( B ) High - magnification view of single mitotic stained for p - MLC ( purple ) E - cadherin ( green ) , with metaphase plate chromosomes stained by DAPI ( blue ) . Scale bar ~ 1µm . n > 10 independent biological replicates . ( C ) High - magnification view of single UAS . Rok - RNAi expressing mitotic stained for p - MLC ( purple ) E - cadherin ( green ) , with metaphase plate chromosomes stained by DAPI ( blue ) . Note loss of cortical p - MLC ( purple ) . Scale bar ~ 1µm . n > 10 independent biological replicates .

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(B) High-magnification view of single mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Scale bar ~1µm. n>10 independent biological replicates. (C) High-magnification view of single UAS.Rok-RNAi expressing mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Note loss of cortical p-MLC staining (purple). Scale bar ~1µm. n>10 independent biological replicates.


( D ) Expression of constitutively active Rho - kinase ( UAS . Rok - CA ) in posterior compartment of wing epithelium is sufficient to elevate p - MLC reduce E - cadherin immunostaining . Zoom ( right ) shows high p - MLC low E - cad in both a mitotic its neighbours . Scale bar ~ 10µm . n > 10 independent biological replicates .

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(D) Expression of constitutively active Rho-kinase (UAS.Rok-CA) in the posterior compartment of the wing epithelium is sufficient to elevate p-MLC and reduce E-cadherin immunostaining. Zoom (right) shows high p-MLC and low E-cad in both a mitotic cell and its neighbours. Scale bar ~10µm. n>10 independent biological replicates.


( E ) MyoII RLC - GFP ( MyoII - GFP ) accumulates at cortex as round up for mitosis later appears at cleavage furrow during cytokinesis ( basal section , diagrammed left ) . Treatment with Rho - kinase inhibitor Y - 27632 inhibits mitotic accumulation of MyoII - GFP . shown right ( n = 8 independent samples per genotype ) . Scale bar ~ 1µm . Mean + / - 1 S . D . shown .

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(E) MyoII RLC-GFP (MyoII-GFP) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis (basal section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic accumulation of MyoII-GFP. Quantification shown right (n = 8 independent samples per genotype). Scale bar ~1µm. Mean +/- 1 S.D. shown.


( F ) E - cad - GFP is downregulated as round up for mitosis ( apical section , diagrammed left ) . Treatment with Rho - kinase inhibitor Y - 27632 inhibits mitotic downregulation of E - cad - GFP . Extended treatment with Y - 27632 leads to a general defect in interphase mitotic in which apical surface areas enlarge E - cad - GFP is generally lost . Arrow indicates mitotic cortex at maximal rounding . shown right ( n = 6 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bar ~ 1µm .

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(F) E-cad-GFP is downregulated as cells round up for mitosis (apical section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic downregulation of E-cad-GFP. Extended treatment with Y-27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E-cad-GFP is generally lost. Arrow indicates mitotic cell cortex at maximal rounding. Quantification shown right (n = 6 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.


( A ) localised Pbl - GFP becomes in mitotic , co - incident with Rok - GFP relocalisation to plasma . Scale bar ~ 1µm . n > 5 independent biological replicates .

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(A) Nuclear localised Pbl-GFP becomes cytoplasmic in mitotic cells, co-incident with Rok-GFP relocalisation to the plasma membrane. Scale bar ~1µm. n > 5 independent biological replicates.


( B ) Phosphorylated Myosin - II Regulatory Chain ( p - MLC ; purple ) detected by antibody reveals high around cortex of rounded mitotic in wild - type wing epithelial , but not those expressing UAS . pbl - RNAi . Phospho - Histone H3 ( pH3 ) ( green ) marks mitotic chromosomes . shown right ( n > 10 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bar ~ 1µm .

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(B) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those expressing UAS.pbl-RNAi. Phospho-Histone H3 (pH3) staining (green) marks mitotic cell chromosomes. Quantification shown right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.


( C ) A stripe of expression of ptc . Gal4 UAS . pbl - RNAi in wing disc leads to both enlarged many basally extruded that undergo apoptosis , marked by pyknotic nuclei loss of aPKC / Dlg . Scale bars ~ 20µm . n > 6 independent biological replicates .

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(C) A stripe of expression of ptc.Gal4 UAS.pbl-RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis, marked by pyknotic nuclei and loss of aPKC/Dlg staining. Scale bars ~20µm. n > 6 independent biological replicates.


( A ) localised Tum - GFP becomes in mitotic , including a distinct plasma localisation ( arrows ) . Note also localisation to cytokinetic ring ( asterisk ) remnant mid - bodies / ring canals in interphase . shown on right ( n > 7 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 2µm .

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(A) Nuclear localised Tum-GFP becomes cytoplasmic in mitotic cells, including a distinct plasma membrane localisation (arrows). Note also localisation to the cytokinetic ring (asterisk) and remnant mid-bodies/ring canals in interphase cells. Quantification shown on the right (n > 7 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~2µm.


( B ) A stripe of expression of ptc . Gal4 UAS . tum - RNAi in wing disc leads to basally extruded that undergo apoptosis , marked by Dcp1 immunostaining , similar to UAS . pbl - RNAi . shown on right ( n > 10 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 20µm .

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(B) A stripe of expression of ptc.Gal4 UAS.tum-RNAi in the wing disc leads to basally extruded cells that undergo apoptosis, marked by Dcp1 immunostaining, similar to UAS.pbl-RNAi. Quantification shown on the right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.


( C ) Phosphorylated Myosin - II Regulatory Chain ( p - MLC ; red ) detected by antibody reveals high around cortex of rounded mitotic in wild - type wing epithelial , but not those homozygous mutant for tum347 , marked by expression of GFP in single - celled clones ( MARCM system ) . pH3 ( blue ) marks mitotic chromosomes . Arrow indicates a GFP - positive single tum347 mutant clone . shown on right ( n > 3 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 20µm .

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(C) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; red) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those cells homozygous mutant for tum347, marked by expression of GFP in single-celled clones (MARCM system). pH3 staining (blue) marks mitotic cell chromosomes. Arrow indicates a GFP-positive single cell tum347 mutant clone. Quantification shown on the right (n > 3 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.


( A ) Phosphorylated Myosin - II Regulatory Chain ( p - MLC ; purple ) detected by antibody reveals high around cortex of rounded mitotic ( pH3 , red ) in wild - type wing discs , aurA homozygous mutant wing discs , but not upon inhibition of both AurA AurB by treatment with VX - 680 . Note increased numbers of mitotic in aurA homozygous mutant wing discs , due to delayed mitotic progression . Scale bars ~ 20µm ( low mag . ) ~ 1µm ( high mag . ) . n > 15 independent biological replicates .

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(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells (pH3, red) in wild-type wing discs, and aurA homozygous mutant wing discs, but not upon inhibition of both AurA and AurB by treatment with VX-680. Note increased numbers of mitotic cells in aurA homozygous mutant wing discs, due to delayed mitotic progression. Scale bars ~20µm (low mag.) and ~1µm (high mag.). n > 15 independent biological replicates.


( B ) Live - imaging of MyoII - GFP reveals delayed mitosis in aurA homozygous mutant wing discs , while inhibition of both AurA AurB by treatment with VX - 680 leads to complete loss of mitotic rounding MyoII - GFP localisation . Asterisks indicate a single mitotic . Scale bars ~ 1µm . shown on bottom right ( n = 5 independent samples per genotype ) .

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(B) Live-imaging of MyoII-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, while inhibition of both AurA and AurB by treatment with VX-680 leads to complete loss of mitotic rounding and MyoII-GFP membrane localisation. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 5 independent samples per genotype).


( C ) Live - imaging of E - cad - GFP reveals delayed mitosis in aurA homozygous mutant wing discs , cytokinesis failure in UAS . aurB - RNAi expressing wing discs , complete failure of mitotic rounding E - cad - GFP downregulation upon inhibition of both AurA AurB by treatment with VX - 680 . Asterisks indicate a single mitotic . Scale bars ~ 1µm . shown on bottom right ( n = 9 independent samples per genotype ) .

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(C) Live-imaging of E-cad-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, cytokinesis failure in UAS.aurB-RNAi expressing wing discs, and complete failure of mitotic rounding and E-cad-GFP downregulation upon inhibition of both AurA and AurB by treatment with VX-680. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 9 independent samples per genotype).


( A ) Striped ptc . Gal4 - driven expression of UAS . GFP in a control wing imaginal disc . Scale bars ~ 20µm . n > 6 independent biological replicates . ( B ) Striped ptc . Gal4 - driven expression of UAS . GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic . n > 7 independent biological replicates . ( C ) Striped ptc . Gal4 - driven expression of UAS . GFP UAS . aurB - RNAi in a control wing imaginal disc leads to enlarged with large nuclei ( marked by DAPI ; arrowhead ) . n > 6 independent biological replicates . ( D ) Striped ptc . Gal4 - driven expression of UAS . GFP UAS . aurB - RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion apoptosis of ( marked by Dcp1 pyknotic nuclei ) . n > 8 independent biological replicates . ( E ) Striped ptc . Gal4 - driven expression of UAS . GFP UAS . pbl - RNAi in wing imaginal disc leads to extrusion apoptosis of ( marked by Dcp1 pyknotic nuclei ) . n > 8 independent biological replicates . ( F ) ptc . Gal4 - driven expression of UAS . aurB - RNAi in an aurA homozygous mutant endoreplicating salivary has no phenotypic consequence . n > 6 independent biological replicates .

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(A) Striped ptc.Gal4-driven expression of UAS.GFP in a control wing imaginal disc. Scale bars ~20µm. n > 6 independent biological replicates. (B) Striped ptc.Gal4-driven expression of UAS.GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells. n > 7 independent biological replicates. (C) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB-RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei (marked by DAPI; white arrowhead). n > 6 independent biological replicates. (D) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB -RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (E) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.pbl-RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (F) ptc.Gal4-driven expression of UAS.aurB-RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence. n > 6 independent biological replicates.


( G ) MARCM clones ( GFP - positive ) expressing UAS . aurB - RNAi in an aurA homozygous mutant result in single clones ( i . e . : no division ) , while loss of AurA or AurB allows some proliferation . shown on right ( n > 7 independent samples per genotype ; Mean + / - 1 S . D . shown ) . Scale bar ~ 20µm .

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(G) MARCM clones (GFP-positive) expressing UAS.aurB-RNAi in an aurA homozygous mutant cells result in single cell clones (i.e.: no cell division), while individual loss of AurA or AurB allows some cell proliferation. Quantification shown on the right (n > 7 independent samples per genotype; Mean +/- 1 S.D. shown). Scale bar ~20µm.


( A ) Live - imaging of MyoII - GFP in a control wing disc epithelium . Asterisk indicates location of a single mitotic about to initiate rounding . Scale bar ~ 1µm . n > 5 independent biological replicates . ( B ) Live - imaging of MyoII - GFP upon treatment with dual AurA / B inhibitor VX - 680 after initiation of mitotic rounding , which rapidly arrests progression of rounding prevents cytokinesis . Scale bar ~ 1µm . n > 9 independent biological replicates .

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(A) Live-imaging of MyoII-GFP in a control wing disc epithelium. Asterisk indicates the location of a single mitotic cell about to initiate rounding. Scale bar ~1µm. n > 5 independent biological replicates. (B) Live-imaging of MyoII-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 9 independent biological replicates.


( C ) Live - imaging of E - cad - GFP upon treatment with dual AurA / B inhibitor VX - 680 after initiation of mitotic rounding , which rapidly arrests progression of rounding prevents cytokinesis . Scale bar ~ 1µm . n > 6 independent biological replicates .

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(C) Live-imaging of E-cad-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 6 independent biological replicates.


( A ) Early stage Drosophila egg chambers showing giant germ - nurse surrounded by E - cadherin - positive follicle epithelium . A follicle undergoing mitotic rounding is highlighted with an asterisk ( * ) . Scale bar ~ 3µm . n > 3 independent biological replicates .

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(A) Early stage Drosophila egg chambers showing giant germ-line nurse cells surrounded by E-cadherin-positive follicle cell epithelium. A follicle cell undergoing mitotic rounding is highlighted with an asterisk (*). Scale bar ~3µm. n > 3 independent biological replicates.


( B ) High resolution imaging of F - actin cortex , E - cadherin , phospho - MyoII in a mitotically rounded follicle . Note maintenance of adherens junctions including both E - cad contractile actomyosin ring during mitotic rounding . Asterisks or pH3 indicate a single mitotic . Scale bar ~ 3µm . n > 4 independent biological replicates .

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(B) High resolution imaging of the F-actin cortex, E-cadherin, and phospho-MyoII in a mitotically rounded follicle cell. Note maintenance of adherens junctions including both E-cad and the contractile actomyosin ring during mitotic rounding. Asterisks or pH3 staining indicate a single mitotic cell. Scale bar ~3µm. n > 4 independent biological replicates.


( A ) damage induces H2AX ADP - ribosylation . 293T were treated with H2O2 ( 2 mM in PBS , 5 minutes ) , MMS ( 1 mM in medium , 30 minutes ) or mock ( PBS , 30 minutes ) . ADP - ribosylated were IPed with anti - ADP - ribose ( anti - ADPR ) antibody . ADP - ribosylated H2AX was examined by Western blotting using anti - H2AX antibody .

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(A) DNA damage induces H2AX ADP-ribosylation. 293T cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes). ADP-ribosylated proteins were IPed with anti-ADP-ribose (anti-ADPR) antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.


( B ) H2AX is ADP - ribosylated at glutamate 141 ( E141 ) . shPARG HCT116 were treated with H2O2 ( 2 mM in PBS , 5 minutes ) ADP - ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15 . 0109 Da , an increment that can be readily distinguished by mass spectrometry . Fragmentation of NH2OH - derivatized peptides yielded typical b - y - ion series , allowing easy localization of ADP - ribosylation sites . Glutamate 141 ( E141 ) is indicated by an asterisk .

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(B) H2AX is ADP-ribosylated at glutamate 141 (E141). shPARG HCT116 cells were treated with H2O2 (2 mM in PBS, 5 minutes) and ADP-ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15.0109 Da, an increment that can be readily distinguished by mass spectrometry. Fragmentation of the NH2OH-derivatized peptides yielded typical b- and y-ion series, allowing easy localization of ADP-ribosylation sites. Glutamate 141 (E141) is indicated by an asterisk.


( C ) E141A mutation abolishes ADP - ribosylation of H2AX . Empty vector ( EV ) , wild type ( WT ) or E141A mutant ( E141A ) of H2AX was expressed in U2OS H2AX knockout that were treated with H2O2 . ADP - ribosylated were IPed with anti - ADPR antibody . ADP - ribosylated H2AX was examined by Western blotting using anti - H2AX antibody .

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(C) The E141A mutation abolishes the ADP-ribosylation of H2AX. Empty vector (EV), wild type (WT) or the E141A mutant (E141A) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2. ADP-ribosylated proteins were IPed with anti-ADPR antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.


( D ) ADP - ribosylation on H2AX is suppressed by olaparib treatment ( 1 µM , 1 hour ) . 0 . 1 % DMSO in medium for 1 hour as mock . samples were examined by IP Western blotting .

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(D) ADP-ribosylation on H2AX is suppressed by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. The samples were examined by IP and Western blotting.


( E ) PARP1 , but not PARP2 , mediates ADP - ribosylation on H2AX . Wild type ( WT ) , PARP1 - null or PARP2 - null were treated with H2O2 .

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(E) PARP1, but not PARP2, mediates the ADP-ribosylation on H2AX. Wild type (WT) cells, PARP1-null cells or PARP2-null cells were treated with H2O2.


( A ) U2OS ( WT ) U2OS H2AX knockout reconstituted with empty vector ( H2AX KO + EV ) , wild type H2AX ( H2AX KO + WT ) , or E141A mutant H2AX ( E141A ) were treated with H2O2 or MMS . viability was measured after 24 hours using MTT . Values are mean ± SD of three assays .

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(A) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 or MMS. Cell viability was measured after 24 hours using MTT assay. Values are mean ± SD of three assays.


( B ) U2OS ( WT ) U2OS H2AX knockout reconstituted with empty vector ( H2AX KO + EV ) , wild type H2AX ( H2AX KO + WT ) , or E141A mutant ( E141A ) H2AX were treated with H2O2 ( 2 mM in PBS , 5 minutes ) or MMS ( 1 mM in medium , 30 minutes ) , subsequently ; were cultured in fresh DMEM medium for 2 . numbers of colony formation were counted . Values are mean ± SD of three assays .

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(B) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant (E141A) H2AX were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes), subsequently; the cells were cultured in fresh DMEM medium for 2 weeks. The numbers of colony formation were counted. Values are mean ± SD of three assays.


( C ) Damaged bases repair are suppressed in U2OS expressing E141A mutant ( E141A ) . U2OS ( WT ) U2OS H2AX knockout reconstituted with wild type H2AX ( H2AX KO + WT ) , or E141A mutant H2AX ( E141A ) were treated with H2O2 ( 2 mM in PBS , 5 minutes ) or MMS ( 1 mM in medium , 30 minutes ) harvested at indicated recovery time points . Then FPG - modified comet was performed . NT : no treatment . Representative comet tails were shown . olive tail moments ( OTM ) were summarized from at least 50 in each experiment . Values are mean ± SD of three assays .

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(C) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant (E141A). U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes) and harvested at the indicated recovery time points. Then FPG-modified comet assay was performed. NT: no treatment. Representative comet tails were shown. The olive tail moments (OTM) were summarized from at least 50 cells in each experiment. Values are mean ± SD of three assays.


( D ) replication in U2OS expressing E141A mutant ( E141A ) is suppressed . were treated with H2O2 ( 2 mM in PBS , 5 minutes ) , MMS ( 1 mM in medium , 30 minutes ) or mock ( PBS , 30 minutes ) , followed by BrdU incorporation ( 30 μM in medium , 30 minutes ) . Representative show BrdU - positive in expressing WT H2AX or E141A following genotoxic stress . Nuclei were stained with DAPI ( blue ) . bottom panel histogram shows of BrdU incorporation in each sample , Values are mean ± SD of three assays .

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(D) DNA replication in U2OS cells expressing the E141A mutant (E141A) is suppressed. Cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes), followed by BrdU incorporation (30 μM in medium, 30 minutes). Representative images show BrdU-positive cells in cells expressing WT H2AX or E141A following genotoxic stress. Nuclei were stained with DAPI (blue). The bottom panel histogram shows the percentage of BrdU incorporation in each sample, Values are mean ± SD of three assays.


( E ) Generation of AP sites is suppressed in expressing E141A . were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes . AP sites were calculated according to standard curve . Values are mean ± SD of three assays .

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(E) Generation of AP sites is suppressed in cells expressing E141A. Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes. AP sites were calculated according to the standard curve. Values are mean ± SD of three assays.


( A ) ADP - ribosylation mediates recruitment of Neil3 . GFP - Neil3 was expressed in U2OS , were treated with olaparib ( 1 µM , 1 hour ) or mock ( 0 . 1 % DMSO in medium , 1 hour ) . Following microirradiation treatment , recruitment of Neil3 was examined with live - imaging at indicated time points ( left panel ) . stripe is indicated with yellow arrowheads . relocation kinetics is shown in right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 were included in each experiment .

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(A) ADP-ribosylation mediates the recruitment of Neil3. GFP-Neil3 was expressed in U2OS cells, and cells were treated with olaparib (1 µM, 1 hour) or mock (0.1 % DMSO in medium, 1 hour). Following laser microirradiation treatment, the recruitment of Neil3 was examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( B ) GRFs of Neil3 alone is sufficient to be recruited to sites of damage . Truncated mutants of Neil3 were generated fused with a GFP tag . Following microirradiation treatment , recruitment of truncated mutants Neil3 were examined with live - imaging at indicated time points ( left panel ) . stripe is indicated with yellow arrowheads . relocation kinetics is shown in right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 were included in each experiment .

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(B) The GRFs of Neil3 alone is sufficient to be recruited to the sites of DNA damage. Truncated mutants of Neil3 were generated and fused with a GFP tag. Following laser microirradiation treatment, the recruitment of truncated mutants Neil3 were examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( C ) PARP inhibitor treatment ( olaparib : 1 µM , 1 hour ) or lack of PARP1 suppresses relocation of GRFs of Neil3 to lesions . stripe is indicated with yellow arrowheads . relocation kinetics is shown in right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 were included in each experiment .

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(C) PARP inhibitor treatment (olaparib: 1 µM, 1 hour) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions. The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( D ) recruitment of Neil3 to oxidative lesions is inhibited by olaparib treatment ( 1 µM , 1 hour ) . 0 . 1 % DMSO in medium for 1 hour as mock . Oxidative damage was induced by KillerRed ( KR ) system with exposure to a 15 - W SYLVANIA cool bulb for 10 minutes . Scale bar , 2 μm . foci were indicated with arrowheads . Right panel : of Neil3 foci co - localized with KR was quantified .

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(D) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. Oxidative damage was induced by KillerRed (KR) system with the exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 minutes. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of Neil3 foci co-localized with KR was quantified.


( A ) GRFs of Neil3 recognizes PAR . recombinant GST fusion were incubated with PAR - PAR was pulled down by glutathione agarose beads . Samples were serially diluted three - fold , spotted onto nitrocellulose , subjected to dot blotting using anti - PAR antibody ( top panel ) . FL represents full Neil3 , N - ter represents N - terminal Neil3 ( 1 - 300 aa ) , Mid represents middle Neil3 ( 300 - 404 aa ) , GRFs represents C - terminal Neil3 GRFs domain ( 480 - 606 aa ) . Recombinant GST was used as negative control ( NC ) . GST fusion were also examined by SDS - PAGE followed with Coomassie blue ( middle panel ) . relative PAR - binding in pull down was summarized in graph ( bottom panel ) . Values are mean ± SD of three assays .

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(A) The GRFs of Neil3 recognizes PAR. The recombinant GST fusion proteins were incubated with PAR and the protein-PAR complex was pulled down by glutathione agarose beads. Samples were serially diluted three-fold, spotted onto the nitrocellulose membrane, and subjected to dot blotting using anti-PAR antibody (top panel). FL represents full length Neil3, N-ter represents N-terminal Neil3 (1-300 aa), Mid represents middle Neil3 (300-404 aa), and GRFs represents C-terminal Neil3 GRFs domain (480-606 aa). Recombinant GST protein was used as the negative control (NC). The GST fusion proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (middle panel). The relative PAR-binding in the pull down assay was summarized in the graph (bottom panel). Values are mean ± SD of three assays.


( B ) Tandem co - immunoprecipitation Western blotting assays demonstrate that Neil3 interacts with ADP - ribosylated H2AX but not E141A mutant H2AX .

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(B) Tandem co-immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP-ribosylated H2AX but not the E141A mutant H2AX.


( C , D ) GRFs of Neil3 is sufficient required for interaction with ADP - ribosylated H2AX . Tandem co - immunoprecipitation Western blotting assays were

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(C,D) The GRFs of Neil3 is sufficient and required for the interaction with ADP-ribosylated H2AX. Tandem co-immunoprecipitation and Western blotting assays were used


( E , F ) relocation kinetic of full - Neil3 or GRFs of Neil3 to lesions was analyzed . GFP - tagged full Neil3 or GRFs of Neil3 was expressed in indicated , relocation kinetics was monitored in a time course following microirradiation ( left panel ) . stripe is indicated with yellow arrowheads . relocation kinetics is shown in right panel . Data represent mean ± SD from three biologically independent experiments ( right panel ) . At least 20 were included in each experiment .

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(E,F) The relocation kinetic of full-length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed. GFP-tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells, and relocation kinetics was monitored in a time course following laser microirradiation (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( G , H ) recruitment of full - Neil3 or GRFs of Neil3 to KR - induced oxidative lesions is mediated by H2AX ADP - ribosylation ( left panel ) . Scale bars , 2 μm . foci were indicated with arrowheads . Scale bars , 0 . 5 μm . Right panel : of Neil3 or GRFs Neil3 foci co - localized with KR was quantified . Mean value with SD is from 20 .

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(G,H) The recruitment of full-length Neil3 or the GRFs of Neil3 to the KR-induced oxidative lesions is mediated by H2AX ADP-ribosylation (left panel). Scale bars, 2 μm. The foci were indicated with white arrowheads. Scale bars, 0.5 μm. Right panel: the percentage of Neil3 or GRFs Neil3 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( A ) PARP inhibition facilitates γH2AX following oxidative stress . were pre - treated with olaparib ( 1 µM , 1 hour ) prior to H2O2 stimulation . 0 . 1 % DMSO in medium for 1 hour as mock . γH2AX or H2AX was detected using Western blotting immunostaining assays . of γH2AX were examined by Western blotting normalized by β - actin averaged . Values are mean ± SD of three assays . P - values were calculated using Student ' s t - . * * P < 0 . 01 ( left panel ) . number of γH2AX foci examined in immunostaining was counted by Image J software graphed by GraphPad Prism 8 .

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(A) PARP inhibition facilitates γH2AX following oxidative stress. The cells were pre-treated with olaparib (1 µM, 1 hour) prior to H2O2 stimulation. 0.1 % DMSO in medium for 1 hour as mock. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.


( B ) effect of H2AX E141 ADP - ribosylation on γH2AX following oxidative stress . γH2AX or H2AX was detected using Western blotting immunostaining assays . of γH2AX were examined by Western blotting normalized by β - actin averaged . Values are mean ± SD of three assays . P - values were calculated using Student ' s t - . * * P < 0 . 01 ( left panel ) . number of γH2AX foci examined in immunostaining was counted by Image J software graphed by GraphPad Prism 8 .

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(B) The effect of H2AX E141 ADP-ribosylation on γH2AX following oxidative stress. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.


( C ) of γH2AX is increased in expressing E141A mutant following KillerRed - induced oxidative lesions ( left panel ) . Scale bar , 2 μm . foci were indicated with arrowheads . Scale bar , 0 . 5 μm . Right panel : relative of γH2AX at site of tetR ( of γH2AX at site of tetR / of γH2AX away from tetR ) was quantified . Mean value with SD is relative of γH2AX in 20 .

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(C) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed-induced oxidative DNA lesions (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Scale bar, 0.5 μm. Right panel: the relative intensity of γH2AX at the site of tetR (intensity of γH2AX at the site of tetR/intensity of γH2AX area away from tetR) was quantified. Mean value with SD is the relative intensity of γH2AX in 20 cells.


( D ) level of MDC1 at KillerRed - induced oxidative lesions is increased in expressing E141A mutant ( left panel ) . Scale bar , 2 μm . foci were indicated with arrowheads . Right panel : of MDC1 foci co - localized with KR was quantified . Mean value with SD is from 20 .

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(D) The level of MDC1 at KillerRed-induced oxidative DNA lesions is increased in the cells expressing the E141A mutant (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of MDC1 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( E ) recruitment of Neil3 / MDC1 chimera but not wild type Neil3 to KR - induced oxidative lesions in expressing E141A mutant ( left panel ) . Neil3 / MDC1 chimera : replaced GRFs of Neil3 with tandem BRCT domains of MDC1 . Scale bar , 2 μm . foci were indicated with arrowheads . Right panel : of detected foci co - localized with KR was quantified . Mean value with SD is from 20 .

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(E) The recruitment of Neil3/MDC1 chimera but not wild type Neil3 to the KR-induced oxidative lesions in cells expressing the E141A mutant (left panel). Neil3/MDC1 chimera: replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: The percentage of detected protein foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( F ) AP sites were measured in expressing E141A mutant or Neil3 / MDC1 chimera , according to standard curve . Values are mean ± SD of three assays .

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(F) AP sites were measured in cells expressing the E141A mutant or the Neil3/MDC1 chimera, according to the standard curve. Values are mean ± SD of three assays.


( G ) replication of U2OS expression with wild type ( WT ) , H2AX KO + WT , E141A mutant ( E141A ) or Neil3 / MDC1 Chimera ( Chimera ) were analyzed by BrdU incorporation following treatment with H2O2 , MMS or mock ( PBS ) . Represented are shown in left panel . graph shows percentages of BrdU - positive expressing WT , E141A or Chimera ( right panel ) .

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(G) DNA replication of U2OS cells expression with individual wild type (WT), the H2AX KO + WT, the E141A mutant (E141A) or the Neil3/MDC1 Chimera (Chimera) were analyzed by BrdU incorporation following treatment with H2O2, MMS or mock (PBS). Represented images are shown in the left panel. The graph shows percentages of BrdU-positive cells expressing WT, E141A or Chimera (right panel).


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

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

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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 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 stained with GRA7 for PVM Hoechst 33258 for nuclei . number of PVs in 5 - 6 fields of coverslips were counted for each condition normalized with number of host in each field . Experiments were performed with RH ( n = 3 ) , Pru ( n = 3 ) . Representative of infected for each condition are provided . Scale bar is 100 µm .

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

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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 n = 7 for plaque ) , PruΔgra15 ( n = 20 for plaque number n = 7 for plaque ) , PruΔgra15 + GRA15 complemented ( n = 12 for plaque number n = 7 for plaque ) .

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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 n = 3 for plaque ) RH + GRA15II ( n = 5 for plaque number n = 3 for plaque ) .

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

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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 ) RH + GRA15II ( n = 3 ) normalized with number of host in each field .

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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 subsequently infected with RH , Pru or PruΔgra15 for 3 h . of vacuoles that stained positive for ( A ) Total ubiquitin ( n = 3 for RH , n = 5 for Pru n = 3 for PruΔgra15 ) , is shown in left bar diagram . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as inset pictures with magnification .

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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 subsequently infected with RH , Pru or PruΔgra15 for 3 h . of vacuoles that stained positive for ( B ) K63 - linked ubiquitin ( n = 3 for RH , n = 3 for Pru n = 3 for PruΔgra15 ) , is shown in left bar diagram . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as inset pictures with magnification .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 subsequently infected with RH , Pru or PruΔgra15 for 3 h . of vacuoles that stained positive for ( C ) p62 ( n = 3 for RH , n = 10 for Pru n = 7 for PruΔgra15 , n = 4 for Pru Δgra15 + GRA15 ) , is shown in left bar diagram . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as inset pictures with magnification .

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

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 subsequently infected with RH , Pru or PruΔgra15 for 3 h . of vacuoles that stained positive for ( D ) NDP52 ( n = 3 for RH , n = 3 for Pru n = 3 for PruΔgra15 ) , is shown in left bar diagram . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as inset pictures with magnification .

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

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 subsequently infected with RH , Pru or PruΔgra15 for 3 h . of vacuoles that stained positive for ( E ) LC3B ( n = 3 for RH , n = 3 for Pru n = 3 for PruΔgra15 , n = 3 for Pru Δgra15 + GRA15 ) , is shown in left bar diagram . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . 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 subsequently infected with RH , Pru or PruΔgra15 for 3 h . of vacuoles that stained positive for ( F ) GABARAP ( n = 3 for RH , n = 3 for Pru n = 3 for PruΔgra15 ) is shown in left bar diagram . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as inset pictures with magnification .

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

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 subsequently infected with RH , Pru or PruΔgra15 for 3 h . of vacuoles that stained positive for ( G ) LAMP1 ( n = 3 for RH , n = 3 for Pru n = 3 for PruΔgra15 ) is shown in left bar diagram . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . 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 ) number of PVs per 20X objective field was counted compared between IFNγ - stimulated IFNγ + bafilomycin A1 ( 100 nM ) treated HFFs 24 h p . i . with Pru . from at least 6 fields were taken for each condition ( n = 3 ) .

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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 subsequently treated with PYR41 ( 1 µM ) for 2 h prior to infection . were washed subsequently infected with Pru ( MOI = 3 ) for 3 h . of vacuoles that stained positive for total ubiquitin , p62 or LC3B was determined ( n = 3 ) .

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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 co - experiments IFNγ - stimulated HFFs infected for 3 h with Pru were used . For each at least 50 vacuoles were scored ( n = 3 ) . On right - side , a representative image is shown for Toxoplasma Pru . Scale bar is 20 µm . yellow box inside each representative image is shown as inset pictures with higher magnification . total number of coated vacuoles was set at 100 % 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 B-MEASURED_VAR 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 co - experiments IFNγ - stimulated HFFs infected for 3 h with Pru were used . For each at least 50 vacuoles were scored ( n = 3 ) . On right - side , a representative image is shown for Toxoplasma Pru . Scale bar is 20 µm . yellow box inside each representative image is shown as inset pictures with higher magnification . total number of coated vacuoles was set at 100 % 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 co - experiments IFNγ - stimulated HFFs infected for 3 h with Pru were used . For each at least 50 vacuoles were scored ( n = 3 ) . On right - side , a representative image is shown for Toxoplasma Pru . Scale bar is 20 µm . yellow box inside each representative image is shown as inset pictures with higher magnification . total number of coated vacuoles was set at 100 % 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 co - experiments IFNγ - stimulated HFFs infected for 3 h with Pru were used . For each at least 50 vacuoles were scored ( n = 3 ) . On right - side , a representative image is shown for Toxoplasma Pru . Scale bar is 20 µm . yellow box inside each representative image is shown as inset pictures with higher magnification . total number of coated vacuoles was set at 100 % 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 B-MEASURED_VAR I-MEASURED_VAR 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 co - experiments IFNγ - stimulated HFFs infected for 3 h with Pru were used . For each at least 50 vacuoles were scored ( n = 3 ) . On right - side , a representative image is shown for Toxoplasma Pru . Scale bar is 20 µm . yellow box inside each representative image is shown as inset pictures with higher magnification . total number of coated vacuoles was set at 100 % 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 . NF - κB inhibitor BAY11 - 7082 ( 1µM ) was added 2 h pre - infection HFFs were subsequently infected with Pru . growth ( using luciferase ) was measured 24 h p . i . Means from unstimulated were set at 100 % . Experiments were performed three times .

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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 ) translocation of 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 . Experiments were done 4 times where each dot represents one experimental mean of at least 15 nuclei . In right panel , representative are shown . were expressing GFP , nuclei are stained with Hoechst 33258 . Scale bar is 20 µm .

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

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

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

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

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

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

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

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 of p62 , with without IFNγ in RH , RH + GRA15WT . experiments were done 3 times with each of strains .

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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 of LC3B with without IFNγ in RH , RH + GRA15WT . experiments were done 3 times with each of strains .

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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 of LAMP1 with without IFNγ in RH , RH + GRA15WT . experiments were done 3 times with each of 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 TRAF6 - specific siRNA transfected HFFs with without IFNγ .

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

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 growth was measured in scrambled siRNA transfected HFFs TRAF6 knockdown HFFs using luciferase - with indicated strains with without IFNγ ( n = 3 ) . antibody against TRAF6 used here was from Abcam ( Appendix Table S2 ) . Full 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 of p62 was done in scrambled siRNA transfected HFFs TRAF6 knockdown HFFs with without IFNγ using indicated strains ( n = 3 ) .

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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 of LC3B was done in scrambled siRNA transfected HFFs TRAF6 knockdown HFFs with 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 subsequently fixed , permeabilized stained for ( A ) IRGB6 For , at least 100 vacuoles were scored . experiments were performed 3 times . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as an inset picture with magnification . 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 (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 subsequently fixed , permeabilized stained for ( B ) GBPs , For , at least 100 vacuoles were scored . experiments were performed 3 times . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as an inset picture with magnification . experiments were performed 3 independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 subsequently fixed , permeabilized stained for ( C ) total ubiquitin , For , at least 100 vacuoles were scored . experiments were performed 3 times . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as an inset picture with magnification . experiments were performed 3 independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 subsequently fixed , permeabilized stained for ( D ) K63 - linked ubiquitin , For , at least 100 vacuoles were scored . experiments were performed 3 times . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as an inset picture with magnification . experiments were performed 3 independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 subsequently fixed , permeabilized stained for ( E ) K48 - linked ubiquitin For , at least 100 vacuoles were scored . experiments were performed 3 times . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as an inset picture with magnification . experiments were performed 3 independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 subsequently fixed , permeabilized stained for ( F ) LC3B . For , at least 100 vacuoles were scored . experiments were performed 3 times . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as an inset picture with magnification . experiments were performed 3 independent 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 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

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 subsequently infected with Pru , PruΔgra15 GRA15 complemented for 24 h for measuring growth by luciferase . 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 ) GRA15 complemented ( 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 subsequently infected with RH or RH expressing type II GRA15 . Plaque numbers were counted ( n = 3 ) , 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 Western blots were performed on MEFs stimulated or not with IFNγ ( 100 U / ml ) infected with an RH expressing type II GRA15 - HA as a control RH expressing GRA45 - HA . blots using antibodies against TRAF6 TRAF2 were made after stripping first blot . Left panel right panel were run on a single gel ; vertical lines indicate excision of irrelevant lanes .

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

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 RH for 3 h subsequently stained for TRAF6 . On right - side , a representative image is shown for Toxoplasma Pru , which expresses GFP . was stained with Hoechst 33258 . Scale bar is 10 µm . yellow box inside each representative image is shown as an inset picture with magnification . Experiments were performed 3 times .

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

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

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

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

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

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

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

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

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

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


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

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

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


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

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

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 . A positive control experiment . SARS - CoV - 2 readily targets ciliated human respiratory epithelial ( hRECs ) . Acetylated α - tubulin labels cilia . Arrows point SARS - CoV - 2 - positive labeled by AB4 ( green ) . Figures display scale bars . Bar diagram at right quantifies frequencies of SARS - CoV - 2 - positive in hRECs . At least six hREC sections from three ( n = 3 ) independent samples were examined . Data presented as mean + .

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 I-MEASURED_VAR I-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

A. A positive control experiment. SARS-CoV-2 readily targets ciliated human respiratory epithelial cells (hRECs). Acetylated α-tubulin labels cilia. Arrows point SARS-CoV-2-positive cells labeled by AB4 (green). Figures display scale bars. Bar diagram at right quantifies frequencies of SARS-CoV-2-positive cells in hRECs. At least six hREC sections from three (n=3) independent samples were examined. Data presented as mean + SEM.


B . Mock organoids of two groups Day - 15 ( i ) - 60 ( ii ) display typical cytoarchitecture of brain organoids . L , Lumen , VZ , Ventricular Zone is containing compact palisade - like elongated nuclei of Neural Progenitor ( NPCs , blue ) CP , a cortical plate containing TUJ - 1 - positive neurons ( magenta ) . Note a distinct difference TUJ - 1 labeling pattern between younger ( Day - 15 ) older ( Day - 60 ) brain organoid . Figures display scale bars . Representative from eight organoids cultured in at least three independent batches ( n = 3 ) derived from donor - 1 ( IMR90 ) iPSC .

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

B. Mock organoids of two age groups Day-15 (i) and-60 (ii) display typical cytoarchitecture of brain organoids. L, Lumen, VZ, Ventricular Zone is containing compact and palisade-like elongated nuclei of Neural Progenitor Cells (NPCs, blue) and CP, a cortical plate containing TUJ-1-positive neurons (magenta). Note a distinct difference TUJ-1 labeling pattern between younger (Day-15) and older (Day-60) brain organoid. Figures display scale bars. Representative images from eight organoids cultured in at least three independent batches (n=3) derived from donor-1 (IMR90) iPSC line.


C . Compared to mock organoids ( i ) , SARS - CoV - 2 - exposed Day - 15 organoids display SARS - CoV - 2 - positive ( AB4 , green ) in their outer periphery , a region of cortical plate ( ii ) that is specified by TUJ - 1 - positive neurons ( magenta ) . L , lumen of a VZ , inner of an organoid where NPCs are located , is free from SARS - CoV - 2 - positive . Magnified region ( dotted while box ) is given below . At least ten organoids from five different batches ( n = 5 ) are tested . Figures display scale bars .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-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

C. Compared to mock organoids (i), SARS-CoV-2-exposed Day-15 organoids display SARS-CoV-2-positive cells (AB4, green) in their outer periphery, a region of the cortical plate (ii) that is specified by TUJ-1-positive neurons (magenta). L, the lumen of a VZ, the inner area of an organoid where NPCs are located, is free from SARS-CoV-2-positive cells. Magnified region (dotted while box) is given below. At least ten organoids from five different batches (n=5) are tested. Figures display scale bars.


D . SARS - CoV - 2 - exposed Day - 60 organoids . Compared to Day 15 organoids mock ( i ) , Day - 60 organoids display an increased number of SARS - CoV - 2 - positive ( AB4 , green ) in their cortical plate that is specified by TUJ - 1 - positive neurons ( magenta ) ( ii ) . Magnified region ( dotted while box ) is given below , showing peri - location of SARS - CoV - 2 in cortical neurons . At least ten organoids from five different batches ( n = 5 ) are tested . Figures display scale bars .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-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

D. SARS-CoV-2-exposed Day-60 organoids. Compared to Day 15 organoids and mock (i), Day-60 organoids display an increased number of SARS-CoV-2-positive cells (AB4, green) in their cortical plate that is specified by TUJ-1-positive neurons (magenta) (ii). Magnified region (dotted while box) is given below, showing the peri-nuclear location of SARS-CoV-2 in cortical neurons. At least ten organoids from five different batches (n=5) are tested. Figures display scale bars.