SENP3 knockdown abolishes DFP - induced mitophagic autolysosomes . Nsi or SENP3i ( I ) together with mito - pHfluorin were transfected into HeLa cells in the absence or presence of DFP ( 24 h ) , and were analyzed using the mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Magenta : SENP3 ; Scale bar , 10 μm ; n = 28 cells per condition from two individual experiments ; * * * * p < 0 . 0001 ; unpaired t - test ) .

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 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 O O O O O O O O O O

SENP3 knockdown abolishes DFP-induced mitophagic autolysosomes. Nsi or SENP3i (I) together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), and were analyzed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Magenta: SENP3; Scale bar, 10 μm; n=28 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test).


Fis1 knockdown abolishes DFP - induced LC3 - II . Fis1 siRNA was introduced into HeLa cells for 48 h , and the cells were treated with DFP for further 24 h ( Nsi , non - specific siRNA ; Fis1i , Fis1 siRNA ; siRNA concentration , 20nM ; DFP , 1mM ) . Whole cell lysate samples were blotted as indicated . Values are presented as mean ± SEM and are normalized to the control value ( n = 3 , biological replicates ; * * , p < 0 . 01 ; paired t - test ) .

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 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 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

Fis1 knockdown abolishes DFP-induced LC3-II. Fis1 siRNA was introduced into HeLa cells for 48 h, and the cells were treated with DFP for further 24 h (Nsi, non-specific siRNA; Fis1i, Fis1 siRNA; siRNA concentration, 20nM; DFP, 1mM). Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=3, biological replicates; **, p<0.01; paired t-test).


Fis1 knockout abolishes DFP - induced LC3 - II . Fis1 + / + or Fis1 - / - cells were treated with DFP for 24 h . Whole cell lysate samples were blotted as indicated . Values are presented as mean ± SEM and are normalized to the control value ( n = 6 , biological replicates ; * , p < 0 . 05 ; paired t - test ) .

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 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

Fis1 knockout abolishes DFP-induced LC3-II. Fis1+/+ or Fis1-/- cells were treated with DFP for 24 h. Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=6, biological replicates; *, p<0.05; paired t-test).


Fis1 knockdown prevents DFP - induced mitophagic autolysosomes . Nsi or Fis1i together with mito - pHfluorin were transfected into HeLa cells in the absence or presence of DFP ( 24 h ) , which were analysed using the mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Blue : Fis1 ; Scale bar , 10 μm ; n = 25 cells per condition from three individual experiments ; * * * p < 0 . 001 ; unpaired - t test ) .

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 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 O O O O O O O O O

Fis1 knockdown prevents DFP-induced mitophagic autolysosomes. Nsi or Fis1i together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), which were analysed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Blue: Fis1; Scale bar, 10 μm; n=25 cells per condition from three individual experiments; *** p<0.001; unpaired-t test).


Flag - Fis1 is deSUMOylated by SENP3 . SENP3 knockdown enhances Fis1 SUMOylation . Nsi or SENP3i was transfected into HEK293 cells expressing Flag - Fis1 , His - SUMO - 2 and Ubc9 for 48 h . His - pulldown and lysate samples were detected by immunoblotting for Flag , SENP3 or β - actin . Values are presented as mean ± SEM ( n = 3 , biological replicates ; * p < 0 . 05 ; paired - t test ) .

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

Flag-Fis1 is deSUMOylated by SENP3. SENP3 knockdown enhances Fis1 SUMOylation. Nsi or SENP3i was transfected into HEK293 cells expressing Flag-Fis1, His-SUMO-2 and Ubc9 for 48 h. His-pulldown and lysate samples were detected by immunoblotting for Flag, SENP3 or β-actin. Values are presented as mean ± SEM (n=3, biological replicates; * p<0.05; paired-t test).


The SUMOylation status of Fis1 influences its mitochondrial localization . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG was expressed in HeLa cells for 48 h . Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated ( the upper panel ) . Histograms ( the right panel ) show normalized levels of Flag - Fis1 WT or Flag - Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction ( Value are presented as mean ± SEM and are normalized to the control value ; n = 7 , biological replicates ; * , p < 0 . 05 ; paired t - test ) .

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

The SUMOylation status of Fis1 influences its mitochondrial localization. Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG was expressed in HeLa cells for 48 h. Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated (the upper panel). Histograms (the right panel) show normalized levels of Flag-Fis1 WT or Flag-Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction (Value are presented as mean ± SEM and are normalized to the control value; n=7, biological replicates; *, p<0.05; paired t-test).


The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum ( ER ) . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG along with GFP - Cb5 as an ER marker were expressed in HeLa cells for 48 h . Immunocytochemistry was then performed ( Green : ER labelled by GFP - Cb5 ; Blue : different Flag - Fis1 forms ; Scale bar , 10 μm )

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

The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum (ER). Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG along with GFP-Cb5 as an ER marker were expressed in HeLa cells for 48 h. Immunocytochemistry was then performed (Green: ER labelled by GFP-Cb5; Blue: different Flag-Fis1 forms; Scale bar, 10 μm)


Expressing a SUMOylation - deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP - induced LC3 - II induction . pcDNA3 , Flag - Fis1 WT or Flag - Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ( I ) ; siRNA concentration , 20nM ) , and the cells were treated with DFP for a further 24 h ( DFP , 1mM ) . Lysate samples were blotted as indicated .

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

Expressing a SUMOylation-deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP-induced LC3-II induction. pcDNA3, Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA (I); siRNA concentration, 20nM), and the cells were treated with DFP for a further 24 h (DFP, 1mM). Lysate samples were blotted as indicated.


SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP - Fis1 K149R in the presence of DFP . CFP - Fis1 WT or CFP - Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h , and the cells were treated with DFP for a further 24 h ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Cyan / blue : CFP - Fis1 ; Magenta , SENP3 ; Scale bar , 10 μm ) ; Values are presented as mean ± SEM ( n = 21 cells per condition from two individual experiments ; * * * * p < 0 . 0001 ; unpaired t - test ) . Knockdown of SENP3 was further confirmed by immunoblotting ( the lower right panel ) .

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

SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP-Fis1 K149R in the presence of DFP. CFP-Fis1 WT or CFP-Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h, and the cells were treated with DFP for a further 24 h (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Cyan/blue: CFP-Fis1; Magenta, SENP3; Scale bar, 10 μm); Values are presented as mean ± SEM (n=21 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test). Knockdown of SENP3 was further confirmed by immunoblotting (the lower right panel).


A ) oxLDL plasma concentration in ACM patients and HC ( n = 36 ; Mann - Whitney test ) . B ) oxLDL plasma concentration in mutated ACM ( n = 7 ) and NON ACM relatives , carriers of the same causative mutation ( n = 9 ; Mann - Whitney test ) . C ) oxLDL plasma concentration in ACM patients carriers of a PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations , or gene elusive ( n = 10 vs . n = 26 ; One - Way ANOVA ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 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 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

A) oxLDL plasma concentration in ACM patients and HC (n=36; Mann-Whitney test). B) oxLDL plasma concentration in mutated ACM (n=7) and NON ACM relatives, carriers of the same causative mutation (n=9; Mann-Whitney test). C) oxLDL plasma concentration in ACM patients carriers of a PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations, or gene elusive (n=10 vs. n=26; One-Way ANOVA). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


D ) Representative images of MDA immunostaining ( green ) on ACM and HC ventricular tissue sections and relative quantification ( n = 4 biological replicates ; Two - tailed Student ' s t - test ) . Nuclei are counterstained with Hoechst33342 ( blue ) . E ) Representative images of CD36 immunostaining on ACM and HC ventricular tissue sections and relative quantification ( n = 4 biological replicates ; Two - tailed Student ' s t - test ) . Nuclei are counterstained with Hoechst33342 ( blue ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 O O O O O O O O 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) Representative images of MDA immunostaining (green) on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). E) Representative images of CD36 immunostaining on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


B ) Left panels : representative images of three cases of ACM in Steady State Free Procession sequences at cardiac MRI . On the left a case with RV dilation , regional bulging of the RV wall without fat infiltration , and oxLDL levels below the cut - off ; in the center and on the right , two cases with fat infiltration in the right or left ventricle wall ( white arrow ) , respectively , and oxLDL levels above the cut - off . Right panel : quantification of the myocardial fat mass of the two ACM subpopulations ( above or below the cut - off ) whose MRI was available for re - analysis ( n = 14 oxLDL < 86ng / ml , n = 25 oxLDL > 86ng / ml ; Two - tailed Student ' s t - test ) .

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

B) Left panels: representative images of three cases of ACM in Steady State Free Procession sequences at cardiac MRI. On the left a case with RV dilation, regional bulging of the RV wall without fat infiltration, and oxLDL levels below the cut-off; in the center and on the right, two cases with fat infiltration in the right or left ventricle wall (white arrow), respectively, and oxLDL levels above the cut-off. Right panel: quantification of the myocardial fat mass of the two ACM subpopulations (above or below the cut-off) whose MRI was available for re-analysis (n=14 oxLDL<86ng/ml, n=25 oxLDL>86ng/ml; Two-tailed Student's t-test).


G ) Kaplan - Meier analysis of actual MAE free survival of patient belonging to the two ACM cohort subgroups in the first 5 - year follow - up ( n = 26 oxLDL < 86ng / ml vs . n = 41oxLDL > 86ng / ml ; Log - Rank p < 0 . 0001 ; HR = 0 . 223 [ 0 . 116 - 0 . 428 ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier analysis of actual MAE free survival of patient belonging to the two ACM cohort subgroups in the first 5-year follow-up (n=26 oxLDL<86ng/ml vs. n=41oxLDL>86ng/ml; Log-Rank p<0.0001; HR=0.223[0.116-0.428]).


B ) Left panels : representative images of MDA immunostaining ( green ) on ACM and HC C - MSC in GM . Nuclei are counterstained with Hoechst33342 ( blue ) . Right panel : image quantification ( n = 4 biological replicates ACM , n = 5 biological replicates HC ; Two - tailed Student ' s t - test ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 .

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

B) Left panels: representative images of MDA immunostaining (green) on ACM and HC C-MSC in GM. Nuclei are counterstained with Hoechst33342 (blue). Right panel: image quantification (n=4 biological replicates ACM, n=5 biological replicates HC; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05, ** p<0.01.


D ) Top panel : representative images of Western Blot analysis of proteins extracted from ACM and HC C - MSC cultured in GM , hybridized with anti - CD36 and anti - PPARγ antibodies . Immunostaining of the housekeeping GAPDH is shown for normalization . Bottom panel : d . a . of PPARγ ( n = 18 biological replicates ) and CD36 ( n = 17 vs . n = 16 biological replicates ) levels , normalized on GAPDH ( Mann - Whitney test ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top panel: representative images of Western Blot analysis of proteins extracted from ACM and HC C-MSC cultured in GM, hybridized with anti-CD36 and anti-PPARγ antibodies. Immunostaining of the housekeeping GAPDH is shown for normalization. Bottom panel: d.a. of PPARγ (n=18 biological replicates) and CD36 (n=17 vs. n=16 biological replicates) levels, normalized on GAPDH (Mann-Whitney test). Data information: mean ± SEM. * p<0.05, ** p<0.01.


F ) Top panels : representative images of internalization of oxLDL ( red ) in HC and ACM cells , cultured either in GM or in AM , and subjected to 10μg / ml DiI - oxLDL treatment . Nuclei are counterstained with Hoechst33342 ( blue ) . Bottom panel : quantification of the relative mean DiI fluorescence for each sample , measured by FACS analysis ( n = 3 biological replicates ; Two - Way Anova ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 .

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 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

F) Top panels: representative images of internalization of oxLDL (red) in HC and ACM cells, cultured either in GM or in AM, and subjected to 10μg/ml DiI-oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Bottom panel: quantification of the relative mean DiI fluorescence for each sample, measured by FACS analysis (n=3 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01.


A ) Left panels : representative images of ORO staining on ACM and HC C - MSC in AM supplemented or not with 150µg / ml oxLDL . Middle panel : image quantification ( n = 11 biological replicates ; Two - Way Anova ) . Right panels : representative images of Western Blot of CD36 , PPARγ and GAPDH protein expression of ACM and HC C - MSC protein extracts in AM supplemented or not with 150µg / ml oxLDL ( n = 5 biological replicates ) and d . a . normalized on the house - keeping GAPDH ( Two - Way Anova ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O 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 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

A) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 150µg/ml oxLDL. Middle panel: image quantification (n=11 biological replicates; Two-Way Anova). Right panels: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 150µg/ml oxLDL (n=5 biological replicates) and d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.


B ) Left panels : representative images of ORO staining on ACM and HC C - MSC in AM supplemented or not with 20μg / ml 13HODE , 5mmol / L NAC or both . Right panel : image quantification ( n = 13 biological replicates ; Two - Way Anova ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O 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 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) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both. Right panel: image quantification (n=13 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.


C ) Left panel : representative images of Western Blot of CD36 , PPARγ and GAPDH protein expression of ACM and HC C - MSC protein extracts in AM supplemented or not with 20μg / ml 13HODE , 5mmol / L NAC or both ( n = 8 biological replicates ) . Right panels : d . a . normalized on the house - keeping GAPDH ( Two - Way Anova ) . Data information : mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

C) Left panel: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both (n=8 biological replicates). Right panels: d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.


D ) Left panel : representative images of internalization of oxLDL ( red ) in ACM C - MSC treated with scramble siRNA or CD36 siRNA , cultured in AM and subjected to 10μg / ml DiI oxLDL treatment . Right panel : quantification of the DiI fluorescence normalized on nuclei number ( n = 3 biological replicates , arbitrary units ; Two - tailed Student ' s t - test ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 .

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 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

D) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC treated with scramble siRNA or CD36 siRNA, cultured in AM and subjected to 10μg/ml DiI oxLDL treatment. Right panel: quantification of the DiI fluorescence normalized on nuclei number (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.


E ) Left panel : representative images of internalization of oxLDL ( red ) in ACM C - MSC cultured in AM or AM + 5μM GW9662 and subjected to 10μg / ml DiI oxLDL treatment . Nuclei are counterstained with Hoechst33342 ( blue ) . Right panel : quantification of the DiI fluorescence normalized on nuclei number for each sample ( n = 3 biological replicates , arbitrary units ; Two - tailed Student ' s t - test ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 .

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 B-CONTROLLED_VAR 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

E) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC cultured in AM or AM+5μM GW9662 and subjected to 10μg/ml DiI oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Right panel: quantification of the DiI fluorescence normalized on nuclei number for each sample (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.


F ) Left panel : representative images of Western Blot of CD36 and GAPDH expression of protein extracts of ACM C - MSC cultured in AM or AM + 5μM GW9662 . Right panels : d . a . normalized on the house - keeping GAPDH ( n = 7 biological replicates ; Two - tailed Student ' s t - test ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 .

O O O O O O 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

F) Left panel: representative images of Western Blot of CD36 and GAPDH expression of protein extracts of ACM C-MSC cultured in AM or AM+5μM GW9662. Right panels: d.a. normalized on the house-keeping GAPDH (n=7 biological replicates; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.


C ) Left panels : representative images of ORO staining of HFD - fed WT and Pkp2 + / - cardiac sections . Right panel : quantification of ORO positive area percentage ( n = 10 ) . For comparison , quantification of ORO positive area of cardiac sections of CD - fed WT and Pkp2 + / - mice ( n = 9 ; Two - Way Anova ) is shown ( Appendix Figure S3 ) . D ) Representative images of PPARγ ( green ) immunostaining on HFD - fed WT and Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . Quantification of the PPARγ staining in HFD is shown relative to the values of CD ( n = 9 ) . E ) Representative images of MDA ( green ) immunostaining on HFD - fed WT and Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . Quantification of the MDA staining in HFD is shown relative to the values of CD ( n = 9 ) . F ) Representative images of CD36 immunostaining ( green ) on HFD - fed WT and Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . Nuclei are counterstained with Hoechst33342 ( blue ) . Quantification of the staining in HFD is shown relative to the values of CD ( n = 9 ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O 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) Left panels: representative images of ORO staining of HFD-fed WT and Pkp2+/- cardiac sections. Right panel: quantification of ORO positive area percentage (n=10). For comparison, quantification of ORO positive area of cardiac sections of CD-fed WT and Pkp2+/- mice (n=9; Two-Way Anova) is shown (Appendix Figure S3). D) Representative images of PPARγ (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the PPARγ staining in HFD is shown relative to the values of CD (n=9). E) Representative images of MDA (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the MDA staining in HFD is shown relative to the values of CD (n=9). F) Representative images of CD36 immunostaining (green) on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Nuclei are counterstained with Hoechst33342 (blue). Quantification of the staining in HFD is shown relative to the values of CD (n=9). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


C ) Left panel : representative images of ORO staining of cardiac sections of Pkp2 + / - mice , fed a 3 - month HFD plus atorvastatin . Right panel : quantification of the percentage of ORO positive area ( n = 9 ) is compared to that in HFD ( as in Figure 6C ; Two - tailed Student ' s t - test ) . D ) Representative images of PPARγ immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . Quantification is compared to the values of Pkp2 + / - in HFD and relative to WT in CD ( as in Figure 6D ; Two - tailed Student ' s t - test ) . E ) Representative images of MDA immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . Quantification is compared to the values of Pkp2 + / - in HFD and relative to WT in CD ( as in Figure 6E ; Two - tailed Student ' s t - test ) . F ) Representative images of CD36 immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . Quantification is compared to the values of Pkp2 + / - HFD and relative to WT in CD ( as in Figure 6F ; Two - tailed Student ' s t - test ) . Nuclei are counterstained with Hoechst33342 ( blue ) . Data information : mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O 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 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 B-MEASURED_VAR 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 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 O

C) Left panel: representative images of ORO staining of cardiac sections of Pkp2+/- mice, fed a 3-month HFD plus atorvastatin. Right panel: quantification of the percentage of ORO positive area (n=9) is compared to that in HFD (as in Figure 6C; Two-tailed Student's t-test). D) Representative images of PPARγ immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6D; Two-tailed Student's t-test). E) Representative images of MDA immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6E; Two-tailed Student's t-test). F) Representative images of CD36 immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- HFD and relative to WT in CD (as in Figure 6F; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


A ) Hek293T cells expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) and schematic of BRET assay , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm . B ) Lamplight - driven response to increasing intensity 1s 405nm light C ) 405nm irradiance response curve D ) Response to 1s 405nm ( blue line ) and 525nm light ( green line ) E ) Response to 405nm ( blue line ) followed by 525nm light of different intensities ( green 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm. B) Lamplight-driven response to increasing intensity 1s 405nm light C) 405nm irradiance response curve D) Response to 1s 405nm (blue line) and 525nm light (green line) E) Response to 405nm (blue line) followed by 525nm light of different intensities (green line)


A ) Hek293T cells expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) and schematic of BRET assay , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm.


G ) Response to 405nm alone or with 525nm

G) Response to 405nm alone or with 525nm light


H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) dark or ( I ) after 8s 405nm light ( blue bar ) J ) Sustained Lamplight - driven activity ( mean BRET ratio 20 - 80s after stimulus ) after exposure to mixed stimuli from dark ( ON ) or 405nm light - adapted state ( OFF ) . Lamplight data fit with linear trendline . Best fitting parameters for ON , slope = 0 . 311 , Y - intercept = 0 . 04 , R2 = 0 . 85 . For OFF , slope = 0 . 30 , Y - intercept = 0 . 07 , R2 = 0 . 69 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar) J) Sustained Lamplight-driven activity (mean BRET ratio 20-80s after stimulus) after exposure to mixed stimuli from dark (ON) or 405nm light-adapted state (OFF). Lamplight data fit with linear trendline. Best fitting parameters for ON, slope = 0.311, Y-intercept = 0.04, R2 = 0.85. For OFF, slope = 0.30, Y-intercept = 0.07, R2 = 0.69).


H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) dark or ( I ) after 8s 405nm light ( blue bar )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar)


B ) Representative trace of hyperpolarisation to 405nm light in a Lamplight - expressing SCN neuron C ) Hyperpolarisation to 405nm was observed over a range of intensities . Recordings from 3 different cells across 2 SCN slices

O O O O O O O O O O O O O O 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) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron C) Hyperpolarisation to 405nm was observed over a range of intensities. Recordings from 3 different cells across 2 SCN slices


B ) Representative trace of hyperpolarisation to 405nm light in a Lamplight - expressing SCN

B) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron


D ) Representative trace showing hyperpolarisation was sustained during dark after 405nm light ( 5s ) and reversed by 525nm light ( 15s )

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D) Representative trace showing hyperpolarisation was sustained during dark after 405nm light (5s) and reversed by 525nm light (15s)


E ) Hyperpolarisation and depolarisation of a Lamplight - expressing neuron by 405nm and 525nm light , respectively , across multiple stimulus presentations

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E) Hyperpolarisation and depolarisation of a Lamplight-expressing neuron by 405nm and 525nm light, respectively, across multiple stimulus presentations


E ) Hyperpolarisation and depolarisation of a Lamplight - expressing neuron by 405nm and 525nm light , respectively , across multiple stimulus

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


F ) Three example traces from a single cell exposed to 405nm followed by 525nm light . Individual traces were fit with exponential decay ( 405nm , blue ) or association curves ( 525nm , green ) for 6 trials across 2 cells

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F) Three example traces from a single cell exposed to 405nm followed by 525nm light. Individual traces were fit with exponential decay (405nm, blue) or association curves (525nm, green) for 6 trials across 2 cells


F ) Three example traces from a single cell exposed to 405nm followed by 525nm light . Individual traces were fit with exponential decay ( 405nm , blue ) or association curves ( 525nm , green ) for 6 trials across 2

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G , H ) Parameters from best - fit curves used to calculate ( G ) latency ( ms ) and ( H ) half - life ( ms ) .

O O O O O O O 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, H) Parameters from best-fit curves used to calculate (G) latency (ms) and (H) half-life (ms).


I ) Simultaneous 525nm and 405nm light prevents spike silencing J ) Constant 405nm light ( 12 . 9 log photons ) presented with 525nm light of decreasing intensity from 14 . 9 - 12 . 9log photons in 6 steps of 10s ( left ) or 20s ( right ) . 525nm light antagonised the 405nm - driven hyperpolarisation for all intensities except 12 . 9log photons

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Simultaneous 525nm and 405nm light prevents spike silencing J) Constant 405nm light (12.9 log photons) presented with 525nm light of decreasing intensity from 14.9 - 12.9log photons in 6 steps of 10s (left) or 20s (right). 525nm light antagonised the 405nm-driven hyperpolarisation for all intensities except 12.9log photons


I ) Simultaneous 525nm and 405nm light prevents spike silencing

I) Simultaneous 525nm and 405nm light prevents spike silencing


A ) L7Cre rd1 retina expressing Lamplight - T2A - mCherry AAV . Anti - mCherry staining ( white ) was widespread ( Left , scale bar = 500µm ) . Fluorescent cell bodies found in different focal planes consistent with expression in ganglion ( Top Right , scale bar = 50µm ) and bipolar cells ( Bottom Right , scale bar = 50µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) L7Cre rd1 retina expressing Lamplight-T2A-mCherry AAV. Anti-mCherry staining (white) was widespread (Left, scale bar = 500µm). Fluorescent cell bodies found in different focal planes consistent with expression in ganglion (Top Right, scale bar = 50µm) and bipolar cells (Bottom Right, scale bar = 50µm).


B ) Representative single unit showing characteristic ipRGC response .

B) Representative single unit showing characteristic ipRGC response.


C ) Three Lamplight response categories - transient excitation ( N = 35 biological replicates ) , transient inhibition ( N = 6 biological replicates ) , and sustained inhibition ( N = 8 biological replicates ) . Left panel shows representative single unit response to 2s 405nm or 525nm light . Mean baseline normalised firing ( grey shading shows SEM ) is shown in centre left ( for entire recording , 500ms bins ) , centre right and far right panels ( during 405nm and 525nm stimuli , respectively 100ms bins , N as above , except N = 5 biological replicates for sustained response to 525nm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Three Lamplight response categories - transient excitation (N = 35 biological replicates), transient inhibition (N = 6 biological replicates), and sustained inhibition (N = 8 biological replicates). Left panel shows representative single unit response to 2s 405nm or 525nm light. Mean baseline normalised firing (grey shading shows SEM) is shown in centre left (for entire recording, 500ms bins), centre right and far right panels (during 405nm and 525nm stimuli, respectively 100ms bins, N as above, except N = 5 biological replicates for sustained response to 525nm).


D ) Representative single unit showing light responses over 20 stimulus repeats .

D) Representative single unit showing light responses over 20 stimulus repeats.


E ) Distribution of response latencies for 3 Lamplight - driven responses to 405nm , as well as reversal caused by 525nm in sustained inhibition units . Two outliers with slow latency ( > 2 . 5s ) in transient excitation group are likely responding to stimulus offset ( N = 35 for Transient Excitation , N = 6 for Transient Inhibition , N = 7 for Sustained Inhibition and N = 5 for Sustained Excitation , all biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of response latencies for 3 Lamplight-driven responses to 405nm, as well as reversal caused by 525nm in sustained inhibition units. Two outliers with slow latency (>2.5s) in transient excitation group are likely responding to stimulus offset (N = 35 for Transient Excitation, N = 6 for Transient Inhibition, N = 7 for Sustained Inhibition and N = 5 for Sustained Excitation, all biological replicates).


F ) Inhibition responses were smaller and less sustained for mixed stimuli ( 50 % 405nm , 50 % 525nm ) compared to 405nm only ( N = 14 biological replicates ) . G ) Excitation responses were attenuated for mixed stimuli compared to 405nm only . Left panel shows representative retinal single unit ( N = 35 biological replicates ) . Data information : For ( F - G ) Left panel shows representative single unit . Right panel shows distribution of difference in firing rate for 405nm vs mixed stimulus . All representative single units show perievent rasters ( first trial at top ) and firing rate histograms ( Bin size = 500ms ) . Timing of light stimuli shown by shaded vertical bars . All light stimuli are 16 log total Lamplight effective photons / cm2 / s . Error bars show SEM . * p < 0 . 05 , * * * p < 0 . 001 ( One - sample t - test compared to zero ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates). G) Excitation responses were attenuated for mixed stimuli compared to 405nm only. Left panel shows representative retinal single unit (N = 35 biological replicates). Data information: For (F-G) Left panel shows representative single unit. Right panel shows distribution of difference in firing rate for 405nm vs mixed stimulus. All representative single units show perievent rasters (first trial at top) and firing rate histograms (Bin size = 500ms). Timing of light stimuli shown by shaded vertical bars. All light stimuli are 16 log total Lamplight effective photons/cm2/s. Error bars show SEM. * p<0.05, *** p < 0.001 (One-sample t-test compared to zero).


F ) Inhibition responses were smaller and less sustained for mixed stimuli ( 50 % 405nm , 50 % 525nm ) compared to 405nm only ( N = 14 biological replicates ) .

O O O O O O O O O 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) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates).


A , B . Aggregate number of detected and undetected cysteines ( A ) and lysines ( B ) in 3 , 840 CpDAA - containing proteins .

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

A, B. Aggregate number of detected and undetected cysteines (A) and lysines (B) in 3,840 CpDAA-containing proteins.


C . Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220 - 479 of CASP8 ( UniProt ID Q14790 ) . Dashed horizontal line marks deleterious threshold of 25 .

O O O O O O O O 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. Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220-479 of CASP8 (UniProt ID Q14790). Dashed horizontal line marks deleterious threshold of 25.


E . Activity of recombinant caspase - 8 protein assayed using fluorogenic IETD - AFC substrate . Percentage activity shown relative to wild - type ( WT ) protein for three replicate experiments , bars and error bars as mean ± SD .

E. Activity of recombinant caspase-8 protein assayed using fluorogenic IETD-AFC substrate. Percentage activity shown relative to wild-type (WT) protein for three replicate experiments, bars and error bars as mean ± SD.


( B - E ) Immunostaining of germaria from 7 day - old females with anti - Vasa ( green , B - D ) or anti - GFP ( green , E ) , and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . ( B ) aubHN2 / + was used as a control . ( C , D ) examples of aubHN2 / QC42 germ cell loss and tumor , respectively . ( E ) Phenotypic rescue of aubHN2 / QC42 with UASp - GFP - Aub expressed using nos - Gal4 . White arrowheads indicate GSCs ; the white arrow indicates GSC loss .

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

(B-E) Immunostaining of germaria from 7 day-old females with anti-Vasa (green, B-D) or anti-GFP (green, E), and anti-Hts (red). DAPI (blue) was used to visualize DNA. (B) aubHN2/+ was used as a control. (C, D) examples of aubHN2/QC42 germ cell loss and tumor, respectively. (E) Phenotypic rescue of aubHN2/QC42 with UASp-GFP-Aub expressed using nos-Gal4. White arrowheads indicate GSCs; the white arrow indicates GSC loss.


( F ) Quantification of mutant germaria with 0 to 1 GSC , or with GSC tumors shown in ( B - E ) . The number of scored germaria ( n ) is indicated on the right graph . * * * p - value < 0 . 001 , ns : non significant , using the χ2 test .

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(F) Quantification of mutant germaria with 0 to 1 GSC, or with GSC tumors shown in (B-E). The number of scored germaria (n) is indicated on the right graph. *** p-value <0.001, ns: non significant, using the χ2 test.


( G - H ' ) Germaria containing control ( G , G ' ) or aubHN2 mutant ( H , H ' ) clonal GSCs stained with anti - GFP ( green ) and anti - Hts ( red ) , 14 days after clone induction . DAPI ( blue ) was used to stain DNA . Clonal cells are marked by the lack of GFP . Clonal GSCs and cysts are outlined with dashed line . White arrowheads show clonal GSCs in the control . aub mutant clonal GSCs have been lost ( H , H ' ) . Scale bar : 10 μm in B - E and G - H ' .

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

(G-H') Germaria containing control (G, G') or aubHN2 mutant (H, H') clonal GSCs stained with anti-GFP (green) and anti-Hts (red), 14 days after clone induction. DAPI (blue) was used to stain DNA. Clonal cells are marked by the lack of GFP. Clonal GSCs and cysts are outlined with dashed line. White arrowheads show clonal GSCs in the control. aub mutant clonal GSCs have been lost (H, H'). Scale bar: 10 μm in B-E and G-H'.


( I ) Quantification of germaria containing at least one clonal GSC at 7 , 14 and 21 days after clonal induction .

(I) Quantification of germaria containing at least one clonal GSC at 7, 14 and 21 days after clonal induction.


( J ) Division rate of wild - type and aubHN2 clonal GSCs . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the two - tailed Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O 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) Division rate of wild-type and aubHN2 clonal GSCs. The number of scored germaria (n) is indicated. *** p-value <0.001 using the two-tailed Student's t test.


( A , B ) Immunostaining of germaria with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . Examples of mnkP6 and mnkP6 aubHN2 / QC42 germaria are shown . White arrowheads indicate GSCs ; the white arrow indicates GSC loss .

O O O O O O O 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

(A, B) Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of mnkP6 and mnkP6 aubHN2/QC42 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss.


( C ) Quantification of the number of GSCs per non - tumorous germarium , and of germaria with GSC tumors , in the indicated genotypes . The number of scored germaria ( n ) is indicated . The two - tailed Student ' s t test and the χ2 test were used in the left and right panels , respectively . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 .

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

(C) Quantification of the number of GSCs per non-tumorous germarium, and of germaria with GSC tumors, in the indicated genotypes. The number of scored germaria (n) is indicated. The two-tailed Student's t test and the χ2 test were used in the left and right panels, respectively. *** p-value <0.001, ** p-value <0.01, * p-value <0.05.


( D - F ) Immunostaining of germaria from 7 day - old females with anti - Vasa or anti - GFP ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . aubHN2 / QC42 ; nos - Gal4 / + was used as a negative control . Examples of rescue in aubHN2 / QC42 ; nos - Gal4 / UASp - GFP - Aub germarium ( E ) , and of lack of rescue in aubHN2 / QC42 ; nos - Gal4 / UASp - GFP - AubAA germarium ( F ) . White arrowheads indicate GSCs ; white arrows indicate GSC loss in ( D ) and germ cell loss in ( F ) . Scale bar : 10 μm in A , B and D - F .

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

(D-F) Immunostaining of germaria from 7 day-old females with anti-Vasa or anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. aubHN2/QC42; nos-Gal4/+ was used as a negative control. Examples of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-Aub germarium (E), and of lack of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-AubAA germarium (F). White arrowheads indicate GSCs; white arrows indicate GSC loss in (D) and germ cell loss in (F). Scale bar: 10 μm in A, B and D-F.


( G ) Quantification of mutant germaria with 0 to 1 GSC shown in ( D - F ) . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , ns : non - significant , using the χ2 test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of mutant germaria with 0 to 1 GSC shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001, ns: non-significant, using the χ2 test.


( A - B ' ' ) Immunostaining of wild - type ( A - A ' ' ) or nos - Gal / UASp - CCR4 - HA ( B - B ' ' ) germaria with anti - Aub ( red ) , and anti - CCR4 or anti - HA ( green ) . GSCs are shown in ( A - A ' ' ) . White arrowheads in ( B - B ' ' ) indicate cytoplasmic accumulation of CCR4 - HA colocalized with Aub in GSCs .

(A-B'') Immunostaining of wild-type (A-A'') or nos-Gal/UASp-CCR4-HA (B-B'') germaria with anti-Aub (red), and anti-CCR4 or anti-HA (green). GSCs are shown in (A-A''). White arrowheads in (B-B'') indicate cytoplasmic accumulation of CCR4-HA colocalized with Aub in GSCs.


( C ) Co - immunoprecipitation ( IP ) of NOT1 , NOT3 and CCR4 with GFP - Aub in ovaries . Wild - type ( WT , mock IP ) or nos - Gal4 / UASp - GFP - Aub ( GFP - Aub ) ovarian extracts were immunoprecipitated with anti - GFP , either in the absence or the presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 , anti - NOT3 and anti - CCR4 . Inputs are extracts prior to IP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation (IP) of NOT1, NOT3 and CCR4 with GFP-Aub in ovaries. Wild-type (WT, mock IP) or nos-Gal4/UASp-GFP-Aub (GFP-Aub) ovarian extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1, anti-NOT3 and anti-CCR4. Inputs are extracts prior to IP.


( D ) Co - IP of NOT1 and NOT3 with GFP - Aub or GFP - AubAA in ovaries . Wild - type ( WT , mock IP ) , nos - Gal4 / UASp - GFP - Aub ( GFP - Aub ) or nos - Gal4 / UASp - GFP - AubAA ( GFP - AubAA ) ovarian extracts were immunoprecipitated with anti - GFP in the presence of RNase A . Western blots were revealed with anti - GFP , anti - NOT1 and anti - NOT3 . Inputs are extracts prior to IP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-IP of NOT1 and NOT3 with GFP-Aub or GFP-AubAA in ovaries. Wild-type (WT, mock IP), nos-Gal4/UASp-GFP-Aub (GFP-Aub) or nos-Gal4/UASp-GFP-AubAA (GFP-AubAA) ovarian extracts were immunoprecipitated with anti-GFP in the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1 and anti-NOT3. Inputs are extracts prior to IP.


( E - G ) Genetic interaction between aub and twin in Vasa self - renewal . Immunostaining of germaria with anti - DNA ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . Examples of aubHN2 / + , twinDG24102 and aubHN2 / + ; twinDG24102 germaria are shown . White arrowheads indicate GSCs ; the white arrow indicates GSC loss . Scale bar : 10 μm in A - B ' ' and E - G .

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 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

(E-G) Genetic interaction between aub and twin in Vasa self-renewal. Immunostaining of germaria with anti-DNA (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of aubHN2/+, twinDG24102 and aubHN2/+; twinDG24102 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. Scale bar: 10 μm in A-B'' and E-G.


( H ) Quantification of mutant germaria with no GSC in 3 , 7 and 14 day - old females of the genotypes shown in ( D - F ) . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the χ2 test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of mutant germaria with no GSC in 3, 7 and 14 day-old females of the genotypes shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test.


( B - C ' ' ) Immunostaining of mosaic germaria with anti - GFP ( green ) , to identify clonal cells by the lack of GFP , and either 8C4 ( B - B ' ' ) or 10F1 ( C - C ' ' ) monoclonal anti - Cbl ( red ) . DAPI was used to visualize DNA . White arrowheads indicate aubHN2 / + control GSCs ; yellow arrowheads indicate clonal mutant aubHN2 GSCs . Scale bar : 10 μm .

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

(B-C'') Immunostaining of mosaic germaria with anti-GFP (green), to identify clonal cells by the lack of GFP, and either 8C4 (B-B'') or 10F1 (C-C'') monoclonal anti-Cbl (red). DAPI was used to visualize DNA. White arrowheads indicate aubHN2/+ control GSCs; yellow arrowheads indicate clonal mutant aubHN2 GSCs. Scale bar: 10 μm.


( D - E ) Quantification of Cbl protein levels in aubHN2 / + and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1 . Fluorescence intensity was measured in arbitrary units using the ImageJ software . Horizontal bars correspond to the mean and standard deviation . * * * p - value < 0 . 001 using the two - tailed Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of Cbl protein levels in aubHN2/+ and aubHN2 mutant GSCs using fluorescence intensity of immunostaining with 8C4 or 10F1. Fluorescence intensity was measured in arbitrary units using the ImageJ software. Horizontal bars correspond to the mean and standard deviation. *** p-value <0.001 using the two-tailed Student's t test.


( F ) RNA immunoprecitation ( IP ) with anti - GFP antibody in wild - type ( mock IP ) and nos - Gal4 / UASp - GFP - Aub ovarian extracts . Cbl mRNA was quantified using RT - qPCR . Normalization was with RpL32 mRNA . Mean of three biological replicates . The error bar represents standard error to the mean . * p - value < 0 . 05 using the two - tailed Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNA immunoprecitation (IP) with anti-GFP antibody in wild-type (mock IP) and nos-Gal4/UASp-GFP-Aub ovarian extracts. Cbl mRNA was quantified using RT-qPCR. Normalization was with RpL32 mRNA. Mean of three biological replicates. The error bar represents standard error to the mean. * p-value <0.05 using the two-tailed Student's t test.


( G ) ePAT assay of CblL mRNA . Ovaries from 1 - day - old ( germarium to stage 8 ) wild - type , aub and twin mutant females , and from 4 - to 7 - day - old bam mutant females were used .

O O O O O O O O O O O O O O O O O O O O O O O O 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) ePAT assay of CblL mRNA. Ovaries from 1-day-old (germarium to stage 8) wild-type, aub and twin mutant females, and from 4- to 7-day-old bam mutant females were used.


( D , E ) Coverage of CblL ( D ) and CblS ( E ) mRNAs with ovarian and embryonic piRNAs . Significant crosslink clusters are indicated in red at the top of the graphs .

O O O O O O O O O O O O O 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) Coverage of CblL (D) and CblS (E) mRNAs with ovarian and embryonic piRNAs. Significant crosslink clusters are indicated in red at the top of the graphs.


( A - E ) Cbl expression in germaria . ( A ) Immunostaining of wild - type germaria with anti - Cbl 8C4 .

O 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-E) Cbl expression in germaria. (A) Immunostaining of wild-type germaria with anti-Cbl 8C4.


( A - E ) Cbl expression in germaria . ( B - B ' ' ) Immunostaining of bamP - GFP germaria that express GFP under the bam promoter , with anti - GFP ( green ) and anti - Cbl 8C4 ( red ) .

O O O O O O O O O O O O O O O O O O O O O O O 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-E) Cbl expression in germaria. (B-B'') Immunostaining of bamP-GFP germaria that express GFP under the bam promoter, with anti-GFP (green) and anti-Cbl 8C4 (red).


( A - E ) Immunostaining expression in germaria . ( C - C ' ) Immunostaining of germaria expressing GFP - Par1 to label spectrosomes with anti - GFP ( green ) and anti - Cbl 8C4 ( red ) . DAPI ( blue ) was used to visualize DNA . White arrowheads indicate GSCs ; yellow arrowheads indicate cystoblasts .

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

(A-E) Immunostaining expression in germaria. (C-C') Immunostaining of germaria expressing GFP-Par1 to label spectrosomes with anti-GFP (green) and anti-Cbl 8C4 (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs; yellow arrowheads indicate cystoblasts.


( F ) Western blots of protein extracts from wild - type and Cbl mutant ovaries showing CblL ( left panel ) and CblS ( right panel ) revealed with 8C4 and 10F1 antibodies , respectively . A low level of CblS remained , while no CblL was present in the CblEY11427 / MB05683 combination . The 10F1 antibody recognizes very poorly CblL in western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blots of protein extracts from wild-type and Cbl mutant ovaries showing CblL (left panel) and CblS (right panel) revealed with 8C4 and 10F1 antibodies, respectively. A low level of CblS remained, while no CblL was present in the CblEY11427/MB05683 combination. The 10F1 antibody recognizes very poorly CblL in western blot.


( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes .

O O O O O O O O O O O O O O O O O O O O O O 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-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes.


( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes . ( I ) Quantification of germaria with increased number of

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes. (I) Quantification of germaria with increased number of spectrosomes


( G - J ) Cbl is required for germline differentiation . Immunostaining of wild - type and CblEY11427 / MB05683 germaria with anti - Hts to visualize spectrosomes and fusomes . White arrowheads indicate GSCs ; the white arrow indicates increased number of spectrosomes . ( J ) quantification of spectrosomes per germarium . The number of scored germaria ( n ) is indicated in ( I ) . * * * p - value < 0 . 001 using the χ2 test in ( I ) , and the two - tailed Student ' s t test in ( J ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Cbl is required for germline differentiation. Immunostaining of wild-type and CblEY11427/MB05683 germaria with anti-Hts to visualize spectrosomes and fusomes. White arrowheads indicate GSCs; the white arrow indicates increased number of spectrosomes. (J) quantification of spectrosomes per germarium. The number of scored germaria (n) is indicated in (I). ***p-value <0.001 using the χ2 test in (I), and the two-tailed Student's t test in (J).


( K - M ) Cbl induces GSC differentiation . Immunostaining of germaria overexpressing Cbl with Hsp83 - CblL ( K ) White arrows indicate a cyst in the GSC niche ( K , outlined )

O O O O O O O O O O O O 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-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblL (K)White arrows indicate a cyst in the GSC niche (K, outlined)


( K - M ) Cbl induces GSC differentiation . Immunostaining of germaria overexpressing Cbl with Hsp83 - CblS ( L ) with anti - Vasa ( green ) and anti - Hts ( red ) . DNA ( blue ) was revealed with DAPI . White arrows indicatethe loss of GSCs and germ cells ( L ) .

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

(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblS (L) with anti-Vasa (green) and anti-Hts (red). DNA (blue) was revealed with DAPI. White arrows indicatethe loss of GSCs and germ cells (L).


( K - M ) Cbl induces GSC differentiation . ( M ) Quantification of germaria with 0 to 1 GSC in the indicated genotypes . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 using the χ2 test . Scale bar : 10 μm in A - C ' , G , H , K , L .

(K-M) Cbl induces GSC differentiation.(M) Quantification of germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test. Scale bar: 10 μm in A-C', G, H, K, L.


( A - B ) Immunostaining of germaria from aubHN2 / QC42 ( A ) females with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . The white arrow indicates the lack of GSCs ; white arrowheads indicate GSCs . Scale bar : 10 μm .

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

(A-B) Immunostaining of germaria from aubHN2/QC42 (A) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.


( A - B ) Immunostaining of germaria from aubHN2 / QC42 ; CblMB / + ( B ) females with anti - Vasa ( green ) and anti - Hts ( red ) . DAPI ( blue ) was used to visualize DNA . The white arrow indicates the lack of GSCs ; white arrowheads indicate GSCs . Scale bar : 10 μm .

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

(A-B) Immunostaining of germaria from aubHN2/QC42; CblMB/+ (B) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.


( C ) Quantification of mutant germaria with 0 to 1 GSC in the indicated genotypes . The number of scored germaria ( n ) is indicated . * * * p - value < 0 . 001 , * * p - value < 0 . 01 , * p - value < 0 . 05 , using the χ2 test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of mutant germaria with 0 to 1 GSC in the indicated genotypes. The number of scored germaria (n) is indicated. *** p-value <0.001, ** p-value <0.01, * p-value <0.05, using the χ2 test.


A . Stills depicting actin flow in extracellular RH , myoA KO and adf KD parasites expressing Cb EmeraldFP . Continuous F - actin flow can be seen from the apical tip , to the Golgi region towards the posterior pole of the parasite . No difference can be observed between RH and myoA KO . For adf KD , the flow is completely abrogated . Red arrowhead marks the Golgi region , while the yellow arrowhead marks the apical nucleation centre . B . Skeletonisation processing of ( A ) depicting areas where F - actin dynamics / flow is prevalent . Individual signals for F - actin form a continuous , dynamic network that connects apical and posterior pole of the parasite . Red arrowhead marks the Golgi region nucleation centre , while the yellow arrowhead marks the apical nucleation centre . The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F - actin . C . Kymograph analysis of ( A ) and ( B ) . The kymograph was generated by tracing a line in the middle of the parasite ' s body ( red line ) . The colour - coded kymograph represents forward movement ( red ) , backwards movement ( green ) and static F - actin ( blue ) . The results demonstrate continuous exchange of F - actin from the apical to the posterior pole of the parasite within the cytosol of the parasite . This cytosolic exchange is similar in RH and myoA KO parasites , while completely abolished in the case of adf KD . Data information : Numbers indicate minutes : seconds ; and Scale bars represent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Stills depicting actin flow in extracellular RH, myoA KO and adf KD parasites expressing Cb EmeraldFP. Continuous F-actin flow can be seen from the apical tip, to the Golgi region towards the posterior pole of the parasite. No difference can be observed between RH and myoA KO. For adf KD, the flow is completely abrogated. Red arrowhead marks the Golgi region, while the yellow arrowhead marks the apical nucleation centre. B. Skeletonisation processing of (A) depicting areas where F-actin dynamics/flow is prevalent. Individual signals for F-actin form a continuous, dynamic network that connects apical and posterior pole of the parasite. Red arrowhead marks the Golgi region nucleation centre, while the yellow arrowhead marks the apical nucleation centre. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin. C. Kymograph analysis of (A) and (B). The kymograph was generated by tracing a line in the middle of the parasite's body (red line). The colour-coded kymograph represents forward movement (red), backwards movement (green) and static F-actin (blue). The results demonstrate continuous exchange of F-actin from the apical to the posterior pole of the parasite within the cytosol of the parasite. This cytosolic exchange is similar in RH and myoA KO parasites, while completely abolished in the case of adf KD. Data information: Numbers indicate minutes:seconds; and Scale bars represent 5 µm.


D . Top three rows : Parasites expressing Cb EmeraldFP along with myoA - SNAP before and after Ca2 + - Ionophore ( A23187 ) or BIPPO treatment . The parasites were also treated with 2 µM of Cytochalasin D or DMSO ( as control ) for 30 min . After addition of A23187 , preferential relocalisation of actin can be observed at the apical tip , while addition of BIPPO caused F - actin accumulation at the basal end . 4th row : myoAKO parasites expressing Cb - EmeraldFP before and after treatment with A23187 and BIPPO . While no apical or basal accumulation of F - actin is observed , some peripheral location of F - actin occurs in presence of A23187 or BIPPO , preferentially in the apical half of the parasite . Bottom row : adf cKD parasites expressing Cb - EmeraldFP before and after treatment with Ca2 + Ionophore or BIPPO . No apparent change in F - actin localisation can be seen upon treatment . E . Skeletonization of movies shown in D . Before addition of A23187 or BIPPO , RH and myoA KO parasites behave similar with no accumulation of F - actin at the apical tip . After treatment with calcium ionophore A23187 , preferential accumulation of F - actin at the apical tip can be observed for RH ( red arrowhead ) , while BIPPO presented preferential accumulation in the basal end ( red arrowhead ) . In myoA KO , some relocalisation of actin is observed at the periphery of the parasite ( yellow arrowhead ) . In the case of adf cKD , no relocalisation occurs . The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F - actin .

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

D. Top three rows: Parasites expressing Cb EmeraldFP along with myoA-SNAP before and after Ca2+-Ionophore (A23187) or BIPPO treatment. The parasites were also treated with 2 µM of Cytochalasin D or DMSO (as control) for 30 min. After addition of A23187, preferential relocalisation of actin can be observed at the apical tip, while addition of BIPPO caused F-actin accumulation at the basal end. 4th row: myoAKO parasites expressing Cb-EmeraldFP before and after treatment with A23187 and BIPPO. While no apical or basal accumulation of F-actin is observed, some peripheral location of F-actin occurs in presence of A23187 or BIPPO, preferentially in the apical half of the parasite. Bottom row: adf cKD parasites expressing Cb-EmeraldFP before and after treatment with Ca2+ Ionophore or BIPPO. No apparent change in F-actin localisation can be seen upon treatment. E. Skeletonization of movies shown in D. Before addition of A23187 or BIPPO, RH and myoA KO parasites behave similar with no accumulation of F-actin at the apical tip. After treatment with calcium ionophore A23187, preferential accumulation of F-actin at the apical tip can be observed for RH (red arrowhead), while BIPPO presented preferential accumulation in the basal end (red arrowhead). In myoA KO, some relocalisation of actin is observed at the periphery of the parasite (yellow arrowhead). In the case of adf cKD, no relocalisation occurs. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin.


F . Quantification of actin accumulation before and after treatment with Ca2 + Ionophore or BIPPO . The parasites were counted for F - actin accumulation after adding Ca2 + Ionophore or BIPPO . Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip . A minimum of 200 parasites were counted upon 3 biological replicates . Two - way ANOVA was used for statistical analysis and Tukey ' s multiple comparisons test . * * * * p value < 0 . 0001 . Error bars represent standard deviation .

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 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

F. Quantification of actin accumulation before and after treatment with Ca2+ Ionophore or BIPPO. The parasites were counted for F-actin accumulation after adding Ca2+ Ionophore or BIPPO. Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip. A minimum of 200 parasites were counted upon 3 biological replicates. Two-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. Error bars represent standard deviation.


A . Wild Type RH parasites mid - invasion . Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes , fixed with 4 % PFA before labelling the TJ with anti - Ron2 ( yellow ) and DAPI ( magenta ) for nuclear staining . White arrow points at invasion direction .

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A. Wild Type RH parasites mid-invasion. Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes, fixed with 4% PFA before labelling the TJ with anti-Ron2 (yellow) and DAPI (magenta) for nuclear staining. White arrow points at invasion direction.


B . Graph showing deformation of the whole parasite and its nucleus between invading and non - invading parasites . 100 parasites found mid - invasion were counted in triplicate and compared to extracellular , freshly egressed parasites . One - way ANOVA was used for statistical analysis . * * * * p value < 0 . 0001 . Data information : Error bars represent standard deviation . and Scale bars represent 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 O O O O O O O O O O O O O O O O O O O O O

B. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. 100 parasites found mid-invasion were counted in triplicate and compared to extracellular, freshly egressed parasites. One-way ANOVA was used for statistical analysis. **** p value <0.0001. Data information: Error bars represent standard deviation. and Scale bars represent 5 µm.


C . Graph showing deformation of the whole parasite and its nucleus between invading and non - invading parasites . Indicated parasites were analysed . For adf cKD induced and non - induced conditions were compared , as indicated . 30 parasites were counted for each condition in triplicate . One - way ANOVA was used for statistical analysis and Tukey ' s multiple comparisons test . * * * * p value < 0 . 0001 . * * * p value 0 . 0009 . Data information : Error bars represent standard deviation . ; and Scale bars represent 5 µm .

C. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. Indicated parasites were analysed. For adf cKD induced and non-induced conditions were compared, as indicated. 30 parasites were counted for each condition in triplicate. One-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. *** p value 0.0009. Data information: Error bars represent standard deviation. ; and Scale bars represent 5 µm.


D . Time - lapse microscopy analysis of indicated parasites invading HFFs cells . Time was determined from the onset of invasion . White arrow points at invasion direction ; yellow arrow points at tight junction ; red dot follows the apical pole of the parasite . Numbers indicate minutes :

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D. Time-lapse microscopy analysis of indicated parasites invading HFFs cells. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds


E . Speed profiles of invading parasites The relative displacement of the apical tip over time allowed speed calculations . Individual stages were named in regard to the invasion step ( Penetration initiation , Speed decrease and finalisation ) . Invasion was analysed with Icy Image Processing Software ( Pasteur Institut ) using the Manual Tracking plugin by analysing each movie 10 times and averaging the

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E. Speed profiles of invading parasites The relative displacement of the apical tip over time allowed speed calculations. Individual stages were named in regard to the invasion step (Penetration initiation, Speed decrease and finalisation). Invasion was analysed with Icy Image Processing Software (Pasteur Institut) using the Manual Tracking plugin by analysing each movie 10 times and averaging the measurements


F . Time - lapse analysis demonstrating abortive invasion of indicated parasites . Time was determined from the onset of invasion . White arrow points at invasion direction ; yellow arrow points at tight junction ; red dot follows the apical pole of the parasite . Numbers indicate minutes :

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F. Time-lapse analysis demonstrating abortive invasion of indicated parasites. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds


A . Time - lapse analysis depicting invading RH adfcKD parasites expressing Cb - EmeraldFP . A F - actin ring can be observed at the TJ in the majority of cases ( red arrowheads , first panel ) . Invasion can also occur without formation of an F - actin ring ( second panel ) . During capped invasion , an F - actin ring is formed ( third panel ) . Invasion of adf cKD parasites occurs without formation of an F - actin ring ( fourth panel ) . F - actin accumulation is observed at the posterior pole of the parasite in all cases ( orange arrows ) . Green segmented lines depict the area that was measured for generation of intensity plot profiles shown in ( B ) . Yellow and purple dotted lines indicate the extra - and intracellular part of the parasite respectively . B . Intensity plot profiles of the movies shown in ( A ) . Depicted plots correlate to half - invaded parasites . In the case of F - actin ring formation , two distinct peaks are detected , correlating to the F - actin ring at the TJ ( red arrowhead ) and the posterior pole of the parasite ( orange arrow ) . In the case of no F - actin ring , no accumulation at the TJ can be observed ; only the peak correlating to posterior F - actin can be detected . For adf cKD , only one peak can be seen corresponding to the posterior pole of the parasite . C . Quantification of F - actin accumulation at the TJ for RH Cb - EmeraldFP parasites as detected in time lapse analysis . The values are expressed as percentage . 28 total parasite invasion events were captured across three different biological replicates . Two - tailed unpaired t - test analysis was performed . * * * * p value < 0 . 0001 . Data information : Error bars represent standard deviation . White arrows point to direction of invasion . Scale bars represent 5 µm . * * * * p value < 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time-lapse analysis depicting invading RH adfcKD parasites expressing Cb-EmeraldFP. A F-actin ring can be observed at the TJ in the majority of cases (red arrowheads, first panel). Invasion can also occur without formation of an F-actin ring (second panel). During capped invasion, an F-actin ring is formed (third panel). Invasion of adf cKD parasites occurs without formation of an F-actin ring (fourth panel). F-actin accumulation is observed at the posterior pole of the parasite in all cases (orange arrows). Green segmented lines depict the area that was measured for generation of intensity plot profiles shown in (B). Yellow and purple dotted lines indicate the extra- and intracellular part of the parasite respectively. B. Intensity plot profiles of the movies shown in (A). Depicted plots correlate to half-invaded parasites. In the case of F-actin ring formation, two distinct peaks are detected, correlating to the F-actin ring at the TJ (red arrowhead) and the posterior pole of the parasite (orange arrow). In the case of no F-actin ring, no accumulation at the TJ can be observed; only the peak correlating to posterior F-actin can be detected. For adf cKD, only one peak can be seen corresponding to the posterior pole of the parasite. C. Quantification of F-actin accumulation at the TJ for RH Cb-EmeraldFP parasites as detected in time lapse analysis. The values are expressed as percentage. 28 total parasite invasion events were captured across three different biological replicates. Two-tailed unpaired t-test analysis was performed. **** p value <0.0001. Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm. ****p value <0.0001.


D . Representative immunofluorescence assays on indicated parasite strains during host cell penetration . Parasites were labelled with indicated antibodies . SAG1 staining was performed prior to permeabilisation to stain only the extracellular portion of the parasite . Antibodies against Ron2 were used to stain the TJ . Red arrowhead : position of the TJ . Orange arrow : posterior pole of the parasite . Data information : Error bars represent standard deviation . White arrows point to direction of invasion . Scale bars represent 5 µm .

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D. Representative immunofluorescence assays on indicated parasite strains during host cell penetration. Parasites were labelled with indicated antibodies. SAG1 staining was performed prior to permeabilisation to stain only the extracellular portion of the parasite. Antibodies against Ron2 were used to stain the TJ. Red arrowhead: position of the TJ. Orange arrow: posterior pole of the parasite. Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm.


E . Tight Junction assay on fixed samples depicting invasion rates of indicated parasites . While in the majority of control parasites F - actin can be detected at the junction ( ~ 80 % ) , no junctional ring can be detected in ~ 20 % of all invasion events . In contrast , adf cKD , act1 cKO or myoA KO only show an overall invasion rate of ~ 20 % compared to controls . In these cases junctional F - actin accumulation could never be detected . Note that the number of total invasion events for adf cKD , act1cKO and myoAKO correlates to the number of invasion events , seen for RH without F - actin ring formation . Data information : Error bars represent standard deviation . One - way ANOVA analysis was performed for each graph * * * * p value < 0 . 0001 .

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E. Tight Junction assay on fixed samples depicting invasion rates of indicated parasites. While in the majority of control parasites F-actin can be detected at the junction (~80%), no junctional ring can be detected in ~20% of all invasion events. In contrast, adf cKD, act1 cKO or myoA KO only show an overall invasion rate of ~20% compared to controls. In these cases junctional F-actin accumulation could never be detected. Note that the number of total invasion events for adf cKD, act1cKO and myoAKO correlates to the number of invasion events, seen for RH without F-actin ring formation. Data information: Error bars represent standard deviation. One-way ANOVA analysis was performed for each graph ****p value <0.0001.


F . Quantification of posterior F - actin accumulation during invasion of indicated parasite lines . One - way ANOVA analysis was performed for each graph * * * * p value < 0 . 0001 .

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F. Quantification of posterior F-actin accumulation during invasion of indicated parasite lines. One-way ANOVA analysis was performed for each graph ****p value <0.0001.


A . Time - lapse analysis of invading RH Cb - EmeraldFP parasites . During penetration , an F - actin ring is formed at the TJ ( orange arrow ) . The nucleus ( purple ) is squeezed through the TJ ( red arrowhead ) and posterior F - actin appears to be directly connected to the nucleus and TJ ( blue arrow ) . B . Analysis of nucleus deformation during live imaging shown in A . The nucleus constricts while its passing through the TJ ( red arrowheads ) . C . The posterior end of the parasite deforms during the invasion process . F - actin is accumulated during invasion , with the posterior end contracting ( blue arrow ) . Data information : Numbers indicate minutes : seconds . Scale bar represents 5 µm .

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A. Time-lapse analysis of invading RH Cb-EmeraldFP parasites. During penetration, an F-actin ring is formed at the TJ (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) and posterior F-actin appears to be directly connected to the nucleus and TJ (blue arrow). B. Analysis of nucleus deformation during live imaging shown in A. The nucleus constricts while its passing through the TJ (red arrowheads). C. The posterior end of the parasite deforms during the invasion process. F-actin is accumulated during invasion, with the posterior end contracting (blue arrow). Data information: Numbers indicate minutes:seconds. Scale bar represents 5 µm.


D . Time - lapse stills depicting RH Cb - EmeraldFP parasites invading in absence of detectable F - actin at the junction ( orange arrow ) . The nucleus ( purple ) is squeezed through the TJ ( red arrowhead ) during invasion . Actin accumulation at the posterior pole of the parasite is strongly detected in all cases ( blue arrow ) . E . Analysis of nucleus deformation during live imaging shown in ( D ) . The nucleus appears to not show constriction once its passing through the TJ when the F - actin ring is not present . F . The posterior end of the parasite also deforms during the invasion process .

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D. Time-lapse stills depicting RH Cb-EmeraldFP parasites invading in absence of detectable F-actin at the junction (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) during invasion. Actin accumulation at the posterior pole of the parasite is strongly detected in all cases (blue arrow). E. Analysis of nucleus deformation during live imaging shown in (D). The nucleus appears to not show constriction once its passing through the TJ when the F-actin ring is not present. F. The posterior end of the parasite also deforms during the invasion process.


G . Z - stack gallery depicting mid - invading myoA KO parasites . Note that the nucleus is located located at the back during invasion events . Data information : Scale bar represents 5 µm . White arrow points to direction of invasion .

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G. Z-stack gallery depicting mid-invading myoA KO parasites. Note that the nucleus is located located at the back during invasion events. Data information: Scale bar represents 5 µm. White arrow points to direction of invasion.


A . SR - SIM images depicting invading parasites with and without the presence of the F - actin ring at the tight junction and invading adf cKD Cb - EmeraldFP parasites . SAG1 staining ( in red ) was performed prior to permeabilisation to specifically label extracellular part of the parasite . Note the accumulation of actin ( blue arrow ) around the nucleus and the posterior pole of the parasite , irrespective of F - actin ring formation at the TJ ( orange arrowhead ) . 3D rendering is shown for each presented case ( inlet ) . Data information : Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion .

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A. SR-SIM images depicting invading parasites with and without the presence of the F-actin ring at the tight junction and invading adf cKD Cb-EmeraldFP parasites. SAG1 staining (in red) was performed prior to permeabilisation to specifically label extracellular part of the parasite. Note the accumulation of actin (blue arrow) around the nucleus and the posterior pole of the parasite, irrespective of F-actin ring formation at the TJ (orange arrowhead). 3D rendering is shown for each presented case (inlet). Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


B . SR image of an invading parasite before nuclear entry . F - actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole . C . F - actin localisation in relation to the TJ during invasion . F - actin ( white arrow ) is in close vicinity to the Ron2 ring ( light blue arrow ) during invasion . Data information : cale bar represents 5 µm for SR - SIM images hite arrow points to direction of invasion .

B. SR image of an invading parasite before nuclear entry. F-actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole. C. F-actin localisation in relation to the TJ during invasion. F-actin (white arrow) is in close vicinity to the Ron2 ring (light blue arrow) during invasion. Data information: cale bar represents 5 µm for SR-SIM images hite arrow points to direction of invasion.


D . Quantification of perinuclear actin in indicated parasites . Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus . Still images to the right represent what was considered as F - actin - nucleus association . The orange arrowhead points to F - actin surrounding the nucleus in the posterior end of the parasite , the red and yellow arrowhead shows F - actin in the vicinity of the nucleus , hinting at a close interaction between the two . Three biological replicates were analysed with a minimum of 25 parasites per replicate . Data information : Error bars represent standard deviation . Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion .

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D. Quantification of perinuclear actin in indicated parasites. Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus. Still images to the right represent what was considered as F-actin - nucleus association. The orange arrowhead points to F-actin surrounding the nucleus in the posterior end of the parasite, the red and yellow arrowhead shows F-actin in the vicinity of the nucleus, hinting at a close interaction between the two. Three biological replicates were analysed with a minimum of 25 parasites per replicate. Data information: Error bars represent standard deviation. Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.