GC - MS - based quantification of ( C ) lactate levels in A375 cells treated with PLX - 4032 ( 1 μM ) for 16 hr . Data information : Statistical analysis was performed by unpaired t - test for the comparison of two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

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GC-MS-based quantification of (C) lactate levels in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.


D Immunoblotting of the indicated proteins in A375 cells treated with PLX - 4032 ( 1 μM ) for 2 , 6 , or 24 hr .

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D Immunoblotting of the indicated proteins in A375 cells treated with PLX-4032 (1 μM) for 2, 6, or 24 hr.


E , F Heatmap for expression fold - change of ASNS , GPT2 , PSAT1 , and MITF between ( E ) post - treatment ( BRAF inhibitor , BRAF - i ; MEK inhibitor , MEK - i ) and pre - treatment , or ( F ) post - treatment ( BRAF - i ) and pre - treatment tumor in each of the patients ( y - axis ) . The p - value of paired Wilcoxon test following the adjustment multiple hypothesis correction ( FDR ) are also displayed ; post - treatment expression significantly greater than pre - treatment except in the case of MITF , where post - treatment expression is significantly less than pre - treatment .

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E, F Heatmap for expression fold-change of ASNS, GPT2, PSAT1, and MITF between (E) post-treatment (BRAF inhibitor, BRAF-i; MEK inhibitor, MEK-i) and pre-treatment, or (F) post-treatment (BRAF-i) and pre-treatment tumor in each of the patients (y-axis). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR) are also displayed; post-treatment expression significantly greater than pre-treatment except in the case of MITF, where post-treatment expression is significantly less than pre-treatment.


( A ) Top : SERA6 architecture . Positions of the papain - like domain , catalytic triad residues , SUB1 cleavage sites and epitopes for the antibodies used are indicated . Positions of the inserted mTAP or myc3 epitope tags ( between codons Asn886 - Val887 ) introduced in SERA6 - mTAP : loxP parasites are shown . Bottom : time - course analysis of egress by western blot , showing processing of SERA6 - mTAP into multiple protein species ( representative of 2 independent experiments ) . Schizonts were sampled at the indicated times following washout of C2 - mediated arrest . Nomenclature for each protein species was based on apparent molecular mass or predicted composition . Note that the predicted mass of the p40 species is ~ 27 kDa , but its migration on SDS PAGE may be aberrant due to its acidic nature ; the predicted pI of sequence between SUB1 site 1 and the central papain - like domain ( Asp371 - Lys605 ) is ~ 4 . 4 ( see https : / / web . expasy . org / compute _ pi / ) .

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(A) Top: SERA6 architecture. Positions of the papain-like domain, catalytic triad residues, SUB1 cleavage sites and epitopes for the antibodies used are indicated. Positions of the inserted mTAP or myc3 epitope tags (between codons Asn886-Val887) introduced in SERA6-mTAP:loxP parasites are shown. Bottom: time-course analysis of egress by western blot, showing processing of SERA6-mTAP into multiple protein species (representative of 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2-mediated arrest. Nomenclature for each protein species was based on apparent molecular mass or predicted composition. Note that the predicted mass of the p40 species is ~27 kDa, but its migration on SDS PAGE may be aberrant due to its acidic nature; the predicted pI of sequence between SUB1 site 1 and the central papain-like domain (Asp371-Lys605) is ~4.4 (see https://web.expasy.org/compute_pi/).


( C ) Giemsa - stained thin films and western blot of DMSO - and RAP - treated SERA6 - WT - myc3 [ SERA6 - mTAP : loxP ] and SERA6 - Cys644Ala - myc3 [ SERA6 - mTAP : loxP ] parasites showing that cleavage of host RBC β - spectrin ( red arrowhead ) does not occur unless a functional copy of SERA6 is present , and is tightly associated with egress ( reproducible in 2 independent experiments ) . Schizonts were sampled at the indicated times following washout of C2 . Scale bar , 5 μm . Also see Fig EV2 .

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(C) Giemsa-stained thin films and western blot of DMSO- and RAP-treated SERA6-WT-myc3 [SERA6-mTAP:loxP] and SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites showing that cleavage of host RBC β-spectrin (red arrowhead) does not occur unless a functional copy of SERA6 is present, and is tightly associated with egress (reproducible in 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2. Scale bar, 5 μm. Also see Fig EV2.


( F ) Representative IFA images confirming loss of MSA180 - HA3 expression following RAP - treatment ( reproducible in 2 independent experiments ) . Mature C2 - arrested cycle 0 schizonts were co - stained with anti - HA ( red ) and anti - SERA5 ( green ) . Merged signals include that of the DNA dye 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar , 5 μm .

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(F) Representative IFA images confirming loss of MSA180-HA3 expression following RAP-treatment (reproducible in 2 independent experiments). Mature C2-arrested cycle 0 schizonts were co-stained with anti-HA (red) and anti-SERA5 (green). Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 5 μm.


( G ) Western blots showing MSA180 is a predominantly soluble PV protein ( representative of 2 independent experiments ) . Mature C2 - arrested MSA180 - HA3 : loxP schizonts were sampled prior to ( Total ) and following saponin fractionation to separate RBC cytosol plus soluble PV proteins ( Supernatant ) from other parasite proteins and membrane - associated proteins ( Pellet ) . The same samples were iprobed with anti - SERA5 and anti - MSP1 ( mAb 89 . 1 ) as positive controls for soluble PV proteins and merozoite surface parasite proteins respectively . The presence of MSA180 p45 - HA3 is likely a post - lysis artefact , perhaps due to SUB1 release during sample manipulation . Note the less efficient saponin - mediated release of MSA180 - HA3 compared to SERA5 , indicating limited membrane association of a fraction of MSA180 . See also Fig EV3 .

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(G) Western blots showing MSA180 is a predominantly soluble PV protein (representative of 2 independent experiments). Mature C2-arrested MSA180-HA3:loxP schizonts were sampled prior to (Total) and following saponin fractionation to separate RBC cytosol plus soluble PV proteins (Supernatant) from other parasite proteins and membrane-associated proteins (Pellet). The same samples were iprobed with anti-SERA5 and anti-MSP1 (mAb 89.1) as positive controls for soluble PV proteins and merozoite surface parasite proteins respectively. The presence of MSA180 p45-HA3 is likely a post-lysis artefact, perhaps due to SUB1 release during sample manipulation. Note the less efficient saponin-mediated release of MSA180-HA3 compared to SERA5, indicating limited membrane association of a fraction of MSA180. See also Fig EV3.


( A ) Replication of DMSO - and RAP - treated MSA180 - HA3 : loxP parasites over three erythrocytic cycles . Parasitaemia values are averages from replicates in different blood sources . The similar growth rate of DMSO - treated parasites and the parental B11 clone shows that the genetic modifications to generate MSA180 - HA3 : loxP parasites did not affect parasite viability . Error bars , ± S . D ( B11 : n = 3 ; DMSO - and RAP - treated MSA180 - HA3 : loxP : n = 6 each ) .

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(A) Replication of DMSO- and RAP-treated MSA180-HA3:loxP parasites over three erythrocytic cycles. Parasitaemia values are averages from replicates in different blood sources. The similar growth rate of DMSO-treated parasites and the parental B11 clone shows that the genetic modifications to generate MSA180-HA3:loxP parasites did not affect parasite viability. Error bars, ± S.D (B11: n=3; DMSO- and RAP-treated MSA180-HA3:loxP: n=6 each).


( B ) Stills from simultaneous time - lapse DIC and fluorescence microscopic examination of DMSO - and RAP - treated MSA180 - HA3 : loxP cycle 0 schizonts following removal of C2 - arrest ( representative of 2 independent experiments ) . Note the defect in RBCM rupture and merozoite release in RAP - treated parasites despite normal PVM rupture ( indicated by a sudden increased visibility and motility of merozoites ) and RBCM poration ( green ring indicating access and binding of phalloidin to the host RBC cytoskeleton ) . Scale bars , 5 μm .

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(B) Stills from simultaneous time-lapse DIC and fluorescence microscopic examination of DMSO- and RAP-treated MSA180-HA3:loxP cycle 0 schizonts following removal of C2-arrest (representative of 2 independent experiments). Note the defect in RBCM rupture and merozoite release in RAP-treated parasites despite normal PVM rupture (indicated by a sudden increased visibility and motility of merozoites) and RBCM poration (green ring indicating access and binding of phalloidin to the host RBC cytoskeleton). Scale bars, 5 μm.


( D ) Time - course comparing fate of SERA6 from DMSO - and RAP - treated MSA180 - HA3 : loxP parasites ( representative of 2 independent experiments ) . Schizonts were sampled at the indicated times following removal of C2 - arrest , or arrested with E64 - d ( 50 μM ) . RAP - treated SERA6 - Cys644Ala - myc3 [ SERA6 - mTAP : loxP ] parasites were included in the blot to allow comparison with processing of catalytically - dead SERA6 ( SERA6 - Cys644Ala - myc3 ) . Note the accumulation of SERA6 p65 and poor conversion to SERA6 p40 in the RAP - treated ( ΔMSA180 ) parasites .

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

(D) Time-course comparing fate of SERA6 from DMSO- and RAP-treated MSA180-HA3:loxP parasites (representative of 2 independent experiments). Schizonts were sampled at the indicated times following removal of C2-arrest, or arrested with E64-d (50 μM). RAP-treated SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites were included in the blot to allow comparison with processing of catalytically-dead SERA6 (SERA6-Cys644Ala-myc3). Note the accumulation of SERA6 p65 and poor conversion to SERA6 p40 in the RAP-treated (ΔMSA180) parasites.


( A ) Representative IFA images demonstrating the dynamic changes in localisation of SERA6 - mTAP during egress ( reproducible in 4 independent experiments ) . SERA6 - mTAP : loxP schizonts were sampled at the indicated times following removal of C2 - arrest , fixed and co - stained with DAPI ( blue ) , anti - HA ( red ) and anti - ankyrin ( green ; ankyrin is a major component of the RBC cytoskeleton ) . Scale bar , 5 μm .

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(A) Representative IFA images demonstrating the dynamic changes in localisation of SERA6-mTAP during egress (reproducible in 4 independent experiments). SERA6-mTAP:loxP schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green; ankyrin is a major component of the RBC cytoskeleton). Scale bar, 5 μm.


( C ) Representative IFA images demonstrating localisation of MSA180 - HA3 during egress . MSA180 - HA3 [ B11 ] schizonts were sampled at the indicated times following removal of C2 - arrest , fixed and co - stained with DAPI ( blue ) , anti - HA ( red ) and anti - ankyrin ( green ) . Scale bar , 5 μm . See also Fig EV5 .

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(C) Representative IFA images demonstrating localisation of MSA180-HA3 during egress. MSA180-HA3 [B11] schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green). Scale bar, 5 μm. See also Fig EV5.


( B ) E64 - d , MMV676881 and Compound 31 inhibit RBCM rupture and merozoite release despite normal PVM rupture , indicated by loss of a continuous ring of EXP2 - mNeon signal ( green ) and RBCM poration ( red ring indicating access and binding of phalloidin to the host RBC cytoskeleton ) ( reproducible in 2 independent experiments ) . P . falciparum EXP2 - mNeon schizonts were allowed to reach the point of egress in the presence of C2 ( 1 µM ) , E64 - d ( 10 µM ) , MMV676881 ( 2 µM ) , Compound 31 ( 2 µM ) or DMSO ( 0 . 1 % v / v ; control ) then examined by DIC and fluorescence microscopy . Scale bar , 5 μm .

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(B) E64-d, MMV676881 and Compound 31 inhibit RBCM rupture and merozoite release despite normal PVM rupture, indicated by loss of a continuous ring of EXP2-mNeon signal (green) and RBCM poration (red ring indicating access and binding of phalloidin to the host RBC cytoskeleton) (reproducible in 2 independent experiments). P. falciparum EXP2-mNeon schizonts were allowed to reach the point of egress in the presence of C2 (1 µM), E64-d (10 µM), MMV676881 (2 µM), Compound 31 (2 µM) or DMSO (0.1% v/v; control) then examined by DIC and fluorescence microscopy. Scale bar, 5 μm.


( C ) Western blot comparing dose - response of E64 - d , MMV676881 and Compound 31 - mediated inhibition of maturation of SERA6 - mTAP from SERA6 - mTAP : loxP parasites ( reproducible in 3 independent experiments ) .

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(C) Western blot comparing dose-response of E64-d, MMV676881 and Compound 31-mediated inhibition of maturation of SERA6-mTAP from SERA6-mTAP:loxP parasites (reproducible in 3 independent experiments).


( C ) Immunoblots analysis of TOMM34 expression in parental and metformin adaptive HCC cells .

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(C) Immunoblots analysis of TOMM34 expression in parental and metformin adaptive HCC cells.


( G ) The morphology ( Left ) , migration ( Middle ) and invasion ( Right ) of metformin adaptive Huh7 cells with ( shTOM ) or without ( shNC ) knockdown of TOMM34 are shown ( 5 × 104 cells for Transwell assay ) . Scale bars , ( Left ) 25 μm , ( Middle ) and ( Right ) 100 μm . ( n = 3 biological replicates , Two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(G) The morphology (Left), migration (Middle) and invasion (Right) of metformin adaptive Huh7 cells with (shTOM) or without (shNC) knockdown of TOMM34 are shown (5×104 cells for Transwell assay). Scale bars, (Left) 25 μm, (Middle) and (Right) 100 μm. (n=3 biological replicates, Two-way ANOVA). Data information: Data are presented as means ± S.D.


( I ) A tail vein injection model was established using adaptive Huh7 cells to assess cell migratory ability . Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 6 mice for each group , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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(I) A tail vein injection model was established using adaptive Huh7 cells to assess cell migratory ability. Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=6 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model ( shTOM : shTOMM34 , TOMM34 knockdown cells ; Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 5 mice for each group , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model (shTOM: shTOMM34, TOMM34 knockdown cells; Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=5 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) . Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 5 mice for each group , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=5 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


( L ) Western blot analysis showing the effects of TOMM34 on the expression of EMT markers in HCC cells ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) .

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(L) Western blot analysis showing the effects of TOMM34 on the expression of EMT markers in HCC cells (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).


( M ) The protein expression of TOMM34 in clinical HCC samples with or without metastasis . Scale bars , ( Left ) 100 μm , ( Right ) 25 μm . ( n = 33 in metastasis present group , n = 53 in metastasis absent group , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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(M) The protein expression of TOMM34 in clinical HCC samples with or without metastasis. Scale bars, (Left) 100 μm, (Right) 25 μm. (n = 33 in metastasis present group, n = 53 in metastasis absent group, Student's t-test). Data information: Data are presented as means ± S.D.


( C ) Transmission electron microscopy ( TEM ) of Huh7 shNC and shTOM ( shTOMM34 ) cells . Yellow arrowheads indicate mitochondria , bule arrowheads indicate nucleus , red arrowheads indicate endoplasmic reticulum . Scale bars , ( Left ) 20 μm , ( Middle ) 4 μm , ( Right ) 1 μm . ( n = 5 biological replicates , One - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(C) Transmission electron microscopy (TEM) of Huh7 shNC and shTOM (shTOMM34) cells. Yellow arrowheads indicate mitochondria, bule arrowheads indicate nucleus, red arrowheads indicate endoplasmic reticulum. Scale bars, (Left) 20 μm, (Middle) 4 μm, (Right) 1 μm. (n=5 biological replicates, One-way ANOVA). Data information: Data are presented as means ± S.D.


( G ) Western blot analysis showing the protein expression of the main mitochondrial respiratory chain complex subunit ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) .

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(G) Western blot analysis showing the protein expression of the main mitochondrial respiratory chain complex subunit (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).


( B ) Co - IP assays indicate the interaction between exogenous TOMM34 and ATP5B in HEK293T cells .

(B) Co-IP assays indicate the interaction between exogenous TOMM34 and ATP5B in HEK293T cells.


( C ) Co - IP assays indicate the interaction between endogenous TOMM34 and ATP5B in Huh7 HCC cells .

(C) Co-IP assays indicate the interaction between endogenous TOMM34 and ATP5B in Huh7 HCC cells.


( E ) The mRNA and protein expression of ATP5B in indicated cells were determined by qPCR ( n = 3 technical replicates , Two - way ANOVA ) or immunoblotting ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 - overexpressed cells ) . Data information : Data are presented as means ± S . D .

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(E) The mRNA and protein expression of ATP5B in indicated cells were determined by qPCR (n=3 technical replicates, Two-way ANOVA) or immunoblotting (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34-overexpressed cells). Data information: Data are presented as means ± S.D.


( F - G ) Western blot showing protein level of ATP5B in Huh7 ( F ) or PLC / PRF / 5 ( G ) cells treated with or without 10 mg / mL CHX for 12 hours or 25 μM MG132 for 8 hours . Relative grey value of ATP5B was calculated using Image J ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ; CHX , cyclohexane ) . ( n = 3 technical replicates , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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(F-G) Western blot showing protein level of ATP5B in Huh7 (F) or PLC/PRF/5 (G) cells treated with or without 10 mg/mL CHX for 12 hours or 25 μM MG132 for 8 hours. Relative grey value of ATP5B was calculated using Image J (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells; CHX, cyclohexane). (n=3 technical replicates, Student's t-test). Data information: Data are presented as means ± S.D.


( H ) Tissue immunofluorescence assay showing the co - localization of TOMM34 and ATP5B at the invasive front of clinical HCC tissues . Scale bars , 20 μm .

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(H) Tissue immunofluorescence assay showing the co-localization of TOMM34 and ATP5B at the invasive front of clinical HCC tissues. Scale bars, 20 μm.


( I ) A positive correlation between TOMM34 and ATP5B levels in human HCC samples was shown by representative immunohistochemical images Scale bars , 50 μm . ( n = 72 ) .

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(I) A positive correlation between TOMM34 and ATP5B levels in human HCC samples was shown by representative immunohistochemical images Scale bars, 50 μm. (n=72).


( B - C ) Western blot showing the expression of indicated proteins in adaptive HCC cells 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the expression of EMT markers in cells with ( shTOM ) or without ( shNC ) knockdown of TOMM34 or overexpression of ATP5B .

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 cells treated with or without 200 nM Gboxin for 24 hours ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) .

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

(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 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 .

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

(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 .

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


( 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

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


Steady - state protein levels 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 protein levels 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) staining of cells described above .

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


( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 * * ) .

(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 the absence ( basal respiration ) or presence ( phosphorylating condition ) of ADP .

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

(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 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 .

O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 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 .

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

(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.


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 .

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

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.


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 .

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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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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 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 .

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 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 .

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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 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

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

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

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 the complemented parasites . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * , P < 0 . 01 , * * * , P < 0 . 001 .

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

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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 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 ) .

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 analysis of ISP1 and ISP3 expression dynamic of DTS / Δisp1 and DTS / Δisp3 parasites . 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 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 .

O O O O O O O O O O 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 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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O 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 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 and episomally expressed in zygotes . Right panel shows quantification of palmitoylated protein band from two independent repeats , values are means ± SD .

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 and C7AC8A mutations cause cytoplasmic 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 Membrane association of ISP1 with either G2A or C7AC8A substitutions .

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


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

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

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 the complemented parasites . Scale bar = 5 μm .

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


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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 , 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 .

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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 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 .

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 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 .

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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 and ISP3 in the TTS and dhhc2kd zygotes . Two replicates performed .

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


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 .

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

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

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


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

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


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

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

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


J IFA of DHHC2 expression in the dhhc2 : : 6HA and 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 and dhhc2 : : 6HA ; ∆ isp1 / 3 .

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


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 .

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

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 the complemented dhhc2kd parasites as indicated .

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


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 .

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

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 .

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

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 and three truncation mutants in zygotes and retorts . Scale bar = 5 μm .

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

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 the dhhc2 : : 6HA zygotes .

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


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 .

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

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


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

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

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 the dhhc2kd parasites complemented with either WT DHHC2 or C4A mutant . Values are means ± SD from three independent tests .

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

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 and human codon optimized DHHC2 ectopically expressed in human HEK293T cells . Both C4A and C128A mutations ablate DHHC2 palmitoylation .

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

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 protein could be palmitoylated by the co - transfected DHHC2WT - Myc , but not DHHC2 _ C128A - Myc in HEK293T cells . Representative of two independent repeats .

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

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 the DHHC2 _ WT - HA and DHHC2 _ WT - Myc co - expressed in HEK293T cells .

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 cells . Cter75 is fused with a BFP : : Myc for easy detection via western blot . Cter75 * peptide contains C4A mutation .

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

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 bacteria E . coli .

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


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

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

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


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

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


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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O 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 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 the dhhc2 : : 6HA gametocytes with only either XA or 22 ° C stimulation . Three replicates performed , values are means ± SD .

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

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 the dhhc2 : : 6HA parasite - derived mutants with individual gene disruption of cdpk4 , hap2 , and dozi .

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


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 .

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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 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 .

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

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 ( 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 staining show the SPM structures of WT , dhhc2kd and ∆ isp1 / 3 parasites . Arrowheads point to cell 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 cell lysate of WT ookinetes . Lower panel indicates the Coomassie blue stain of recombinant GST and GST - ISP1 .

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 .

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

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 the TTS ookinetes .

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


D Co - localization of ISP1 : : HA and 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 assay ( PLA ) detecting interaction of ISP1 - HA and 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 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 .

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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 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 .

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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 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 .

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

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

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


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

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


( 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 .

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

(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 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 .

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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 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 .

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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 ) 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

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

(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 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

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(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 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 .

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(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 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 .

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

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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 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 .

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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 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 .

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

(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 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 .

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(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 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 .

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(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 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 .

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(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 - / - 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 .

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(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 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 .

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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 . 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 .

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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 . 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 .

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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 and TNF - α - stimulated cells , and 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 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 .

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 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 .

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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 . 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 .

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.