexpression of ATP5B.


( H ) Transwell assays showing migration invasion of indicated ( 5 × 104 ; shTOM , shTOMM34 , TOMM34 knockdown ) . Scale bars , 100 μm . ( n = 4 biological replicates , two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(H) Transwell assays showing the migration and invasion of indicated cells (5×104 cells; shTOM, shTOMM34, TOMM34 knockdown cells). Scale bars, 100 μm. (n=4 biological replicates, two-way ANOVA). Data information: Data are presented as means ± S.D.


( A - B ) Co - IP was performed to detect interaction between TOMM34 ATP5B treated with or without Gboxin for 24 hours in parental or adaptive PLC / PRF / 5 .

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(A-B) Co-IP assay was performed to detect the interaction between TOMM34 and ATP5B treated with or without Gboxin for 24 hours in parental or adaptive PLC/PRF/5 cells.


( C - D ) Immunoblotting of EMT markers of HCC treated with or without 200 nM Gboxin for 24 hours ( shTOM : shTOMM34 , TOMM34 knockdown ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed ) .

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(C-D) Immunoblotting of EMT markers of HCC cells treated with or without 200 nM Gboxin for 24 hours (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).


( E - F ) Transwell showing migration invasion of HCC treated with or without 200 nM Gboxin for 36 hours ( 5 × 104 ; shTOM : shTOMM34 , TOMM34 knockdown ) . Scale bars , 100 μm . ( n = 3 biological replicates , two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(E-F) Transwell assay showing the migration and invasion of HCC cells treated with or without 200 nM Gboxin for 36 hours (5×104 cells; shTOM: shTOMM34, TOMM34 knockdown cells). Scale bars, 100 μm. (n=3 biological replicates, two-way ANOVA). Data information: Data are presented as means ± S.D.


( D ) Blue - native gel electrophoresis of S . cerevisiae strains with indicated mutations in Cor1 .

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(D) Blue-native gel electrophoresis of S. cerevisiae strains with indicated mutations in Cor1.


( A ) Blue - native gel electrophoresis immunoblots of expressing wild type form of Cor1 ( Cor1WT ) , as well as mutants Cor1N63A , N187A , D192A ( Cor1 * ) Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) in wild type ( WT ) background as well as in lacking CRD1 ( crd1Δ ) . Blots were probed with antibodies against Cor1 to visualize III ( CIII ) , as well as Cox1 , to monitor IV ( CIV ) . CV : V ; Cor1Cor2 : multimer consisting of Cor1

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(A) Blue-native gel electrophoresis and immunoblots of cells expressing the wild type form of Cor1 (Cor1WT), as well as the mutants Cor1N63A, N187A, D192A (Cor1*) and Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**) in wild type (WT) background as well as in cells lacking CRD1 (crd1Δ). Blots were probed with antibodies against Cor1 to visualize Complex III (CIII), as well as Cox1, to monitor Complex IV (CIV). CV: Complex V; Cor1Cor2: multimer consisting of Cor1 and Cor2


( B ) Reduced - minus - oxidized difference spectra of strains described in ( A ) . Heme peaks are indicated for Cor1WT in WT background .

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(B) Reduced-minus-oxidized difference spectra of strains described in (A). Heme peaks are indicated for Cor1WT in WT background.


Steady - state of strains as described in ( A ) cultivated on YP media containing

Steady-state protein levels of strains as described in (A) cultivated on YP media containing glycerol


Steady - state of strains as described in ( A ) cultivated on YP media containing CM media with galactose as carbon source ( D , E ) .

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Steady-state protein levels of strains as described in (A) cultivated on YP media containing CM media with galactose as carbon source (D, E).


( A ) Growth of strains expressing wild type form of Cor1 ( Cor1WT ) or mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) . Growth curves calculated doubling times from exponential phase ( insert ) are presented . were cultivated in media containing either glucose or glycerol as carbon source .

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(A) Growth analysis of strains expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Growth curves and calculated doubling times from the exponential phase (insert) are presented. Cells were cultivated in media containing either glucose or glycerol as carbon source.


( B ) Determination of chronological lifespan via propidium iodide ( PI ) of described above .

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(B) Determination of chronological lifespan via propidium iodide (PI) staining of cells described above.


( D , E ) of competitive fitness as described in ( C ) . Cor1WT strains harbored a chromosomally - integrated hygromycin selection cassette , whereas Cor1 * * contained a clonNAT selection cassette ( D ) . Subsequently , selection markers were switched as a control ( E ) .

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(D,E) Analysis of competitive fitness as described in (C). Cor1WT strains harbored a chromosomally-integrated hygromycin selection cassette, whereas Cor1** contained a clonNAT selection cassette (D). Subsequently, selection markers were switched as a control (E).


( A , B ) Spectrophotometric measurement of Cyt c oxidase ( CIV ; A ) NADH Cyt c reductase ( CIII ; B ) activities in solubilized mitochondrial extracts . Mitochondria were isolated from expressing wild type form of Cor1 ( Cor1WT ) or mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) .

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(A,B) Spectrophotometric measurement of Cyt c oxidase (CIV; A) and NADH Cyt c reductase (CIII; B) activities in solubilized mitochondrial extracts. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**).


( C , D ) Polarographic measurement of KCN - sensitive oxygen consumption , driven by NADH ( C ) or succinate + glycerol - 3 - phosphate ( G3P ; D ) in isolated coupled mitochondria of indicated strains . Measurement was performed in absence ( basal respiration ) or presence ( phosphorylating condition ) of ADP .

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(C,D) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH (C) or succinate + glycerol-3-phosphate (G3P; D) in isolated coupled mitochondria of indicated strains. Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.


( E ) Respiratory control calculated from polarographic measurements of KCN - sensitive oxygen consumption driven by NADH succinate + glycerol - 3 - phosphate ( G3P ) in isolated coupled mitochondria of indicated strains .

(E) Respiratory control ratio calculated from polarographic measurements of KCN-sensitive oxygen consumption driven by NADH and succinate + glycerol-3-phosphate (G3P) in isolated coupled mitochondria of indicated strains.


( F , G ) Polarographic measurement of KCN - sensitive oxygen consumption driven by NADH in mitoplasts in absence ( endog . Cyt c ) or presence ( + exog . Cyt c ) of exogenous oxidized Cyt c . NADH oxidation ( E ) calculated + Cyt c / - Cyt c of substrate oxidation ( F ) are visualized .

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(F,G) Polarographic measurement of KCN-sensitive oxygen consumption driven by NADH in mitoplasts in the absence (endog. Cyt c) or presence (+ exog. Cyt c) of exogenous oxidized Cyt c. NADH oxidation (E) and the calculated ratio +Cyt c/- Cyt c of substrate oxidation (F) are visualized.


( A ) Polarographic measurement of KCN - sensitive oxygen consumption , driven by NADH in isolated coupled mitochondria . Mitochondria were isolated from expressing wild type form of Cor1 ( Cor1WT ) or mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) . Thereby , strains either overexpressed cytochrome c ( Cyt cOE ) or harbored empty plasmid as a control ( Empty ) . Measurement was performed in absence ( basal respiration ) or presence ( phosphorylating condition ) of ADP .

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(A) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH in isolated coupled mitochondria. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Thereby, strains either overexpressed cytochrome c (Cyt cOE) or harbored the empty plasmid as a control (Empty). Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.


of competitive fitness of strains described in ( A ) . Cor1WT strains harbored a chromosomally - integrated clonNAT selection cassette , whereas Cor1 * * contained a hygromycin selection cassette . Strains were cultivated in CM media containing glucose ( B ) as carbon source .

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Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing glucose (B) as carbon source.


of competitive fitness of strains described in ( A ) . Cor1WT strains harbored a chromosomally - integrated clonNAT selection cassette , whereas Cor1 * * contained a hygromycin selection cassette . Strains were cultivated in CM media containing galactose ( C ) or glycerol ( D ) as carbon source .

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Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing galactose (C) or glycerol (D) as carbon source.


A In vitro differentiation to mature ookinete of wildtype ( WT ) , Δisp1 , Δisp3 , Δisp1 / 3 . Values are means ± SD ( n = 4 biological replicates ) ; two - t , * , P < 0 . 05 , * * , P < 0 . 01 , * * * , P < 0 . 001 , ns : not significant .

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A In vitro differentiation to mature ookinete of wildtype (WT), Δisp1, Δisp3, and Δisp1/3 parasites. Values are means ± SD (n=4 biological replicates); two-tailed t test, *, P<0.05, **, P<0.01, ***, P<0.001, ns: not significant.


Giemsa of zygotes / ookinetes from in vitro culture . Upper diagrams indicate morphological changes from zygote to ookinete . Black arrow indicates apical . Scale bar = 5 μm .

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Giemsa staining of zygotes/ookinetes from in vitro culture. Upper diagrams indicate morphological changes from zygote to ookinete. Black arrow indicates the apical. Scale bar = 5 μm.


C Time - course of ookinete differentiation from in vitro culture . Values are means ± ( n = 3 biological replicates ) , two - t , * * * , P < 0 . 001 .

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C Time-course analysis of ookinete differentiation from in vitro culture. Values are means ± SEM (n=3 biological replicates), two-tailed t test, ***, P<0.001.


D In vivo ookinete differentiation in midgut of infected mosquitos . Right panel indicates smear stained with Giemsa solution . Values are means ± ( n = 3 biological replicates ) , two - t , * * , P < 0 . 01 . Scale bar = 5 μm .

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D In vivo ookinete differentiation in midgut of the infected mosquitos. Right panel indicates the parasite smear stained with Giemsa solution. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01. Scale bar = 5 μm.


E Number of oocysts in mosquito midgut 7 days post blood feeding . n is number of mosquitoes in each group . horizontal shows mean value of each group , Mann - Whitney , * * * * , P < 0 . 0001 . Right panel shows mosquito midguts stained with 0 . 5 % mercurochrome . Scale bar = 50 μm . Three biological replicates performed .

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E Number of oocysts in mosquito midgut 7 days post blood feeding. n is the number of mosquitoes in each group. The horizontal line shows the mean value of each group, Mann-Whitney test, ****, P<0.0001. Right panel shows mosquito midguts stained with 0.5% mercurochrome. Scale bar = 50 μm. Three biological replicates performed.


F Formation of salivary sporozoites in mosquitoes 14 days post blood feeding . In each group , at least twenty blood - fed mosquitoes were counted . Values are means ± ( n = 3 biological replicates ) , two - t , * * , P < 0 . 01 .

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F Formation of salivary gland sporozoites in mosquitoes 14 days post blood feeding. In each group, at least twenty blood-fed mosquitoes were counted. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01.


G Western blot of ISP1 ISP3 in zygote of Δisp1 / 3 episomally complemented with 3V5 - tagged isp1 or 6HA - tagged isp3 genes from either P . yoelii or P . falciparum . P28 as loading control . DTS ( isp1 : : 3V5 ; isp3 : : 6HA ) is a doubly - tagged

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G Western blot of ISP1 and ISP3 in zygote of the Δisp1/3 parasites episomally complemented with 3V5-tagged isp1 or 6HA-tagged isp3 genes from either P. yoelii or P. falciparum. P28 as loading control. The DTS (isp1::3V5;isp3::6HA) is a doubly-tagged parasite


H IFA of tagged ISP1 ISP3 in zygotes of complemented . Nuclei are stained with Hoechst 33342 . Scale bar = 5 μm .

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H IFA analysis of tagged ISP1 and ISP3 in zygotes of the complemented parasites. Nuclei are stained with Hoechst 33342. Scale bar = 5 μm.


I In vitro ookinete differentiation of complemented . Values are means ± ( n = 3 biological replicates ) , two - t , * * , P < 0 . 01 , * * * , P < 0 . 001 .

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I In vitro ookinete differentiation of the complemented parasites. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01, ***, P<0.001.


IFA of ISP1 ISP3 expression from zygote to ookinete of isp1 : : 6HA isp3 : : 6HA , respectively . Scale bar = 5 μm .

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IFA of ISP1 and ISP3 expression from zygote to ookinete of the isp1::6HA and isp3::6HA parasites, respectively. Scale bar = 5 μm.


B Co - localization of ISP1 ISP3 from zygote to ookinete in doubly - tagged ( DTS , isp1 : : 3V5 ; isp3 : : 6HA ) . Scale bar = 5 μm .

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B Co-localization of ISP1 and ISP3 from zygote to ookinete in the doubly-tagged strain (DTS, isp1::3V5;isp3::6HA). Scale bar = 5 μm.


C Western blot of ISP1 ISP3 expression in zygotes of isp1 : : 6HA isp3 : : 6HA . numbers are relative intensities of band in blot . Right panel : of band . Values are means ± ( n = 4 biological replicates ) , two - t , * * * * , P < 0 . 0001 .

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C Western blot of ISP1 and ISP3 expression in zygotes of isp1::6HA and isp3::6HA parasites. The numbers are the relative intensities of band in blot. Right panel: the quantification of band intensity. Values are means ± SEM (n=4 biological replicates), two-tailed t test, ****, P<0.0001.


D Western blot of ISP1 ISP3 expression in zygotes of DTS , DTS / Δisp1 , DTS / Δisp3 . numbers are relative intensities of band in blot . Three replicates performed .

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D Western blot of ISP1 and ISP3 expression in zygotes of DTS, DTS/Δisp1, and DTS/Δisp3 parasites. The numbers are the relative intensities of band in blot. Three replicates performed.


E RT - qPCR of isp1 isp3 transcripts in zygotes of WT , Δisp1 , Δisp3 , Δisp1 / 3 . expression is normalized to 18s rRNA . Values are means ± ( n = 4 biological replicates ) .

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E RT-qPCR analysis of isp1 and isp3 transcripts in zygotes of the WT, Δisp1, Δisp3, and Δisp1/3 parasites. The expression is normalized to the 18s rRNA. Values are means ± SEM (n=4 biological replicates).


F IFA of ISP1 ISP3 expression dynamic of DTS / Δisp1 DTS / Δisp3 . Scale bar = 5 μm .

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F IFA analysis of ISP1 and ISP3 expression dynamic of DTS/Δisp1 and DTS/Δisp3 parasites. Scale bar = 5 μm.


B microscopy of ISP its N - terminal 20 residues ( N20 ) fused with a BFP : : 3V5 peptide episomally expressed in zygotes . Scale bar = 5 μm .

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B Fluorescent microscopy of ISP and its N-terminal 20 residues (N20) fused with a BFP::3V5 peptide episomally expressed in zygotes. Scale bar = 5 μm.


C Solubility detecting association of ISP1 _ FL ISP1 _ N20 using different detergents . soluble are in hypotonic buffer ( Hypo ) , peripheral in carbonate buffer ( Carb ) ， integral in Triton X - 100 buffer ( Trx ) , insoluble in pellet ( P ) . BFP : : 3V5 is in soluble fraction , while ISP1 _ FL ISP1 _ N20 are in - associated fractions . ER BiP is a loading control .

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C Solubility assay detecting membrane association of ISP1_FL and ISP1_N20 using different detergents. Cytosolic soluble proteins are in hypotonic buffer (Hypo), peripheral membrane proteins in carbonate buffer (Carb)，integral membrane proteins in Triton X-100 buffer (Trx), and insoluble proteins in pellet (P). BFP::3V5 is in the soluble fraction, while ISP1_FL and ISP1_N20 are in the membrane-associated fractions. ER protein BiP is a loading control.


D Acyl - RAC method detecting palmitoylation of ISP1 ISP3 in zygotes of DTS . from F ( flow - through ) , W ( wash ) , E ( elution ) are analyzed . P28 as a loading control .

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D Acyl-RAC method detecting palmitoylation of ISP1 and ISP3 in zygotes of the DTS strain. Proteins from F (flow-through), W (wash), and E (elution) are analyzed. P28 as a loading control.


E Substitutions of N - terminal cysteine or glycine residues to alanine ( G2A or C7A / C8A ) ablates palmitoylation of ISP1 . ISP1 was fused with BFP : : 3V5 episomally expressed in zygotes . Right panel shows of palmitoylated band from two independent repeats , values are means ± SD .

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E Substitutions of N-terminal cysteine or glycine residues to alanine (G2A or C7A/C8A) ablates palmitoylation of ISP1. ISP1 was fused with BFP::3V5 and episomally expressed in zygotes. Right panel shows quantification of palmitoylated protein band from two independent repeats, values are means ± SD.


F G2A C7AC8A mutations cause distribution of ISP1 during zygote to ookinete differentiation . Scale bar = 5 μm .

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F G2A and C7AC8A mutations cause cytoplasmic distribution of ISP1 during zygote to ookinete differentiation. Scale bar = 5 μm.


G association of ISP1 with either G2A or C7AC8A substitutions .

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G Membrane association of ISP1 with either G2A or C7AC8A substitutions.


H Expression palmitoylation of ISP1 in ∆ isp1 / 3 complemented with 3V5 tagged WT ISP1 or C7AC8A mutant .

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H Expression and palmitoylation of ISP1 in ∆isp1/3 parasites complemented with the 3V5 tagged WT ISP1 or C7AC8A mutant.


I IFA of ISP1 at zygote of complemented . Scale bar = 5 μm .

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I IFA of ISP1 at zygote of the complemented parasites. Scale bar = 5 μm.


In vitro differentiation to mature ookinete of complemented . Values are means ± ( n = 4 replicates ) , two - t , * * * , P < 0 . 001 , ns : not significant .

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In vitro differentiation to mature ookinete of the complemented parasites. Values are means ± SEM (n=4 replicates), two-tailed t test, ***, P<0.001, ns: not significant.


A Two - colored IFA of ISP1 / DHHC2 ISP3 / DHHC2 at zygotes of triple tagged isp1 : : 3V5 / isp3 : : 6HA / dhhc2 : : 4Myc ( TTS ) . Right graph shows overlay of peaks along periphery . Scale bar = 5 μm .

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A Two-colored IFA of ISP1/DHHC2 and ISP3/DHHC2 at zygotes of the triple tagged strain isp1::3V5/isp3::6HA/dhhc2::4Myc (TTS). Right graph shows overlay of fluorescence peaks along cell periphery. Scale bar = 5 μm.


B Western blot of DHHC2 , ISP1 , P28 ( plasma ) of TTS zygotes treated with PBS , trypsin ( Try ) , or heat - inactivated ( HI ) trypsin . Upper panel shows PPM IMC structures .

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B Western blot of DHHC2, ISP1, and P28 (plasma membrane protein) of the TTS zygotes treated with PBS, trypsin (Try), or heat-inactivated (HI) trypsin. Upper panel shows the PPM and IMC structures.


C Knockdown of dhhc2 expression . Upper panel shows promoter swap strategy applied in TTS , generating dhhc2kd mutant with endogenous dhhc2 promoter replaced with clag promoter . Western blot confirming decreased expression of DHHC2 in dhhc2kd zygotes . Three replicates performed .

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C Knockdown of dhhc2 expression. Upper panel shows the promoter swap strategy applied in TTS strain, generating the dhhc2kd mutant with endogenous dhhc2 promoter replaced with clag promoter. Western blot confirming decreased expression of DHHC2 in dhhc2kd zygotes. Three replicates performed.


D Two - colored IFA of DHHC2 P28 in TTS dhhc2kd zygotes . Scale bar = 5 μm . Bottom panel , quantifications of DHHC2 . Values are means ± SD ( n : number of analyzed ) . Mann - Whitney , * * * * , P < 0 . 0001 .

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D Two-colored IFA of DHHC2 and P28 proteins in the TTS and dhhc2kd zygotes. Scale bar = 5 μm. Bottom panel, quantifications of DHHC2 fluorescence. Values are means ± SD (n: the number of cells analyzed). Mann-Whitney test, ****, P<0.0001.


E Western blot of DHHC2 , ISP1 ISP3 in TTS dhhc2kd zygotes . Two replicates performed .

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E Western blot of DHHC2, ISP1 and ISP3 in the TTS and dhhc2kd zygotes. Two replicates performed.


F Palmitoylation of ISP1 ISP3 in TTS dhhc2kd zygotes using Acyl - RAC method . NH : NH2OH . Right panel indicates of band from three replicates , values are means ± SD .

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F Palmitoylation of ISP1 and ISP3 in the TTS and dhhc2kd zygotes using Acyl-RAC method. NH: NH2OH. Right panel indicates quantification of protein band intensity from three replicates, values are means ± SD.


G IFA of ISP1 ISP3 in zygotes of TTS dhhc2kd . Scale bar = 5 μm .

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G IFA of ISP1 and ISP3 in zygotes of TTS and dhhc2kd. Scale bar = 5 μm.


H Giemsa of zygote / ookinete from in vitro cultured TTS dhhc2kd . Scale bar = 5 μm .

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H Giemsa staining of zygote/ookinete from in vitro cultured TTS and dhhc2kd parasites. Scale bar = 5 μm.


I Time - course of in vitro ookinete differentiation . Values are means ± SD from three independent .

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I Time-course analysis of in vitro ookinete differentiation. Values are means ± SD from three independent tests.


J IFA of DHHC2 expression in dhhc2 : : 6HA dhhc2 : : 6HA ; ∆ isp1 / 3 zygotes . Scale bar = 5 μm .

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J IFA of DHHC2 expression in the dhhc2::6HA and dhhc2::6HA;∆isp1/3 zygotes. Scale bar = 5 μm.


K Western blot of DHHC2 in zygotes of dhhc2 : : 6HA dhhc2 : : 6HA ; ∆ isp1 / 3 .

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K Western blot of DHHC2 in zygotes of dhhc2::6HA and dhhc2::6HA;∆isp1/3.


L IFA of DHHC2 ISP1 in zygotes of dhhc2kd complemented with 4Myc tagged WT PyDHHC2 of P . yoelii , PfDHHC2 of P . falciparum , or PyDHHC2 with PAT catalytic deficient mutation C128A . Scale bar = 5 μm .

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L IFA of DHHC2 and ISP1 in zygotes of dhhc2kd parasites complemented with 4Myc tagged WT PyDHHC2 of P. yoelii, PfDHHC2 of P. falciparum, or PyDHHC2 with PAT catalytic deficient mutation C128A. Scale bar = 5 μm.


M Western blot of DHHC2 in complemented dhhc2kd as indicated .

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M Western blot of DHHC2 in the complemented dhhc2kd parasites as indicated.


N In vitro differentiation to mature ookinetes of complemented . Values are means ± SD from three independent . two - t , ns : not significant .

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N In vitro differentiation to mature ookinetes of the complemented parasites. Values are means ±SD from three independent tests. two-tailed t test, ns: not significant.


Western blot of episomally expressed DHHC2 : : 6HA or variants containing truncated segment ( C1 : 210 - 232 , C2 : 233 - 262 , C3 : 263 - 284 residues ) in WT zygotes .

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Western blot of episomally expressed DHHC2::6HA or variants containing truncated segment (C1:210-232, C2:233-262, C3:263-284 residues) in WT zygotes.


C IFA of episomally expressed DHHC2 : : 6HA three truncation mutants in zygotes retorts . Scale bar = 5 μm .

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C IFA of episomally expressed DHHC2::6HA and three truncation mutants in zygotes and retorts. Scale bar = 5 μm.


D Acyl - RAC method detecting palmitoylation of endogenous DHHC2 in dhhc2 : : 6HA zygotes .

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D Acyl-RAC method detecting palmitoylation of endogenous DHHC2 in the dhhc2::6HA zygotes.


E Palmitoylation of DHHC2 : : 6HA C4A mutant episomally expressed in zygotes . C4A : substitutions of C - terminal four cysteine to alanine . Right panel indicates of band from two replicates , values are means ± SD .

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E Palmitoylation of DHHC2::6HA and C4A mutant episomally expressed in zygotes. C4A: substitutions of C-terminal four cysteine to alanine. Right panel indicates the quantification of protein band intensity from two replicates, values are means ± SD.


F distribution of C4A mutant during zygote to ookinete differentiation . Scale bar = 5 μm .

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F Cytoplasmic distribution of C4A mutant during zygote to ookinete differentiation. Scale bar = 5 μm.


G IFA of DHHC2 at dhhc2 : : 6HA zygotes treated with 100 μΜ 2 - BP . Scale bar = 5 μm .

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G IFA of DHHC2 at the dhhc2::6HA zygotes treated with 100 μΜ 2-BP. Scale bar = 5 μm.


H In vitro ookinete differentiation of dhhc2kd complemented with either WT DHHC2 or C4A mutant . Values are means ± SD from three independent .

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H In vitro ookinete differentiation of the dhhc2kd parasites complemented with either WT DHHC2 or C4A mutant. Values are means ± SD from three independent tests.


I Palmitoylation of episomally expressed DHHC2 : : 6HA with mutation in respective cysteine ( C255A , C258A , C260A , C262A ) . Representative of three independent repeats .

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I Palmitoylation of the episomally expressed DHHC2::6HA with mutation in respective individual cysteine (C255A, C258A, C260A, and C262A). Representative of three independent repeats.


J Palmitoylation of HA - tagged human codon optimized DHHC2 ectopically expressed in human HEK293T . Both C4A C128A mutations ablate DHHC2 palmitoylation .

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J Palmitoylation of HA-tagged and human codon optimized DHHC2 ectopically expressed in human HEK293T cells. Both C4A and C128A mutations ablate DHHC2 palmitoylation.


K DHHC2 _ C128A - HA mutant could be palmitoylated by co - transfected DHHC2WT - Myc , but not DHHC2 _ C128A - Myc in HEK293T . Representative of two independent repeats .

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K DHHC2_C128A-HA mutant protein could be palmitoylated by the co-transfected DHHC2WT-Myc, but not DHHC2_C128A-Myc in HEK293T cells. Representative of two independent repeats.


L Co - immunoprecipitation of DHHC2 _ WT - HA DHHC2 _ WT - Myc co - expressed in HEK293T .

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L Co-immunoprecipitation of the DHHC2_WT-HA and DHHC2_WT-Myc co-expressed in HEK293T cells.


M C - terminus 75 residues peptide ( Cter75 ) of DHHC2 could be palmitoylated as an independent substrate by co - transfected DHHC2WT - HA in HEK293T . Cter75 is fused with a BFP : : Myc for easy detection via western blot . Cter75 * peptide contains C4A mutation .

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M C-terminus 75 residues peptide (Cter75) of DHHC2 could be palmitoylated as an independent substrate by co-transfected DHHC2WT-HA in HEK293T cells. Cter75 is fused with a BFP::Myc for easy detection via western blot. Cter75* peptide contains C4A mutation.


N Palmitoylation of tagged DHHC2 ectopically expressed in E . coli .

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N Palmitoylation of tagged DHHC2 ectopically expressed in bacteria E.coli.


O Palmitoylation of DHHC2 ectopically expressed in treated with 2 - BP .

O Palmitoylation of DHHC2 ectopically expressed in bacteria treated with 2-BP.


B Endogenous DHHC2 is palmitoylated in dhhc2 : : 6HA gametocytes .

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B Endogenous DHHC2 is palmitoylated in the dhhc2::6HA gametocytes.


C Episomally expressed DHHC2 driven by promoter of ccp2 gene is palmitoylated in gametocytes , gametes , zygotes of WT . Left panel shows expression of HA - tagged DHHC2 in gametocyte by co - with antibodies of HA α - Tubulin II ( gametocyte specific ) . Scale bar = 5 μm .

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C Episomally expressed DHHC2 driven by promoter of ccp2 gene is palmitoylated in female gametocytes, female gametes, and zygotes of WT parasites. Left panel shows the expression of HA-tagged DHHC2 in female gametocyte by co-staining with antibodies of HA and α-Tubulin II (male gametocyte specific). Scale bar = 5 μm.


D IFA of DHHC2 P28 in gametocytes , gametes , zygotes of dhhc2 : : 6HA . Gametocytes were stimulated with XA 22 ° C in vitro . P28 is expressed specifically in gametes zygotes . Scale bar = 5 μm .

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D IFA of DHHC2 and P28 in female gametocytes, female gametes, and zygotes of the dhhc2::6HA parasite. Gametocytes were stimulated with XA and 22°C in vitro. P28 is expressed specifically in female gametes and zygotes. Scale bar = 5 μm.


E of with DHHC2 polarization in P28 positive during gametocyte to zygote differentiation x / y : number of with DHHC2 polarization number of analyzed . Values are means ± SD from three independent .

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E Quantification of parasites with DHHC2 polarization in P28 positive cells during gametocyte to zygote differentiation x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ±SD from three independent tests.


F No polarization of DHHC2 in dhhc2 : : 6HA gametocytes with only either XA or 22 ° C stimulation . Three replicates performed , values are means ± SD .

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F No polarization of DHHC2 in the dhhc2::6HA gametocytes with only either XA or 22°C stimulation. Three replicates performed, values are means ± SD.


G No change in DHHC2 expression in zygotes of dhhc2 : : 6HA - derived mutants with gene disruption of cdpk4 , hap2 , dozi .

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G No change in DHHC2 expression in zygotes of the dhhc2::6HA parasite-derived mutants with individual gene disruption of cdpk4, hap2, and dozi.


H of with DHHC2 polarization in P28 positive . are indicated in ( G ) or from a genetic cross ( dhhc2 : : 6HA / cdpk4 WT ) . x / y : number of with DHHC2 polarization number of analyzed . Values are means ± SD from three , two - t , * * * , P < 0 . 001 , * * * * , P < 0 . 0001 , ns , not significant .

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H Quantification of parasite with DHHC2 polarization in P28 positive cells. Parasites are indicated in (G) or from a genetic cross (dhhc2::6HA/cdpk4 and WT). x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ± SD from three tests, two-tailed t test, ***, P<0.001, ****, P<0.0001, ns, not significant.


I Expression localization of DHHC2 GAP45 during gamete to ookinete development of dhhc2 : : 6HA . GAP45 is an IMC marker is only detected in zygote after fertilization , but not gametes . Scale bar = 5 μm .

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I Expression and localization of DHHC2 and GAP45 during female gamete to ookinete development of dhhc2::6HA parasite. GAP45 is an IMC marker and is only detected in zygote after fertilization, but not gametes. Scale bar = 5 μm.


A Transmission electron microscopy ( ) cross sections of WT , dhhc2kd ∆ isp1 / 3 showing arrangement of SPM underneath IMC . Approximately 60 recognizable hollow microtubules ( green ) are associated with IMC ( red ) , distributing evenly around circumference with an interval of 100 - 120 nm in WT ookinetes . Microtubules lost association with IMC in ookinetes of dhhc2kd ∆ isp1 / 3 . Left : one representative in each is shown ; Right : two areas are magnified to show details . Lower panel is of between adjacent microtubules associated with IMC . Values are mean ± SD for n = 204 ( WT ) , 189 ( dhhc2kd ) , 211 ( ∆ isp1 / 3 ) measurements in 20 each group from two independent experiments ; Kolmogorov - Smirnov , * * * * , P < 0 . 0001 .

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A Transmission electron microscopy (TEM) cross sections of WT, dhhc2kd and ∆isp1/3 parasites showing the arrangement of SPM underneath IMC. Approximately 60 recognizable hollow microtubules (green) are associated with IMC (red), distributing evenly around circumference with an interval of 100-120 nm in WT ookinetes. Microtubules lost association with IMC in the ookinetes of dhhc2kd and ∆isp1/3 parasites. Left: one representative cell in each parasite is shown; Right: two areas are magnified to show the details. Lower panel is the quantification of distance between adjacent microtubules associated with IMC. Values are mean ± SD for n=204 (WT), 189 (dhhc2kd), and 211 (∆isp1/3) measurements in 20 cells each group from two independent experiments; Kolmogorov-Smirnov test, ****, P<0.0001.


B Apical polar ring emanating SPM in WT , dhhc2kd ∆ isp1 / 3 examined by negative . A dome - like SPM structure radiating from apical polar ring was observed in early ( 3 hours ) or mature ( 12 hours ) WT ookinetes , but impaired in mutant . n is number of analyzed from three independent experiments . More in Appendix Figure S5 . Scale bar = 1μm .

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B Apical polar ring and the emanating SPM in WT, dhhc2kd and ∆isp1/3 parasites examined by negative staining and TEM. A dome-like SPM structure radiating from the apical polar ring was observed in early (3 hours) or mature (12 hours) WT ookinetes, but impaired in mutant parasites. n is the number of cells analyzed from three independent experiments. More images in the Appendix Figure S5. Scale bar = 1μm.


C SiR - tubulin show SPM structures of WT , dhhc2kd ∆ isp1 / 3 . Arrowheads point to apical . Scale bar = 5 μm .

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C SiR-tubulin staining show the SPM structures of WT, dhhc2kd and ∆isp1/3 parasites. Arrowheads point to cell apical. Scale bar = 5 μm.


A GST - ISP1 pulls down endogenous β - Tubulin from lysate of WT ookinetes . Lower panel indicates Coomassie blue stain of recombinant GST GST - ISP1 .

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A GST-ISP1 pulls down endogenous β-Tubulin from cell lysate of WT ookinetes. Lower panel indicates the Coomassie blue stain of recombinant GST and GST-ISP1.


B Episomally expressed ISP1 : : HA immunoprecipitated with β - Tubulin in WT ookinetes . Episomal vector as control .

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B Episomally expressed ISP1::HA immunoprecipitated with β-Tubulin in WT ookinetes. Episomal vector as control.


C Endogenous ISP1 : : 3V5 immunoprecipitated with β - Tubulin in TTS ookinetes .

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C Endogenous ISP1::3V5 immunoprecipitated with β-Tubulin in the TTS ookinetes.


D Co - localization of ISP1 : : HA β - Tubulin at ookinete periphery in IFA . Scale bar = 5 μm .

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D Co-localization of ISP1::HA and β-Tubulin at ookinete periphery in IFA. Scale bar = 5 μm.


E Proximity ligation ( PLA ) detecting interaction of ISP1 - HA β - Tubulin at ookinete . Episomal vector as control . Scale bar = 5 μm .

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E Proximity ligation assay (PLA) detecting interaction of ISP1-HA and β-Tubulin at ookinete. Episomal vector as control. Scale bar = 5 μm.


F Solubility detecting association of α - β - Tubulins in WT , dhhc2kd , ∆ isp1 / 3 . GAPDH is a cytosol soluble , P28 is a plasma integral .

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F Solubility assay detecting membrane association of α- and β-Tubulins in WT, dhhc2kd, and ∆isp1/3 parasites. GAPDH is a cytosol soluble protein, P28 is a plasma membrane integral protein.


G of α - β - Tubulins association in F . association index is defined as of Carb fraction to Hypo fraction in F , normalized to that of WT . Values are means ± from three independent .

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G Quantification of α- and β-Tubulins membrane association in F. The membrane association index is defined as the ratio of Carb fraction to Hypo fraction in F, and normalized to that of WT. Values are means ± SEM from three independent tests.


H Linear correlation of association index of α - Tubulin β - Tubulin with conversion rate to mature ookinete among WT , dhhc2kd ∆ isp1 / 3 . Horizontal vertical values are means ± SD .

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H Linear correlation of membrane association index of α-Tubulin and β-Tubulin with the conversion rate to mature ookinete among WT, dhhc2kd and ∆isp1/3 parasites. Horizontal and vertical values are means ± SD.


I association of α - β - Tubulins in WT ookinete culture after DMSO , 2 - BP , or colchicine treatment .

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I Membrane association of α- and β-Tubulins in WT ookinete culture after DMSO, 2-BP, or colchicine treatment.


J of α - β - Tubulins association Values are means ± from three independent repeats .

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J Quantification of α- and β-Tubulins membrane association Values are means ± SEM from three independent repeats.


( D - E ) Measurements of ciliary cAMP using cilia - Pink Flamindo . ( D ) Exemplary are shown for cilia ( cilia - bPAC or cyto - bPAC ) expressing cilia - Pink Flamindo biosensor after stimulation . Scale bars are indicated ; cAMP are color - coded using a high - low look - up table . ( E ) of data exemplified in ( D ) . cAMP level is determined as mean ciliary during first 60 s after stimulation ( measurement interval 10 s ) ; p value for a Kolmogorov - Smirnov is indicated . Each data point represents an cilium . Bars display median interquartile range ; cilia - bPAC : 40 cilia ( biological replicates ) from n = 14 independent experiments ; cyto - bPAC : 31 cilia ( biological replicates ) from n = 31 independent experiments .

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(D-E) Measurements of ciliary cAMP levels using cilia-Pink Flamindo. (D) Exemplary images are shown for cilia (cilia-bPAC or cyto-bPAC cells) expressing the cilia-Pink Flamindo biosensor after light stimulation. Scale bars are indicated; cAMP levels are color-coded using a high-low look-up table. (E) Quantification of data exemplified in (D). The cAMP level is determined as the mean ciliary fluorescence intensity during the first 60 s after light stimulation (measurement interval 10 s); p value for a Kolmogorov-Smirnov test is indicated. Each data point represents an individual cilium. Bars display median and interquartile range; cilia-bPAC: 40 cilia (biological replicates) from n = 14 independent experiments; cyto-bPAC: 31 cilia (biological replicates) from n = 31 independent experiments.


( A ) Wild - type ( WT ) mIMCD - 3 cultured in a 3D matrix during continuous exposure to DMSO ( control ) or 10 µM of Forskolin . Exemplary are shown ( n = 5 biological replicates ) . of cyst number is shown on right Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment , p values calculated using an unpaired , two - sided Student ' s t - are indicated .

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(A) Wild-type (WT) mIMCD-3 cells cultured in a 3D matrix during continuous exposure to DMSO (control) or 10 µM of Forskolin. Exemplary images are shown (n = 5 biological replicates). Quantification of cyst number and area is shown on the right Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment, p values calculated using an unpaired, two-sided Student's t-test are indicated.


( D ) mIMCD - 3 stably expressing cilia - bPAC or cyto - bPAC , cultured in a 3D matrix in , during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² ) or in incubated with 10 µM of Forskolin . Exemplary are shown ( n > 3 ) . ( E ) of data exemplified in ( D ) . Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment ; p values calculated using an unpaired , two - sided Student ' s t - are indicated .

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(D) mIMCD-3 cells stably expressing cilia-bPAC or cyto-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) or in the dark and incubated with 10 µM of Forskolin. Exemplary images are shown (n > 3). (E) Quantification of the data exemplified in (D). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using an unpaired, two-sided Student's t-test are indicated.


( D ) Immunoblotting of lysates from mIMCD - 3 wild - type ( WT ) mIMCD - 3 stably expressing cilia - bPAC for S6 ribosomal ( S6RP ) phosphorylation at Ser235 / 236 ( pS6RP ) . of total S6RP ( S6RP ) beta - Tubulin have been determined as controls . Cilia - bPAC were treated with DMSO ( control ) or with 10 nM of Rapamycin . were stimulated with ( 1 h / 1 h , 48 h , 465 nm , 38 . 8 µW / cm² ) ( E ) of pS6RP / S6RP . Data are shown as mean ± S . D . , n > 3 ; p values were calculated using a paired , two - sided Student ' s t - . Each data point shows an independent

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

(D) Immunoblotting of lysates from mIMCD-3 wild-type (WT) cells and mIMCD-3 cells stably expressing cilia-bPAC for S6 ribosomal protein (S6RP) phosphorylation at Ser235/236 (pS6RP). Levels of total S6RP protein (S6RP) and beta-Tubulin have been determined as controls. Cilia-bPAC cells were treated with DMSO (control) or with 10 nM of Rapamycin. All cells were stimulated with light (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²) (E) Quantification of the pS6RP/S6RP ratio. Data are shown as mean ± S.D., n > 3; p values were calculated using a paired, two-sided Student's t-test. Each data point shows an independent experiment


( G ) mIMCD - 3 stably expressing cilia - bPAC , cultured in a 3D matrix in , during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² ) incubation with 10 nM rapamycin . exposure started 2 d later than incubation with 10 nM rapamycin . Exemplary are shown ( n = 3 ) ( H ) of data exemplified in ( G ) . Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment ; p values calculated using a paired , two - sided Student ' s t - are

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

(G) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubation with 10 nM rapamycin. Light exposure started 2 d later than incubation with 10 nM rapamycin. Exemplary images are shown (n = 3) (H) Quantification of the data exemplified in (G). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using a paired, two-sided Student's t-test are indicated


( A ) mIMCD - 3 stably expressing cilia - bPAC , cultured in a 3D matrix in or during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² ) incubated with DMSO ( control ) , 250 µM IBMX , a ubiquitous PDE inhibitor , or 10 µM Rolipram , a PDE4 - specific inhibitor . Exemplary are shown ( n = 3 ) . Scale bars are indicated . ( B ) of data exemplified in ( A ) . Data are shown as mean ± S . D . , p values calculated using an unpaired , two - sided Student ' s t - are indicated . Data points show experiments .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark or during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubated with DMSO (control), 250 µM IBMX, a ubiquitous PDE inhibitor, or 10 µM Rolipram, a PDE4-specific inhibitor. Exemplary images are shown (n = 3). Scale bars are indicated. (B) Quantification of data exemplified in (A). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual experiments.


( C ) mIMCD - 3 stably expressing cyto - bPAC , cultured in a 3D matrix during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² , 1 day after pharmacological stimulus ) incubation with DMSO or 10 µM rolipram . Exemplary are shown ( n = 4 - 5 ) . Scale bars are indicated . ( D ) of data exemplified in ( C ) Data are shown as mean ± S . D . , p values calculated using an unpaired Student ' s t - are indicated . Data points show experiments .

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(C) mIMCD-3 cells stably expressing cyto-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO or 10 µM rolipram. Exemplary images are shown (n = 4-5). Scale bars are indicated. (D) Quantification of data exemplified in (C) Data are shown as mean ± S.D., p values calculated using an unpaired Student's t-test are indicated. Data points show individual experiments.


( F ) of Pde4 isoform expression ( long short isoforms ) in wild - type mIMCD - 3 , revealed by immunoblotting against PDE4A , PDE4B , PDE4D , PDE4D .

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(F) Analysis of Pde4 isoform expression (long and short isoforms) in wild-type mIMCD-3 cells, revealed by immunoblotting against PDE4A, PDE4B, PDE4D, and PDE4D.


( H ) mIMCD - 3 stably expressing cilia - bPAC , cultured in a 3D matrix during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² , 1 day after pharmacological stimulus ) incubation with DMSO ( 0 µM ) , 3 µM , or 10 µM of Pde4 - long - isoform activator MR - L8 . Exemplary are shown ( n = 3 ) . Scale bars are indicated . ( I ) of data exemplified in ( H ) . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - are indicated . Data have been normalized to , 0 µM MR - L8 condition ( Set to 100 % ) within each experiment . Data points show experiments .

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(H) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO (0 µM), 3 µM, or 10 µM of the Pde4-long-isoform activator MR-L8. Exemplary images are shown (n = 3). Scale bars are indicated. (I) Quantification of data exemplified in (H). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data have been normalized to the Light, 0 µM MR-L8 condition (Set to 100%) within each experiment. Data points show individual experiments.


( D ) 3D culture of mIMCD - 3 WT continuously exposed to acetone ( control ) or 100 nM PGE2 . Exemplary are shown ( n = 3 biological replicates ) . ( E ) of exemplified in ( D ) . Data are shown as mean ± S . D . , p values calculated using an unpaired , two - sided Student ' s t - are indicated . Data points show biological replicates . ( F ) of cyst in PGE2 - stimulated mIMCD - 3 WT in presence of PDE4 long isoform - specific activator MR - L8 . Data were normalized to PGE2 - control - treated are shown as mean ± S . D . p values calculated using a one - sample t - compared to 100 % are indicated , n = 4 biological replicates .

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(D) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2. Exemplary images are shown (n = 3 biological replicates). (E) Quantification of images exemplified in (D). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual biological replicates. (F) Quantification of the cyst area in PGE2-stimulated mIMCD-3 WT cells in the presence of the PDE4 long isoform-specific activator MR-L8. Data were normalized to PGE2- and control-treated cells and are shown as mean ± S.D. p values calculated using a one-sample t-test compared to 100% are indicated, n = 4 biological replicates.


( G ) 3D culture of mIMCD - 3 WT continuously exposed to acetone ( control ) or 100 nM PGE2 , DMSO ( control ) or 10 µM of EP4 inhibitor L161 , 982 . PGE2 treatment started 2 d after seeding . Exemplary are shown ( n = 3 ) . Scale bars are indicated . ( H ) for exemplified in ( G ) , including addition of 3 µM AH6809 , an EP2 inhibitor Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - are indicated ( p value of n / a indicates that is not applicable because both conditions showed no variance ) . Data points show experiments have been normalized to addition of 100 nM PGE2 ( set to 100 % ) within each experiment .

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

(G) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2, and DMSO (control) or 10 µM of the EP4 inhibitor L161,982. PGE2 treatment started 2 d after seeding. Exemplary images are shown (n = 3). Scale bars are indicated. (H) Quantification for images exemplified in (G), including addition of 3 µM AH6809, an EP2 inhibitor Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated (p value of n/a indicates that the test is not applicable because both conditions showed no variance). Data points show individual experiments and have been normalized to addition of 100 nM PGE2 (set to 100%) within each experiment.


( I ) 3D culture of mIMCD - 3 stably expressing cilia - bPAC , incubated with DMSO ( control ) or EP4 - inhibitor L161 , 982 in or during exposure ( 1 / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² , started 2 d after pre - incubation with DMSO or L161 , 982 ) . Exemplary are shown ( n = 3 ) . ( J ) of exemplified in ( I ) . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - are indicated . Data points show experiments have been normalized to / DMSO condition ( Set to 100 % ) within each experiment .

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

(I) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or the EP4-inhibitor L161,982 in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or L161,982). Exemplary images are shown (n = 3). (J) Quantification of images exemplified in (I). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments and have been normalized to the light/DMSO condition (Set to 100%) within each experiment.


( F ) 3D culture of mIMCD - 3 stably expressing cilia - bPAC , incubated with DMSO ( control ) or 20 µM Celecoxib , a COX - 2 - inhibitor , in or during exposure ( 1 / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² , started 2 d after pre - incubation with DMSO or Celecoxib ) . Exemplary are shown ( n = 3 ) . Scale bars are indicated . ( G ) of exemplified in ( F ) from 3D cultures incubated with 1 μM celecoxib . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - are indicated . Data points show experiments .

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

(F) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or 20 µM Celecoxib, a COX-2-inhibitor, in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or Celecoxib). Exemplary images are shown (n = 3). Scale bars are indicated. (G) Quantification of images exemplified in (F) and from 3D cultures incubated with 1 μM celecoxib. Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments.


( A ) Wild - type ( WT ) , cilia - bPAC , or cyto - bPAC ( magenta ) mIMCD - 3 were stimulated by ( for 1 h prior to fixation , 465 nm , 38 . 8 µW / cm² ) , treated with DMSO ( control ) or 50 µM Ciliobrevin - D , a dynein - inhibitor . were labeled with DAPI ( blue ) to label , an ARL13B antibody ( ) to label cilia , a phospho - specific ( Ser133 ) CREB antibody ( pCREB , green ) . Arrows indicate direction of shift of respective channel . box indicates position of magnified view shown on right of each panel . B ) of ciliary pCREB signal in cilia - bPAC cyto - bPAC . Data are shown as mean ± S . D . , n > 3 . Each data point represents an independent experiment corresponds to median of > 73 cilia ( biological replicates ) ; p values for an unpaired , two - sided Student ' s t - with Welch ' s correction are indicated .

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

(A) Wild-type (WT), cilia-bPAC, or cyto-bPAC (magenta) mIMCD-3 cells were stimulated by light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²), and treated with DMSO (control) or 50 µM Ciliobrevin-D, a dynein-inhibitor. Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. B) Quantification of the ciliary pCREB signal in cilia-bPAC and cyto-bPAC cells. Data are shown as mean ± S.D., n > 3. Each data point represents an independent experiment and corresponds to the median of > 73 cilia (biological replicates); p values for an unpaired, two-sided Student's t-test with Welch's correction are indicated.


( C ) mIMCD - 3 Ift27 - / - with or without stable cilia - bPAC expression ( magenta ) were stimulated with ( for 1 h prior to fixation , 465 nm , 38 . 8 µW / cm² ) . were labeled with DAPI ( blue ) to label , an ARL13B antibody ( ) to label cilia , a phospho - specific ( Ser133 ) CREB antibody ( pCREB , green ) . Arrows indicate direction of shift of respective channel . box indicates position of magnified view shown on right of each panel . ( D ) of ciliary pCREB signal . Data are shown as mean ± S . D . , n > 3 . Each data point represents median of > 90 cilia from same experiment ; p values are indicated for an unpaired , two - sided Student ' s t - with Welch ' s correction .

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

(C) mIMCD-3 Ift27-/- cells with or without stable cilia-bPAC expression (magenta) were stimulated with light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. (D) Quantification of the ciliary pCREB signal. Data are shown as mean ± S.D., n > 3. Each data point represents the median of > 90 cilia from the same experiment; p values are indicated for an unpaired, two-sided Student's t-test with Welch's correction.


B . Validation of TNF - RSC interacting . A549 were stimulated with FLAG - TNF - α for indicated time TNF - RSCs were isolated as described in Figure 3A . TNF - α - stimulation - dependent interaction of indicated was confirmed by isolating TNF - RSC blotting with indicated antibodies . amount of analyzed in input material used for TNF - RSC isolation is also shown .

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B. Validation of the TNF-RSC interacting proteins. A549 cells were stimulated with FLAG-TNF-α for the indicated time and the TNF-RSCs were isolated as described in Figure 3A. TNF-α-stimulation-dependent interaction of the indicated proteins was confirmed by isolating the TNF-RSC and blotting with the indicated antibodies. The amount of the analyzed proteins in the input material used for the TNF-RSC isolation is also shown.


C . of ubiquitin linkage peptides in TNF - RSC . box plot shows relative abundance of K48 , K63 linear ubiquitin linkage - specific peptides in TNF - RSCs isolated from TNF - α - stimulated ( 15 min ) control quantified by SILAC - MS .

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C. Quantification of ubiquitin linkage peptides in the TNF-RSC. The box plot shows the relative abundance of K48, K63 and linear ubiquitin linkage-specific peptides in the TNF-RSCs isolated from TNF-α-stimulated (15 min) and control cells quantified by SILAC-based MS.


E . list of enriched in SPATA2pull - down . table shows that were enriched in GFP - SPATA2pull - down . columns under " Unstimulated " shows SILAC ratios of indicated in SPATA2 pull - downs from unstimulated , whereas columns under " Stimulated ( TNF - α 15 min ) " shows SILAC ratios of indicated in SPATA2 pull - downs from stimulated with TNF - α for 15 minutes .

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E. The list of proteins enriched in SPATA2pull-down. The table shows proteins that were enriched in GFP-SPATA2pull-down. The columns under "Unstimulated" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from unstimulated cells, whereas the columns under "Stimulated (TNF-α 15 min)" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from cells stimulated with TNF-α for 15 minutes.


F . Validation of TNF - α - stimulation - independent association of SPATA2 with CYLD . GFP - SPATA2 was immunoprecipitated from unstimulated TNF - α - stimulated , probed with CYLD antibody .

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F. Validation of TNF-α-stimulation-independent association of SPATA2 with CYLD. GFP-SPATA2 was immunoprecipitated from unstimulated and TNF-α-stimulated cells, and probed with CYLD antibody.


G . HOIP interacts with SPATA2 . FLAG - HOIP expressing A20 . 2J were SILAC labeled FLAG - HOIP was immunoprecipitated from unstimulated associated were quantified using LC - MS / MS . table contains that were enriched in HOIPpull - down compared to control pull - down .

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

G. HOIP interacts with SPATA2. FLAG-HOIP expressing A20.2J cells were SILAC labeled and FLAG-HOIP was immunoprecipitated from unstimulated cells and associated proteins were quantified using LC-MS/MS. The table contains proteins that were enriched in HOIPpull-down compared to control pull-down.


H . Validation of SPATA2 interaction with HOIP . GFP - tagged RNF31 was purified from unstimulated or TNF - a - stimulated ( 15 min ) HCT116 immunoprecipitates were blotted with SPATA2 , CYLD GFP ( HOIP ) antibodies . lower panels show expression of indicated in input . asterisk indicates unspecific band .

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H. Validation of SPATA2 interaction with HOIP. GFP-tagged RNF31 was purified from unstimulated or TNF-a-stimulated (15 min) HCT116 cells and the immunoprecipitates were blotted with SPATA2, CYLD and GFP (HOIP) antibodies. The lower panels show expression of the indicated proteins in the input. The asterisk indicates unspecific band.


B . predicted PUB domain in N - terminus of SPATA2 interacts with CYLD , but not with LUBAC or other TNF - RSC components . GFP - SPATA2 indicated deletion mutants were purified from A549 that were stimulated with TNF - α for 15 minutes , enriched were quantified using SILAC - MS .

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B. The predicted PUB domain in the N-terminus of SPATA2 interacts with CYLD, but not with LUBAC or other TNF-RSC components. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells that were stimulated with TNF-α for 15 minutes, and the enriched proteins were quantified using SILAC-based MS.


C . Validation of SPATA2 N - terminus interaction with CYLD . GFP - SPATA2 indicated deletion mutants were purified from A549 as described in Figure 5A , association of CYLD was analyzed by immunoblotting . lower panels show controls .

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

C. Validation of SPATA2 N-terminus interaction with CYLD. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells as described in Figure 5A, and association of CYLD was analyzed by immunoblotting. The lower panels show controls.


D . USP domain of CYLD interacts with SPATA2 . STREP - FLAG - CYLD indicated deletion mutants were purified from A549 using Step - Tactin sepharose blotted with SPATA2 antibody . lower panels show controls .

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D. The USP domain of CYLD interacts with SPATA2. STREP-FLAG-CYLD and the indicated deletion mutants were purified from A549 cells using Step-Tactin sepharose and blotted with SPATA2 antibody. The lower panels show controls.


E . C - terminus of SPATA2 interacts with HOIP . A549 were transiently transfected with GFP - tagged SPATA2 , SPATA2 D200 SPATA2 200 * , SPATA2 mutants were immunoprecipitated using GFP - trap beads . immunoprecipitates were immunoblotted with HOIP , CYLD GFP ( HOIP ) antibodies . lower panels show input control , * shows unspecific band .

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E. The C-terminus of SPATA2 interacts with HOIP. A549 cells were transiently transfected with GFP-tagged SPATA2, SPATA2 D200 and SPATA2 200*, and SPATA2 and the mutants were immunoprecipitated using GFP-trap beads. The immunoprecipitates were immunoblotted with HOIP, CYLD and GFP (HOIP) antibodies. The lower panels show input control, and the * shows unspecific band.


B . Effect of SPATA2 knockdown on association of with TNF - RSC . list shows SILAC ratios of TNF - RSC - associated in control SPATA2 knockdown . that showed reduced association with TNF - RSC after SPATA2 knockdown are highlighted red .

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B. Effect of SPATA2 knockdown on association of proteins with TNF-RSC. The list shows SILAC ratios of TNF-RSC-associated proteins in control cells and SPATA2 knockdown cells. The proteins that showed reduced association with the TNF-RSC after SPATA2 knockdown are highlighted red.


C . Validation of SPATA2 - dependent association of CYLD with TNF - RSC . TNF - RSC was purified under indicated conditions association of SPATA2 , CYLD , RIPK1 HOIP with TNF - RSC was analyzed by immunoblotting . input controls show expression of analyzed in lysates . asterisk denotes unspecific bands .

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

C. Validation of SPATA2-dependent association of CYLD with the TNF-RSC. The TNF-RSC was purified under the indicated conditions and association of SPATA2, CYLD, RIPK1 and HOIP with the TNF-RSC was analyzed by immunoblotting. The input controls show expression of the analyzed proteins in cell lysates. The asterisk denotes unspecific bands.


A . SPATA2 regulates TNF - a - induced NF - κB activation . HEK293T were transfected with SPATA2 siRNA ( SMARTpool or with to siRNA ) or a non - targeting control siRNA , NF - kB activation was monitored with luciferase - NF - kB reporter assays . Error bars specify standard error of mean ( ) of three independent experiments . p * * < 0 . 05 ( two - , unpaired Student ´ s t - ) .

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A. SPATA2 regulates TNF-a-induced NF-κB activation. HEK293T cells were transfected with the SPATA2 siRNA (SMARTpool or with to individual siRNA) or a non-targeting control siRNA, and NF-kB activation was monitored with luciferase-based NF-kB reporter assays. Error bars specify the standard error of the mean (SEM) of three independent experiments. p** < 0.05 (two-tailed, unpaired Student´s t-test).


B . Knockdown of SPATA2 enhances expression of NF - κB target genes . HEK293T were transfected with SPATA2 siRNA or a non - targeting control siRNA expression of indicated mRNAs was measured in stimulated with TNF - a for 0 , 3 6 hours with qPCR .

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B. Knockdown of SPATA2 enhances expression of the NF-κB target genes. HEK293T cells were transfected with SPATA2 siRNA or a non-targeting control siRNA and the expression of the indicated mRNAs was measured in cells stimulated with TNF-a for 0, 3 and 6 hours with qPCR.


C . SPATA2 is involved in TNF - α - induced necroptosis . SPATA2 was knocked down in L929 using RNAi , were pretreated with DMSO , Z - VAD , or Z - VAD + NEC1 for 1 hour followed by treatment with TNF - α for 2 . 5 hours . viability was assessed with propidium iodide flow cytometry . Knockdown of SPATA2 mRNA was verified using real time PCR . p * * < 0 . 05 ( two - , unpaired Student ´ s t - ) .

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

C. SPATA2 is involved in TNF-α-induced necroptosis. SPATA2 was knocked down in L929 cells using RNAi, and cells were pretreated with DMSO, Z-VAD, or Z-VAD+NEC1 for 1 hour followed by treatment with TNF-α for 2.5 hours. Cell viability was assessed with propidium iodide staining and flow cytometry. Knockdown of SPATA2 mRNA was verified using real time PCR. p** < 0.05 (two-tailed, unpaired Student´s t-test).


D . Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF - α - induced necroptosis . TNF - α - dependent necroptosis was induced in L929 as described in Figure 6C . Phosphorylation of MLKL was analyzed using S358 phospho - specific antibody .

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D. Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF-α-induced necroptosis. TNF-α-dependent necroptosis was induced in L929 as described in Figure 6C. Phosphorylation of MLKL was analyzed using S358 phospho-specific antibody.


( a ) Representative 2D μ - CT scans of RasV12 ; ctrl at day 6 , RasV12 ; scrib - / - larvae at day 6 day 10 . Muscle ( green ) , fat ( blue ) eye - antennal discs / ( red ) are outlined . Scale bar : 1mm . Anterior ( A ) , Right ( R ) .

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(a) Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib-/- larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined.Scale bar: 1mm. Anterior (A), Right (R).


( a ) of growth of RasV12 ; ctrl day 6 ( n = 15 ) RasV12 ; scrib - / - tumors , day 6 ( n = 15 ) , day 7 ( n = 5 ) , day 8 ( n = 4 ) , day 9 ( n = 5 ) day 10 ( n = 5 ) .

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(a) Quantification of tumor growth of RasV12; ctrl day 6 (n=15) RasV12; scrib-/- tumors, day 6 (n=15), day 7 (n=5), day 8 (n=4), day 9 (n=5) and day 10 (n=5).


( b ) Quantifications of muscle volume , of larvae carrying tumors of RasV12 ; ctrl ( n = 15 ) , RasV12 ; scrib - / - at day 6 ( n = 15 ) , day 7 ( n = 5 ) , day 8 ( n = 5 ) , day 9 ( n = 5 ) dat 10 ( n = 5 ) .

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(b) Quantifications of muscle volume, of larvae carrying tumors of RasV12; ctrl (n=15), and RasV12; scrib-/- at day 6(n=15), day 7 (n=5), day 8 (n=5), day 9 (n=5) and dat 10 (n=5).


( c ) of width of Dorsal Oblique 3 ( DO3 ) muscle in larvae carrying tumors of RasV12 ; ctrl at day 6 ( n = 29 ) RasV12 ; scrib - / - at days 6 ( n = 29 ) day 10 ( n = 31 ) .

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(c) Quantification of width of Dorsal Oblique 3 (DO3) muscle in larvae carrying tumors of RasV12; ctrl at day 6 (n=29) and RasV12; scrib-/-at days 6 (n=29) and day 10 (n=31).


( d ) of adipose tissue volume in larvae carrying tumors of RasV12 ; ctrl at day 6 RasV12 ; scrib - / - at days 6 day 10 .

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(d) Quantification of adipose tissue volume in larvae carrying tumors of RasV12; ctrl at day 6 and RasV12; scrib-/-at days 6 and day 10.


( e ) Representative confocal of adipose tissue in RasV12 ; ctrl RasV12 ; scrib - / - tumors bearing at indicated ages . Lipid droplets are highlighted with Lipid Tox . Scale bar :

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(e) Representative confocal images of adipose tissue in RasV12; ctrl and RasV12; scrib-/- tumors bearing animals at indicated ages. Lipid droplets are highlighted with Lipid Tox staining. Scale bar: 50μm


( f ) of dry of RasV12 ; ctrl ( n = 9 ) , RasV12 ; scrib - / - day 6 ( n = 15 ) day 10 ( n = 18 ) bearing larvae excluding .

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(f) Quantification of dry weight of RasV12; ctrl (n=9), RasV12; scrib-/- day 6 (n=15) and day 10 (n=18) tumor bearing larvae excluding the tumor weight.


( g ) of larval motility measure by crawling crawling pattern in RasV12 ; ctrl ( n = 14 ) , RasV12 ; scrib - / - day 6 ( n = 15 ) , day 8 ( n = 16 ) day 10 ( n = 14 ) , each colored represents a single larva .

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(g) Quantification of larval motility measure by crawling distance and crawling pattern in RasV12; ctrl (n=14), RasV12; scrib-/- day 6 (n=15), day 8 (n=16) and day 10 (n=14), each colored line represents a single larva.


( h ) Coomassie feeding to asses food intake of RasV12 ; scrib - / - over time , three repeated measurements of an average food intake of 20 larvae .

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(h) Coomassie feeding assay to asses food intake of RasV12; scrib-/- over time, three repeated measurements of an average food intake of 20 larvae.


( a - e ) Cartoon ( top ) illustrates genotypes of larvae , eye - antennal disc ( EAD , circle : in green microenvironment in black ) , systemic are illustrated as a square ( wild type in brown atg13 mutant in orange ) at indicated ages . Cartoon ( left ) illustrates structure of cephalic attached to mouth hook . Representative of Larva ( image of larva using backlight of microscope ) , cephalic ( green highlights GFP - labelled clones ) , muscle ( Phalloidin in green stains actin Hoechst in blue stains nucleus ) adipose tissue ( Lipid Tox in red stains for lipid droplets , Hoechst stains nucleus ) from top to bottom . ( a ) RasV12 ; ctrl bearing at day 6 . ( b ) RasV12 ; scrib - / - bearing at day 6 . ( c ) RasV12 ; scrib - / - bearing at day 8 . ( d ) RasV12 , scrib - / - , atg13 - / - / / atg13 - / - at day 8 . ( e ) RasV12 , scrib - / - , atg13 - / - / / atg13 - / - complemented with transgenic atg13 , rescuing growth , at day 8 .

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

(a-e) Cartoon (top) illustrates the genotypes of the larvae, eye-antennal disc (EAD, circle: tumor cells in green and microenvironment cell in black), systemic cells are illustrated as a square (wild type cells in light brown and atg13 mutant cells in light orange) at indicated ages. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Representative images of Larva (image of larva using the backlight of microscope), cephalic complex (green highlights the GFP-labelled tumor clones), muscle (Phalloidin in green stains actin and Hoechst in blue stains nucleus) and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains nucleus) from top to bottom. (a) RasV12; ctrl tumor bearing animal at day 6. (b) RasV12; scrib-/- tumor bearing animal at day 6. (c) RasV12; scrib-/- tumor bearing animal at day 8. (d) RasV12, scrib-/-, atg13-/-//atg13-/- at day 8. (e) RasV12, scrib-/-, atg13-/-//atg13-/- animal complemented with transgenic atg13, rescuing the tumor growth, at day 8.


( f ) of of space occupied with lobes of fat within larval segments 4 to 8 ( shown in yellow dashed in 2a ) , of bearing tumors at day 6 RasV12 ; ctrl ( n = 45 ) , RasV12 ; scrib - / - ( n = 30 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 30 ) ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 29 ) at day 10 RasV12 ; scrib - / - ( n = 40 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 25 ) ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 28 ) .

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(f) Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 (shown in yellow dashed line in 2a), of animals bearing tumors at day 6 RasV12; ctrl (n=45), RasV12; scrib-/- (n=30), RasV12, scrib-/-, atg13-/-//atg13-/- (n=30) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=29) and at day 10 RasV12; scrib-/- (n=40), RasV12, scrib-/-, atg13-/-//atg13-/- (n=25) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=28).


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(g) Quantification of tumor volumes day 6 RasV12; ctrl (n=7), RasV12; scrib-/- (n=11) and day 8 RasV12; scrib-/- (n=13), RasV12, scrib-/-, atg13-/-//atg13-/- (n=9) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=9).


( h ) of Ventral Longitudinal muscle 4 ( VL4 ) ( shown in yellow dashed in 2a ) of third segment of larvae carrying at day 6 RasV12 ; ctrl ( n = 15 ) , RasV12 ; scrib - / - ( n = 14 ) , at day8 RasV12 ; scrib - / - ( n = 11 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 12 ) ey3 . 5 - atg13 ; RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - ( n = 14 ) .

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

(h) Quantification of Ventral Longitudinal muscle 4 (VL4) (shown in yellow dashed line in 2a) of third segment of larvae carrying at day 6 RasV12; ctrl (n=15), RasV12; scrib-/- (n=14), and at day8 RasV12; scrib-/- (n=11), RasV12, scrib-/-, atg13-/-//atg13-/- (n=12) and ey3.5-atg13; RasV12; scrib-/-, atg13-/-//atg13-/- (n=14).


( i ) of dry excluding of RasV12 ; scrib - - , atg13 - / - / / atg13 - / - at day 6 ( n = 8 ) day 10 ( n = 7 ) .

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(i) Quantification of dry weight excluding tumor of RasV12; scrib- -, atg13-/-//atg13-/-at day 6 (n=8) and day 10 (n=7).


( j ) of larval motility measured by crawling crawling pattern for RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - at day 6 ( n = 15 ) at day 8 ( n = 15 ) , each colored represents a single larva .

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(j) Quantification of larval motility measured by crawling distance and crawling pattern for RasV12; scrib-/-, atg13-/-//atg13-/- at day 6 (n=15) and at day 8 (n=15), each colored line represents a single larva.