H , Functional assay with WT ( n = 4 ) or Ythdf2 cKO Treg ( n = 5 ) cells isolated from B16F10 tumors on day 16 . H , Percentage of IFNγ producing CD8 T cells after coculture . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired 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

H, Functional assay with WT (n=4) or Ythdf2 cKO Treg (n=5) cells isolated from B16F10 tumors on day 16. H, Percentage of IFNγ producing CD8 T cells after coculture. Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


D . The morphology of oocytes and embryos from female patients with MOS variants . The day of oocyte retrieval was defined as day 0 . Polar bodies were indicated by black arrows . Scale bar = 20 µm .

D. The morphology of oocytes and embryos from female patients with MOS variants. The day of oocyte retrieval was defined as day 0. Polar bodies were indicated by black arrows. Scale bar=20 µm.


B . The immunofluorescence images show the pERK1 / 2 ( red ) dynamics in immature ( GV , n = 5 ) , mature ( MII , n = 10 ) , and fertilized oocytes from unidentified control patients ( n = 6 ) , as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants . pERK1 / 2 activation was inhibited after U0126 treatment ( n = 6 ) . FITC - α - tubulin ( Green ) and DAPI ( blue ) were used for co - staining . 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 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. The immunofluorescence images show the pERK1/2 (red) dynamics in immature (GV, n=5), mature (MII, n=10), and fertilized oocytes from unidentified control patients (n=6), as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants. pERK1/2 activation was inhibited after U0126 treatment (n=6). FITC-α-tubulin (Green) and DAPI (blue) were used for co-staining. Scale bar=10 µm.


C . Western blot analysis of the MEK1 / 2 and ERK1 / 2 activation after transfection of FLAG - tagged wild - type MOS and identified MOS variants in HEK 293 cells .

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

C. Western blot analysis of the MEK1/2 and ERK1/2 activation after transfection of FLAG-tagged wild-type MOS and identified MOS variants in HEK 293 cells.


F . Immunofluorescence of FLAG ( green ) and pERK1 / 2 ( grey ) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs . The signal of mCherry was directly captured after fixation , with DAPI staining for DNA visualization ( blue ) . ( n = over 30 oocytes in each group ) . 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

F. Immunofluorescence of FLAG (green) and pERK1/2 (grey) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs. The signal of mCherry was directly captured after fixation, with DAPI staining for DNA visualization (blue). (n=over 30 oocytes in each group). Scale bar=10 µm.


A . Immunofluorescence of FLAG ( red ) and F - actin ( gray ) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild - type human MOS or MOS variants mRNAs and culture in medium with 2 . 5 µM milrinone for 24 h , followed by release to maturation ( n = 15 - 20 oocytes each group ) . 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

A. Immunofluorescence of FLAG (red) and F-actin (gray) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild-type human MOS or MOS variants mRNAs and culture in medium with 2.5 µM milrinone for 24 h, followed by release to maturation (n=15-20 oocytes each group). Scale bar= 10 µm.


C . The immunofluorescence of F - actin ( red ) and TPX2 ( green ) in WT and Erk1 / 2oo - / - oocytes , using DAPI ( blue ) co - staining ( n = 15 - 20 oocytes for each group ) . The zoomed images of F - actin are displayed in gray . Scale bar = 10 µm . D . The box plot summarizing the relative intensities of F - actin between control and ERK1 / 2oo - / - oocytes ( n = 7 - 10 oocytes for each group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The immunofluorescence of F-actin (red) and TPX2 (green) in WT and Erk1/2oo-/- oocytes, using DAPI (blue) co-staining (n=15-20 oocytes for each group). The zoomed images of F-actin are displayed in gray. Scale bar=10 µm. D. The box plot summarizing the relative intensities of F-actin between control and ERK1/2oo-/- oocytes (n=7-10 oocytes for each group).


E . Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1 / 2oo - / - mice . The MII oocyte , zygote , 2 - cell and blastocyst embryos were harvested in vivo at 14 , 24 , 48 , and 96 h post - hCG , respectively ( n = 3 mice for each timepoints in two groups ) . Scale bar = 100 µm . F , G . Bar graphs shows the blastocyst percentage ( F ) and fragment percentage ( G ) in wild - type ( n = 51 ) and maternal ERK1 / 2 deletion embryos ( n = 63 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1/2oo-/- mice. The MII oocyte, zygote, 2-cell and blastocyst embryos were harvested in vivo at 14, 24, 48, and 96 h post-hCG, respectively (n=3 mice for each timepoints in two groups). Scale bar=100 µm. F, G. Bar graphs shows the blastocyst percentage (F) and fragment percentage (G) in wild-type (n=51) and maternal ERK1/2 deletion embryos (n=63).


H . The bar graphs show the expression levels of selected MOS - regulated decay genes ( n = 2 or 3 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

H. The bar graphs show the expression levels of selected MOS-regulated decay genes (n=2 or 3 biological replicates).


H . Heatmap showing the expression level of selected maternal genes , mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1 / 2 inactivation .

O O O O O O 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. Heatmap showing the expression level of selected maternal genes, mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1/2 inactivation.


A . The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO ( control , Ctrl , n = 11 ) or 20 uM U0126 treatment ( n = 12 ) . B . The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO ( control , Ctrl , n = 19 ) or 20 uM U0126 ( n = 17 ) .

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

A. The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO (control, Ctrl, n=11) or 20 uM U0126 treatment (n=12). B. The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO (control, Ctrl, n=19) or 20 uM U0126 (n=17). C


C . The representative images showing the JC - 1 monomers ( green ) and aggregates ( red ) of mouse oocytes after treatment with DMSO ( control , Ctrl , n = 34 ) or U0126 ( n = 40 ) in in vitro culture medium for 16 h . Scale bar = 100 µm . D . The bar graph of ratio of intensities of JC - 1 aggregates to those of monomers in ( C ) . The intensities of JC - 1 were quantified by Image J software . The oocytes number used was labeled .

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

C. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm. D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled. E


E . Representative images displaying JC - 1 monomers ( green ) , aggregates ( red ) and bright filed images of GV and MII oocytes from wild - type ( WT ) and Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big and multiple polar bodies . Scale bar = 100 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

E. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm.


F . Confocal images of JC - 1 monomers ( green ) and aggregates ( red ) of GV and MII oocytes from wild - type ( WT ) and Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big and multiple polar bodies , indicated by red arrows . Hoechst 33342 ( blue ) was co - stained to represent nucleus or chromosome . 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 O O O O

F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm.


A ) HFFs were pre - stimulated with IFNγ ( 10U / mL ) for 24 h . Plaque assays were performed for each strain and each condition . Plaque number and area loss were calculated 4 days p . i . for RH and 6 days p . i . for Pru . Assays were performed with RH ( n = 4 ) , Pru ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HFFs were pre-stimulated with IFNγ (10U/mL) for 24 h. Plaque assays were performed for each strain and each condition. Plaque number and area loss were calculated 4 days p.i. for RH and 6 days p.i. for Pru. Assays were performed with RH (n=4), Pru (n=4).


B ) Relative parasite growth was measured 24 h p . i . in IFNγ - stimulated and unstimulated HFFs by luciferase assay . Growth of each strain in IFNγ - stimulated HFFs is expressed relative to growth in unstimulated HFFs . Experiments were performed with RH ( n = 4 ) , Pru ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative parasite growth was measured 24 h p.i. in IFNγ-stimulated and unstimulated HFFs by luciferase assay. Growth of each strain in IFNγ-stimulated HFFs is expressed relative to growth in unstimulated HFFs. Experiments were performed with RH (n=4), Pru (n=4).


C ) Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U / mL IFNγ for 24 h . Following stimulation , HFFs were infected with either RH ( MOI = 1 ) or Pru ( MOI = 3 ) for another 24 h . Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei . The number of PVs in 5 - 6 fields of the coverslips were counted for each condition and normalized with the number of host cells in each field . Experiments were performed with RH ( n = 3 ) , Pru ( n = 3 ) . Representative images of percentage infected cells for each strain and condition are provided . Scale bar is 100 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

C) Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U/mL IFNγ for 24 h. Following stimulation, HFFs were infected with either RH (MOI=1) or Pru (MOI=3) for another 24 h. Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei. The number of PVs in 5-6 fields of the coverslips were counted for each condition and normalized with the number of host cells in each field. Experiments were performed with RH (n=3), Pru (n=3). Representative images of percentage infected cells for each strain and condition are provided. Scale bar is 100 µm.


D ) Parasites per vacuole were determined 24 h p . i . with similar conditions and staining as described in ( C ) except MOIs used were 0 . 5 and 1 for RH ( n = 3 ) and Pru ( n = 3 ) , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Parasites per vacuole were determined 24 h p.i. with similar conditions and staining as described in (C) except MOIs used were 0.5 and 1 for RH (n=3) and Pru (n=3), respectively.


E ) Plaque assays were performed similarly as described for ( a ) with Pru WT ( n = 20 for plaque number and n = 7 for plaque area ) , PruΔgra15 ( n = 20 for plaque number and n = 7 for plaque area ) , PruΔgra15 + GRA15 complemented ( n = 12 for plaque number and n = 7 for plaque area ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plaque assays were performed similarly as described for (a) with Pru WT (n=20 for plaque number and n=7 for plaque area), PruΔgra15 (n=20 for plaque number and n=7 for plaque area), PruΔgra15+GRA15 complemented (n=12 for plaque number and n=7 for plaque area).


F ) Plaque assays were performed similarly as described for ( A ) with RH ( n = 5 for plaque number and n = 3 for plaque area ) and RH + GRA15II ( n = 5 for plaque number and n = 3 for plaque area ) .

F) Plaque assays were performed similarly as described for (A) with RH (n=5 for plaque number and n=3 for plaque area) and RH+GRA15II (n=5 for plaque number and n=3 for plaque area).


G ) Relative parasite growth was measured as described in ( B ) with Pru WT ( n = 12 ) , PruΔgra15 ( n = 12 ) , PruΔgra15 + GRA15 complemented ( n = 8 ) .

O O O O O O O O O O O O O O O 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) Relative parasite growth was measured as described in (B) with Pru WT (n=12), PruΔgra15 (n=12), PruΔgra15+GRA15 complemented (n=8).


H ) Number of PVs was calculated as described in ( C ) with Pru WT ( n = 3 ) , PruΔgra15 ( n = 3 ) , RH ( n = 3 ) and RH + GRA15II ( n = 3 ) and normalized with the number of host cells in each field .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of PVs was calculated as described in (C) with Pru WT (n=3), PruΔgra15 (n=3), RH (n=3) and RH+GRA15II (n=3) and normalized with the number of host cells in each field.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( A ) Total ubiquitin ( n = 3 for RH , n = 5 for Pru and n = 3 for PruΔgra15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (A) Total ubiquitin (n=3 for RH, n=5 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( B ) K63 - linked ubiquitin ( n = 3 for RH , n = 3 for Pru and n = 3 for PruΔgra15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (B) K63-linked ubiquitin (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( C ) p62 ( n = 3 for RH , n = 10 for Pru and n = 7 for PruΔgra15 , n = 4 for Pru Δgra15 + GRA15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (C) p62 (n=3 for RH, n=10 for Pru and n=7 for PruΔgra15, n=4 for Pru Δgra15+GRA15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( D ) NDP52 ( n = 3 for RH , n = 3 for Pru and n = 3 for PruΔgra15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (D) NDP52 (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( E ) LC3B ( n = 3 for RH , n = 3 for Pru and n = 3 for PruΔgra15 , n = 3 for Pru Δgra15 + GRA15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (E) LC3B (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15, n=3 for Pru Δgra15+GRA15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( F ) GABARAP ( n = 3 for RH , n = 3 for Pru and n = 3 for PruΔgra15 ) is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (F) GABARAP (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15) is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( G ) LAMP1 ( n = 3 for RH , n = 3 for Pru and n = 3 for PruΔgra15 ) is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (G) LAMP1 (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15) is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


H ) The number of PVs per 20X objective field was counted and compared between IFNγ - stimulated and IFNγ + bafilomycin A1 ( 100 nM ) treated HFFs 24 h p . i . with Pru strain . Images from at least 6 fields were taken for each condition ( n = 3 ) .

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

H) The number of PVs per 20X objective field was counted and compared between IFNγ-stimulated and IFNγ + bafilomycin A1 (100 nM) treated HFFs 24 h p.i. with Pru strain. Images from at least 6 fields were taken for each condition (n=3).


A ) HFFs were stimulated with IFNγ ( 10 U / mL for 24 h ) or left unstimulated and subsequently treated with PYR41 ( 1 µM ) for 2 h prior to infection . Cells were washed and subsequently infected with Pru parasites ( MOI = 3 ) for 3 h . The percentage of vacuoles that stained positive for total ubiquitin , p62 or LC3B was determined ( n = 3 ) .

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

A) HFFs were stimulated with IFNγ (10 U/mL for 24 h) or left unstimulated and subsequently treated with PYR41 (1 µM) for 2 h prior to infection. Cells were washed and subsequently infected with Pru parasites (MOI=3) for 3 h. The percentage of vacuoles that stained positive for total ubiquitin, p62 or LC3B was determined (n=3).


For all the co - staining experiments IFNγ - stimulated HFFs infected for 3 h with the Pru strain were used . For each staining at least 50 vacuoles were scored ( n = 3 ) . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain . Scale bar is 20 µm . The yellow box inside each representative image is shown as inset pictures with higher magnification . The total number of coated vacuoles was set at 100 % and the percentage of vacuoles positive for Ub , p62 was calculated .

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

For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, p62 was calculated.


For all the co - staining experiments IFNγ - stimulated HFFs infected for 3 h with the Pru strain were used . For each staining at least 50 vacuoles were scored ( n = 3 ) . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain . Scale bar is 20 µm . The yellow box inside each representative image is shown as inset pictures with higher magnification . The total number of coated vacuoles was set at 100 % and the percentage of vacuoles positive for Ub , LC3B was calculated .

For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, LC3B was calculated.


For all the co - staining experiments IFNγ - stimulated HFFs infected for 3 h with the Pru strain were used . For each staining at least 50 vacuoles were scored ( n = 3 ) . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain . Scale bar is 20 µm . The yellow box inside each representative image is shown as inset pictures with higher magnification . The total number of coated vacuoles was set at 100 % and the percentage of vacuoles positive for Ub , GABARAP was calculated .

For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, GABARAP was calculated.


For all the co - staining experiments IFNγ - stimulated HFFs infected for 3 h with the Pru strain were used . For each staining at least 50 vacuoles were scored ( n = 3 ) . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain . Scale bar is 20 µm . The yellow box inside each representative image is shown as inset pictures with higher magnification . The total number of coated vacuoles was set at 100 % and the percentage of vacuoles positive for p62 LC3B was calculated .

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

For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for p62 LC3B was calculated.


For all the co - staining experiments IFNγ - stimulated HFFs infected for 3 h with the Pru strain were used . For each staining at least 50 vacuoles were scored ( n = 3 ) . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain . Scale bar is 20 µm . The yellow box inside each representative image is shown as inset pictures with higher magnification . The total number of coated vacuoles was set at 100 % and the percentage of vacuoles positive for p62 GABARAP was calculated .

For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for p62 GABARAP was calculated.


A ) HFFs were stimulated with IFNγ for 24 h ( 10 U / ml ) or left unstimulated . The NF - κB inhibitor BAY11 - 7082 ( 1µM ) was added 2 h pre - infection and HFFs were subsequently infected with Pru parasites . Parasite growth ( using luciferase assay ) was measured 24 h p . i . Means from unstimulated cells were set at 100 % . Experiments were performed three times .

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

A) HFFs were stimulated with IFNγ for 24 h (10 U/ml) or left unstimulated. The NF-κB inhibitor BAY11-7082 (1µM) was added 2 h pre-infection and HFFs were subsequently infected with Pru parasites. Parasite growth (using luciferase assay) was measured 24 h p.i. Means from unstimulated cells were set at 100%. Experiments were performed three times.


B ) Nuclear translocation of the NF - κB p65 subunit was quantified in HFFs or HFFs treated with BAY11 - 7082 ( 1µM , added 2 h pre - infection ) 24 h p . i . with Pru parasites . Experiments were done 4 times where each dot represents one experimental mean of at least 15 nuclei . In the right panel , representative images are shown . Parasites were expressing GFP , nuclei are stained with Hoechst 33258 . Scale bar is 20 µm .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nuclear translocation of the NF-κB p65 subunit was quantified in HFFs or HFFs treated with BAY11-7082 (1µM, added 2 h pre-infection) 24 h p.i. with Pru parasites. Experiments were done 4 times where each dot represents one experimental mean of at least 15 nuclei. In the right panel, representative images are shown. Parasites were expressing GFP, nuclei are stained with Hoechst 33258. Scale bar is 20 µm.


C ) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ ( 10 U / ml ) using an RH strain expressing type II GRA15 - HA and as a control RH expressing GRA35 - HA . The blots using antibodies against TRAF2 and TRAF6 were made after stripping the first blot . The inputs loaded represent 1 % of total lysate prepared for immunoblotting and mass spec ( Table EV1 ) . The antibodies against TRAF2 and TRAF6 were obtained from Santa Cruz Biotechnology ( Appendix Table S2 ) . Full length blots for this figure can be observed in the source data for this figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ (10 U/ml) using an RH strain expressing type II GRA15-HA and as a control RH expressing GRA35-HA. The blots using antibodies against TRAF2 and TRAF6 were made after stripping the first blot. The inputs loaded represent 1% of total lysate prepared for immunoblotting and mass spec (Table EV1). The antibodies against TRAF2 and TRAF6 were obtained from Santa Cruz Biotechnology (Appendix Table S2). Full length blots for this figure can be observed in the source data for this figure.


D ) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ ( 10 U / ml ) using an RH strain expressing type II GRA15 - HA and as a control RH expressing GRA43 - HA . The blots using antibodies against TRAF6 were made after stripping the first blot that is used for HA blotting . The inputs loaded represent 10 % of total lysate prepared for immunoblotting . Following TRAF6 blotting , the membrane was stripped and blotted for TNFAIP3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ (10 U/ml) using an RH strain expressing type II GRA15-HA and as a control RH expressing GRA43-HA. The blots using antibodies against TRAF6 were made after stripping the first blot that is used for HA blotting. The inputs loaded represent 10% of total lysate prepared for immunoblotting. Following TRAF6 blotting, the membrane was stripped and blotted for TNFAIP3.


E ) Immunofluorescence analysis of TRAF6 recruitment to the PVM of HFFs infected for 3 h with RH , Pru and PruΔgra15 strains . On the right - hand side , a representative fluorescent image is shown of TRAF6 recruitment to the PVM where Toxoplasma Pru strain expresses GFP , DNA was stained with Hoechst 33258 . Scale bar is 10 µm . n = 3 for all the strains . The antibody against TRAF6 was purchased from Abnova ( Appendix Table S2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of TRAF6 recruitment to the PVM of HFFs infected for 3 h with RH, Pru and PruΔgra15 strains. On the right-hand side, a representative fluorescent image is shown of TRAF6 recruitment to the PVM where Toxoplasma Pru strain expresses GFP, DNA was stained with Hoechst 33258. Scale bar is 10 µm. n=3 for all the strains. The antibody against TRAF6 was purchased from Abnova (Appendix Table S2).


F ) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ ( 10 U / ml ) using a RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut . For the unstimulated cells , uninfected HFFs were used an additional negative control ( left panel ) . Left panel and right panel were run on a single gel ; vertical white lines indicate excision of irrelevant lanes . Full length blots are in the source data for this figure . The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova , respectively . The asterisks ( * ) in the lower right panel indicates the faint band of TRAF6 in the RH + GRA15TRAF2mut immunoprecipitate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ (10 U/ml) using a RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut. For the unstimulated cells, uninfected HFFs were used an additional negative control (left panel). Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes. Full length blots are in the source data for this figure. The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova, respectively. The asterisks (*) in the lower right panel indicates the faint band of TRAF6 in the RH+GRA15TRAF2mut immunoprecipitate.


Immunofluorescence analysis of p62 , with and without IFNγ in RH , RH + GRA15WT . All the experiments were done 3 times with each of the strains .

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

Immunofluorescence analysis of p62, with and without IFNγ in RH, RH+GRA15WT. All the experiments were done 3 times with each of the strains.


Immunofluorescence analysis of LC3B with and without IFNγ in RH , RH + GRA15WT . All the experiments were done 3 times with each of the strains .

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

Immunofluorescence analysis of LC3B with and without IFNγ in RH, RH+GRA15WT. All the experiments were done 3 times with each of the strains.


Immunofluorescence analysis of LAMP1 with and without IFNγ in RH , RH + GRA15WT . All the experiments were done 3 times with each of the strains .

Immunofluorescence analysis of LAMP1 with and without IFNγ in RH, RH+GRA15WT. All the experiments were done 3 times with each of the strains.


J ) Plaque assays were performed with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) .

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J) Plaque assays were performed with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).


K ) Expression of TRAF6 was detected by Western blotting of lysates from scrambled siRNA transfected and TRAF6 - specific siRNA transfected HFFs with and without IFNγ .

K) Expression of TRAF6 was detected by Western blotting of lysates from scrambled siRNA transfected and TRAF6-specific siRNA transfected HFFs with and without IFNγ.


L ) Relative parasite growth was measured in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs using luciferase - based assay with indicated strains with and without IFNγ ( n = 3 ) . The antibody against TRAF6 used here was from Abcam ( Appendix Table S2 ) . Full length blots are in supplementary figures 4l - m .

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L) Relative parasite growth was measured in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs using luciferase-based assay with indicated strains with and without IFNγ (n=3). The antibody against TRAF6 used here was from Abcam (Appendix Table S2). Full length blots are in supplementary figures 4l-m.


Immunofluorescence analysis of p62 was done in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs with and without IFNγ using indicated strains ( n = 3 ) .

Immunofluorescence analysis of p62 was done in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs with and without IFNγ using indicated strains (n=3).


Immunofluorescence analysis of LC3B was done in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs with and without IFNγ using indicated strains ( n = 3 ) .

Immunofluorescence analysis of LC3B was done in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs with and without IFNγ using indicated strains (n=3).


MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( A ) IRGB6 For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times .

MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (A) IRGB6 For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.


MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( B ) GBPs , For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times .

MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (B) GBPs, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.


MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( C ) total ubiquitin , For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times .

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MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (C) total ubiquitin, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.


MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( D ) K63 - linked ubiquitin , For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times .

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MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (D) K63-linked ubiquitin, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.


MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( E ) K48 - linked ubiquitin For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times .

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MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (E) K48-linked ubiquitin For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.


MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( F ) LC3B . For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times .

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MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (F) LC3B. For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.


G ) MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) or left unstimulated and subsequently infected with Pru , PruΔgra15 and GRA15 complemented parasites for 24 h for measuring parasite growth by luciferase assay . All experiments were performed 3 independent times .

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G) MEFs were stimulated with IFNγ for 24 h (100 U/ml) or left unstimulated and subsequently infected with Pru, PruΔgra15 and GRA15 complemented parasites for 24 h for measuring parasite growth by luciferase assay. All experiments were performed 3 independent times.


H ) Plaque numbers were measured for Pru ( n = 12 ) , PruΔgra15 ( n = 12 ) and GRA15 complemented parasites ( n = 6 ) .

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H) Plaque numbers were measured for Pru (n=12), PruΔgra15 (n=12) and GRA15 complemented parasites (n=6).


I - J ) MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) or left unstimulated and subsequently infected with RH or RH expressing type II GRA15 . Plaque numbers were counted ( n = 3 ) , and areas were measured 4 days p . i . ( n = 3 ) .

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I-J) MEFs were stimulated with IFNγ for 24 h (100 U/ml) or left unstimulated and subsequently infected with RH or RH expressing type II GRA15. Plaque numbers were counted (n=3), and areas were measured 4 days p.i. (n=3).


A ) Immunoprecipitations and Western blots were performed on MEFs stimulated or not with IFNγ ( 100 U / ml ) and infected with an RH strain expressing type II GRA15 - HA and as a control RH expressing GRA45 - HA . The blots using antibodies against TRAF6 and TRAF2 were made after stripping the first blot . Left panel and right panel were run on a single gel ; vertical white lines indicate excision of irrelevant lanes .

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A) Immunoprecipitations and Western blots were performed on MEFs stimulated or not with IFNγ (100 U/ml) and infected with an RH strain expressing type II GRA15-HA and as a control RH expressing GRA45-HA. The blots using antibodies against TRAF6 and TRAF2 were made after stripping the first blot. Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes.


B ) IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 and RH for 3 h and subsequently stained for TRAF6 . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . Experiments were performed 3 times .

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B) IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 and RH for 3 h and subsequently stained for TRAF6. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. Experiments were performed 3 times.


C ) IRGB6 staining on PVM after infection of wild - type or Traf6 - / - MEFs for 3 h with Pru , PruΔgra15 and RH . On the right - hand side , a representative fluorescent image is shown of IRGB6 coating on the PVM of both wild - type and Traf6 - / - MEF . Scale bar is 10 µm . At least 100 different vacuoles were observed and analyzed for each experiment ( n = 3 ) .

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C) IRGB6 staining on PVM after infection of wild-type or Traf6 -/- MEFs for 3 h with Pru, PruΔgra15 and RH. On the right-hand side, a representative fluorescent image is shown of IRGB6 coating on the PVM of both wild-type and Traf6 -/- MEF. Scale bar is 10 µm. At least 100 different vacuoles were observed and analyzed for each experiment (n=3).


D ) Parasite growth was measured 24 h p . i . using luciferase readout from unstimulated and IFNγ - stimulated wild - type , NFκB p65 - / - and Traf6 - / - MEFs . Growth was compared between Pru , PruΔgra15 and PruΔgra15 + GRA15 ( complemented ) strains . Reading from unstimulated cells was considered as 100 % and percentage growth in IFNγ - stimulated cells was expressed relative to unstimulated cells . Experiments were performed three times with each MEF type .

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D) Parasite growth was measured 24 h p.i. using luciferase readout from unstimulated and IFNγ-stimulated wild-type, NFκB p65-/- and Traf6 -/- MEFs. Growth was compared between Pru, PruΔgra15 and PruΔgra15+GRA15 (complemented) strains. Reading from unstimulated cells was considered as 100% and percentage growth in IFNγ-stimulated cells was expressed relative to unstimulated cells. Experiments were performed three times with each MEF type.


E ) Immunoprecipitation and Western blot were performed in MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut and as a control RH expressing GRA45 - HA . Left panel and right panel were run on a single gel ; vertical white lines indicate excision of irrelevant lanes . Full length blots are in the source data for this figure . The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova , respectively .

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E) Immunoprecipitation and Western blot were performed in MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut and as a control RH expressing GRA45-HA. Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes. Full length blots are in the source data for this figure. The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova, respectively.


Immunofluorescence analysis of TRAF6 , was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) .

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Immunofluorescence analysis of TRAF6, was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).


Immunofluorescence analysis of IRGB6 , was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) .

Immunofluorescence analysis of IRGB6, was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).


Immunofluorescence analysis of GBP1 - 5 , was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) .

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Immunofluorescence analysis of GBP1-5, was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).


Immunofluorescence analysis of p62 was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) .

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Immunofluorescence analysis of p62 was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).


Immunofluorescence analysis of ubiquitin was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) .

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Immunofluorescence analysis of ubiquitin was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).


K ) Plaque assays were performed with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 4 ) .

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K) Plaque assays were performed with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=4).


A Representative clinical picture of two patients with CAOP syndrome ( 14 - year - old patient 1 and 5 - year - old patient 2 ) .

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A Representative clinical picture of two patients with CAOP syndrome (14-year-old patient 1 and 5-year-old patient 2).


B Haematoxylin and eosin ( H & E ) staining of skin biopsies from patient 1 and healthy controls . Scale bars : 200 µm .

B Haematoxylin and eosin (H&E) staining of skin biopsies from patient 1 and healthy controls. Scale bars: 200 µm.


A , B Representative transmission electron microscopy ( TEM ) images showing keratinocyte mitochondria ( red arrow ) in patient 1 ( A ) and in the Mbtps1 - conditional knockout ( cKO ) mouse model ( B ) . Scale bars : 2 µm .

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A, B Representative transmission electron microscopy (TEM) images showing keratinocyte mitochondria (red arrow) in patient 1 (A) and in the Mbtps1-conditional knockout (cKO) mouse model (B). Scale bars: 2 µm.


C , D Quantification of the mitochondrial number and morphology in patient 1 ( C ) and the Mbtps1 - cKO mouse model ( D ) . ( C ) Left panel : n = 13 biological replicates of normal individuals , n = 10 biological replicates of patient 1 . Middle and right panel : n = 121 biological samples of normal individuals and patient 1 . ( D ) Left panel : n = 23 biological replicates of Mbtps1 - loxP mice ; n = 15 biological replicates of Mbtps1 - cKO mice . Middle panel and right panel : n = 113 biological replicates of Mbtps1 - loxP mice and Mbtps1 - cKO .

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C, D Quantification of the mitochondrial number and morphology in patient 1 (C) and the Mbtps1-cKO mouse model (D). (C) Left panel: n = 13 biological replicates of normal individuals, n = 10 biological replicates of patient 1. Middle and right panel: n = 121 biological samples of normal individuals and patient 1. (D) Left panel: n = 23 biological replicates of Mbtps1-loxP mice; n = 15 biological replicates of Mbtps1-cKO mice. Middle panel and right panel: n =113 biological replicates of Mbtps1-loxP mice and Mbtps1-cKO.


E Immunofluorescence experiments showed that mutant S1P ( p . Val355Gly and p . Ter1053Arg ) was diffusely localized in the cytosol and showed lower mitochondrial localization compared with wild - type S1P , which suggested that these variants disrupt its mitochondrial import . The white arrowheads indicate the colocation ( yellow ) of S1P ( green ) and the mitochondrial tracer ( red ) . Scale bars : 20 μm ( panels 1 - 4 ) ; scale bars : 2 μm ( panel 5 ) .

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E Immunofluorescence experiments showed that mutant S1P (p.Val355Gly and p.Ter1053Arg) was diffusely localized in the cytosol and showed lower mitochondrial localization compared with wild-type S1P, which suggested that these variants disrupt its mitochondrial import. The white arrowheads indicate the colocation (yellow) of S1P (green) and the mitochondrial tracer (red). Scale bars: 20 μm (panels 1-4); scale bars: 2 μm (panel 5).


F Cellular component separation assay showing that S1P was enriched in mitochondria . Biochemical fractionation of the whole cell lysate ( WCL ) , cytosol ( Cyto , tubulin ) , mitochondria ( Mito , COX IV ) , lysosomes ( Lyso , LAMP1 ) , Golgi apparatus ( Golgi , GM130 ) , endoplasmic reticulum ( ER , ERp72 ) , and nucleus ( Nue , Lamin B ) from HaCaT cells .

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F Cellular component separation assay showing that S1P was enriched in mitochondria. Biochemical fractionation of the whole cell lysate (WCL), cytosol (Cyto, tubulin), mitochondria (Mito, COX IV), lysosomes (Lyso, LAMP1), Golgi apparatus (Golgi, GM130), endoplasmic reticulum (ER, ERp72), and nucleus (Nue, Lamin B) from HaCaT cells.


G A component separation assay revealed a lower expression of mutant S1P ( p . Val355Gly and p . Ter1053Arg ) than wild - type S1P in mitochondria .

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G A component separation assay revealed a lower expression of mutant S1P (p.Val355Gly and p.Ter1053Arg) than wild-type S1P in mitochondria.


H The impaired binding of mutant S1P ( p . Val355Gly and p . Ter1053Arg ) to translocase of the outer membrane ( TOM ) 70 and translocase of the inner membrane ( TIM ) 23 was detected by a coimmunoprecipitation ( Co - IP ) assay .

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H The impaired binding of mutant S1P (p.Val355Gly and p.Ter1053Arg) to translocase of the outer membrane (TOM) 70 and translocase of the inner membrane (TIM) 23 was detected by a coimmunoprecipitation (Co-IP) assay.


A LC - MS / MS analysis of S1P - binding proteins . Total protein lysate was subjected to coimmunoprecipitation ( Co - IP ) with normal IgG or S1P antibody . The purified protein complex was separated by SDS - PAGE and then subjected to silver staining . The arrows indicate the bands containing S1P , ETFA and ETFB . The differential bands were then analysed by liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) .

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A LC-MS/MS analysis of S1P-binding proteins. Total protein lysate was subjected to coimmunoprecipitation (Co-IP) with normal IgG or S1P antibody. The purified protein complex was separated by SDS-PAGE and then subjected to silver staining. The arrows indicate the bands containing S1P, ETFA and ETFB. The differential bands were then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).


C S1P interacts with endogenous ETFA and ETFB in HaCaT cells . Total protein lysate was subjected to Co - IP with normal IgG , ETFA ( left panel ) or ETFB ( right panel ) antibody . The interaction between S1P and ETFA / ETFB was then detected by immunoblotting .

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C S1P interacts with endogenous ETFA and ETFB in HaCaT cells. Total protein lysate was subjected to Co-IP with normal IgG, ETFA (left panel) or ETFB (right panel) antibody. The interaction between S1P and ETFA/ETFB was then detected by immunoblotting.


D The direct interaction between S1P and ETFA - ETFB was validated by a GST pull - down assay . Left panel : in vitro - translated S1P was pulled down by purified GST - ETFA fusion protein . Right panel : in vitro - translated S1P was pulled down by purified GST - ETFB fusion protein .

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D The direct interaction between S1P and ETFA-ETFB was validated by a GST pull-down assay. Left panel: in vitro-translated S1P was pulled down by purified GST-ETFA fusion protein. Right panel: in vitro-translated S1P was pulled down by purified GST-ETFB fusion protein.


A Representative immunohistochemical images of S1P , ETFA and ETFB in normal controls and patient 1 . Scale bars : 100 μm .

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A Representative immunohistochemical images of S1P, ETFA and ETFB in normal controls and patient 1. Scale bars: 100 μm.


B Unchanged mRNA levels and decreased protein levels of ETFA and ETFB were observed in the skin biopsy of patient 1 . Quantitative RT - PCR analysis ( upper panel , n = 6 biological replicates of normal controls ; n = 6 technical replicates of patient 1 ) and immunohistochemistry ( lower panel , n = 6 biological replicates ; n = 3 technical replicates ) of the S1P , ETFA and ETFB levels .

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B Unchanged mRNA levels and decreased protein levels of ETFA and ETFB were observed in the skin biopsy of patient 1. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates of normal controls; n = 6 technical replicates of patient 1) and immunohistochemistry (lower panel, n = 6 biological replicates; n = 3 technical replicates) of the S1P, ETFA and ETFB levels.


C Representative immunohistochemistry images of S1P , ETFA and ETFB in both Mbtps1 - conditional knockout ( cKO ) and Mbtps1 - loxP mice . Scale bars : 100 μm .

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C Representative immunohistochemistry images of S1P, ETFA and ETFB in both Mbtps1-conditional knockout (cKO) and Mbtps1-loxP mice. Scale bars: 100 μm.


D MBTPS1 gene knockout led to decreases in the ETFA and ETFB protein levels in vivo . Quantitative RT - PCR analysis ( upper panel , n = 6 biological replicates ) and immunohistochemistry ( lower panel , n = 6 biological replicates ) of the S1P , ETFA and ETFB levels in Mbtps1 - cKO mice and Mbtps1 - loxP mice .

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D MBTPS1 gene knockout led to decreases in the ETFA and ETFB protein levels in vivo. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates) and immunohistochemistry (lower panel, n = 6 biological replicates) of the S1P, ETFA and ETFB levels in Mbtps1-cKO mice and Mbtps1-loxP mice.


E Cycloheximide ( CHX ) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells . Left panel : Representative western blotting images of the ETFA and ETFB protein levels during CHX chase . Right panel : Quantification of the immunoblotting results corresponding to the left panel ( n = 3 biological replicates ) .

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E Cycloheximide (CHX) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells. Left panel: Representative western blotting images of the ETFA and ETFB protein levels during CHX chase. Right panel: Quantification of the immunoblotting results corresponding to the left panel (n = 3 biological replicates).


F Mutant S1P ( p . Val355Gly and p . Ter1053Arg ) only weakly interacted with ETFA and ETFB . We constructed Flag - tagged wild - type and mutant S1P HaCaT cell lines and then performed a Co - IP assay with Flag antibody , and the interaction between S1P and ETFA / ETFB was detected by immunoblotting .

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F Mutant S1P (p.Val355Gly and p.Ter1053Arg) only weakly interacted with ETFA and ETFB. We constructed Flag-tagged wild-type and mutant S1P HaCaT cell lines and then performed a Co-IP assay with Flag antibody, and the interaction between S1P and ETFA/ETFB was detected by immunoblotting.


G Wild - type S1P , but not mutant S1P ( p . Val355Gly and p . Ter1053Arg ) , decreased the association between ETFA and ETFB . We performed a Co - IP assay with an ETFA antibody , and the interaction between ETFA and ETFB was detected by immunoblotting .

G Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), decreased the association between ETFA and ETFB. We performed a Co-IP assay with an ETFA antibody, and the interaction between ETFA and ETFB was detected by immunoblotting.


H Wild - type S1P , but not mutant S1P ( p . Val355Gly and p . Ter1053Arg ) , enhanced the incorporation of FAD into the ETF complex . The visible spectra of flavin show two shoulder peaks at 420 nm and 460 nm , indicating the incorporation of FAD in the ETF complex . In the presence of the wild - type S1P protein , the two peaks were shifted by 0 . 02 OD units , whereas the additional mutant S1P ( p . Val355Gly and p . Ter1053Arg ) weakly shifted the two peaks by 0 . 005 - 0 . 01 OD units .

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H Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), enhanced the incorporation of FAD into the ETF complex. The visible spectra of flavin show two shoulder peaks at 420 nm and 460 nm, indicating the incorporation of FAD in the ETF complex. In the presence of the wild-type S1P protein, the two peaks were shifted by 0.02 OD units, whereas the additional mutant S1P (p.Val355Gly and p.Ter1053Arg) weakly shifted the two peaks by 0.005-0.01 OD units.


Mitochondrial respiration ( OCR ) in control ( Ctrl ) and MBTPS1 - knockout ( KO ) HaCaT cells was quantified in real time using a Seahorse extracellular flux analyser . Right subpanels : Quantiﬁcation of basal respiration , maximal respiration , and OCR - coupled ATP production in mitochondrial respiration ( n = 5 biological replicates ) .

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Mitochondrial respiration (OCR) in control (Ctrl) and MBTPS1-knockout (KO) HaCaT cells was quantified in real time using a Seahorse extracellular flux analyser. Right subpanels: Quantiﬁcation of basal respiration, maximal respiration, and OCR-coupled ATP production in mitochondrial respiration (n = 5 biological replicates).


B Quantiﬁcation of relevant metabolites in mitochondrial oxidative phosphorylation ( OXPHOS ) and glycolysis in Ctrl and MBTPS1 - KO HaCaT cells ( n = 4 biological replicates ) .

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B Quantiﬁcation of relevant metabolites in mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis in Ctrl and MBTPS1-KO HaCaT cells (n = 4 biological replicates).


C Nonmitochondrial respiration ( ECAR ) was quantified in real time using a Seahorse extracellular flux analyser . Quantification of non - glycolytic acidification , glycolysis , glycolytic capacity , and glycolytic reserve in Ctrl and MBTPS1 - KO HaCaT cells ( n = 4 biological replicates ) .

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C Nonmitochondrial respiration (ECAR) was quantified in real time using a Seahorse extracellular flux analyser. Quantification of non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve in Ctrl and MBTPS1-KO HaCaT cells (n = 4 biological replicates).


D MBTPS1 knockout led to a decrease in global ATP production in HaCaT cells . Control and MBTPS1 - KO HaCaT cells were cultured in 6 - well dishes . A standard curve was generated to calculate the sample ATP concentrations using an ATP Lite Luminescence Assay Kit ( n = 4 biological replicates ) .

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D MBTPS1 knockout led to a decrease in global ATP production in HaCaT cells. Control and MBTPS1-KO HaCaT cells were cultured in 6-well dishes. A standard curve was generated to calculate the sample ATP concentrations using an ATP Lite Luminescence Assay Kit (n = 4 biological replicates).


E MBTPS1 knockout significantly increased the generation of mitochondrial reactive oxygen species ( Mito SOX ) in HaCaT cells . Left panel : Representative immunofluorescence images of Mito SOX in HaCaT cells . Scale bars : 100 µm . Right panel : The immunofluorescence intensity was quantified to calculate the relative Mito ROS level in HaCaT cells ( n = 4 biological replicates ) .

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E MBTPS1 knockout significantly increased the generation of mitochondrial reactive oxygen species (Mito SOX) in HaCaT cells. Left panel: Representative immunofluorescence images of Mito SOX in HaCaT cells. Scale bars: 100 µm. Right panel: The immunofluorescence intensity was quantified to calculate the relative Mito ROS level in HaCaT cells (n = 4 biological replicates).


A The S1P deficiency - induced decrease in ETFA and ETFB was significantly reversed by riboflavin ( Rib ) in a concentration - dependent manner in MBTPS1 - KO HaCaT cells . GAPDH was used as a loading control . MBTPS1 - KO cells were initially cultured in DMEM for 24 h and supplemented with 0 , 2 . 5 , 5 , or 10 µM riboflavin for 3 days . The expression of ETFA and ETFB was then detected by immunoblotting .

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A The S1P deficiency-induced decrease in ETFA and ETFB was significantly reversed by riboflavin (Rib) in a concentration-dependent manner in MBTPS1-KO HaCaT cells. GAPDH was used as a loading control. MBTPS1-KO cells were initially cultured in DMEM for 24 h and supplemented with 0, 2.5, 5, or 10 µM riboflavin for 3 days. The expression of ETFA and ETFB was then detected by immunoblotting.


B The S1P deficiency - induced abnormalities in mitochondrial respiration can be significantly reversed by riboflavin supplementation and ETFA / B overexpression in HaCaT cells . Left panel : Mitochondrial respiration ( OCR ) was quantified in real time using a Seahorse extracellular flux analyser .

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B The S1P deficiency-induced abnormalities in mitochondrial respiration can be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells. Left panel: Mitochondrial respiration (OCR) was quantified in real time using a Seahorse extracellular flux analyser.


C The decreased global ATP production caused by S1P deficiency could be significantly reversed by riboflavin supplementation and ETFA / B overexpression in HaCaT cells ( n = 4 biological replicates ) .

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C The decreased global ATP production caused by S1P deficiency could be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells (n = 4 biological replicates).


D Inflammatory lesions in CAOP syndrome were significantly improved by riboflavin supplementation . The representative head image of patient 1 before therapy is shown in Figure 1A ( panel 1 ) .

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D Inflammatory lesions in CAOP syndrome were significantly improved by riboflavin supplementation. The representative head image of patient 1 before therapy is shown in Figure 1A (panel 1).


B Representative images of Drosophila adult wings that resulted from larvae wing imaginal discs expressing a control RNAi ( mCherry RNAi ) , a lds RNAi , or co - expressing san RNAi with control RNAi ( san RNAi control RNAi ) or with lds RNAi ( san RNAi lds RNAi ) .

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B Representative images of Drosophila adult wings that resulted from larvae wing imaginal discs expressing a control RNAi (mCherry RNAi), a lds RNAi, or co-expressing san RNAi with control RNAi (san RNAi control RNAi) or with lds RNAi (san RNAi lds RNAi).


A Representative images of cells in the wing disc pouch upon nub - Gal4 - mediated RNAi for lds ( bottom ) compared to a nub - Gal4 control ( top ) . Cells also express HisH2AvD - mRFP1 ( magenta ) and centromeric marker cid - EGFP ( green ) ; Times ( min : sec ) are relative to NEBD ; scale bars 5 µm and applies to all images . B Mitotic timing defined from NEBD to anaphase onset ; each dot represents a different cell ( n = 77 cells from at least 6 independent movies per condition ) ; black lines represent mean ; statistical analysis was performed using the non - parametric Mann - Whitney test . Data information : Scale bars are 10 µm and apply to all images .

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A Representative images of cells in the wing disc pouch upon nub-Gal4-mediated RNAi for lds (bottom) compared to a nub-Gal4 control (top). Cells also express HisH2AvD-mRFP1 (magenta) and centromeric marker cid-EGFP (green); Times (min:sec) are relative to NEBD; scale bars 5 µm and applies to all images. B Mitotic timing defined from NEBD to anaphase onset; each dot represents a different cell (n=77 cells from at least 6 independent movies per condition); black lines represent mean; statistical analysis was performed using the non-parametric Mann-Whitney test. Data information: Scale bars are 10 µm and apply to all images.


E Localisation of Lds - EGFP ( green ) throughout mitosis in Drosophila syncytial embryos ( E ) . Times are relative to NEBD , flies also express HisH2AvDmRFP1 ( magenta ) . Data information : Scale bars are 10 µm and apply to all images .

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E Localisation of Lds-EGFP (green) throughout mitosis in Drosophila syncytial embryos (E). Times are relative to NEBD, flies also express HisH2AvDmRFP1 (magenta). Data information: Scale bars are 10 µm and apply to all images.


A Analysis of ongoing transcription monitored by MCP - EGFP labelled nascent transcripts on a reporter containing MS2 loops ( green ) , in the referred experimental conditions . Flies also express H2AvD - mRFP1 ( magenta ) . Scale bar is 10 µm and applies to all images .

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A Analysis of ongoing transcription monitored by MCP-EGFP labelled nascent transcripts on a reporter containing MS2 loops (green), in the referred experimental conditions. Flies also express H2AvD-mRFP1 (magenta). Scale bar is 10 µm and applies to all images.


B Segregation defects upon inhibition of transcription in luciferase and lds RNAi . Graph depicts mean ± s . e . m . of the frequencies of defects scored in 11 ( luciferase RNAi , H2O and lds RNAi , α - amanitin ) or 8 ( lds RNAi , H2O and Luc RNAi , α - amanitin ) independent embryos ; a total of ~ 800 nuclei were scored per condition .

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B Segregation defects upon inhibition of transcription in luciferase and lds RNAi. Graph depicts mean ± s.e.m. of the frequencies of defects scored in 11 (luciferase RNAi, H2O and lds RNAi, α-amanitin) or 8 (lds RNAi, H2O and Luc RNAi, α-amanitin) independent embryos; a total of ~800 nuclei were scored per condition.


A Wing morphology phenotype obtained upon depletion of Lds combined with control co - depletion ( luciferase RNAi ) or expression / depletion of chromosome architecture modulators . Wing morphology defects were classified according to their severity ( 1 = normal wings and 5 = no wing ) ; depicted numbers indicate the average morphology grade and the number of flies counted , from at least 2 independent experiments .

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A Wing morphology phenotype obtained upon depletion of Lds combined with control co-depletion (luciferase RNAi) or expression/depletion of chromosome architecture modulators. Wing morphology defects were classified according to their severity (1= normal wings and 5= no wing); depicted numbers indicate the average morphology grade and the number of flies counted, from at least 2 independent experiments.


B Live cell imaging analysis of the kinetics of Rad21 - EGFP ( green ) removal from mitotic chromosomes in luciferase - and lds - depleted embryos . DNA is marked with H2AvD - mRFP1 ( magenta ) ; times are relative to NEBD ; scale bar is 10 µm and applies to all images .

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B Live cell imaging analysis of the kinetics of Rad21-EGFP (green) removal from mitotic chromosomes in luciferase- and lds-depleted embryos. DNA is marked with H2AvD-mRFP1 (magenta); times are relative to NEBD; scale bar is 10 µm and applies to all images.


( A , Chromatin localisation of chromosome assembly factors ( Top2 ( A ) in green ) upon RNAi for luciferase ( left ) or lds ( right ) in early embryos . DNA is marked with H2AvD - mRFP1 ( magenta ) ; scale bars are 10 µm and apply to all images .

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(A, Chromatin localisation of chromosome assembly factors (Top2 (A) in green) upon RNAi for luciferase (left) or lds (right) in early embryos. DNA is marked with H2AvD-mRFP1 (magenta); scale bars are 10 µm and apply to all images.


C ) Chromatin localisation of Condensin I subunit Barren ( C ) , in green ) upon RNAi for luciferase ( left ) or lds ( right ) in early embryos . DNA is marked with H2AvD - mRFP1 ( magenta ) ; scale bars are 10 µm and apply to all images .

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C) Chromatin localisation of Condensin I subunit Barren (C), in green) upon RNAi for luciferase (left) or lds (right) in early embryos. DNA is marked with H2AvD-mRFP1 (magenta); scale bars are 10 µm and apply to all images.


G Telophase images of embryos depleted for luciferase ( left ) , Cap - D2 ( middle ) and wapl ( right ) RNAi , and expressing Lds - EGFP ( green ) and HisH2AvDmRFP1 ( magenta ) . Bottom panels depict Lds - EGFP alone . Scale bar is 10 µm and applies to all images .

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G Telophase images of embryos depleted for luciferase (left), Cap-D2 (middle) and wapl (right) RNAi, and expressing Lds-EGFP (green) and HisH2AvDmRFP1 (magenta). Bottom panels depict Lds-EGFP alone. Scale bar is 10 µm and applies to all images.