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 .

B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY 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 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 .

B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY 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 .

B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY 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 .

B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY 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 .

B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY 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 (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 .

B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY 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 .

B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY 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 (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 ) .

O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM 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 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 ) .

O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD 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 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD 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 .

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

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 .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY 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 .

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

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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD B-EXP_ASSAY 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 .

B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O B-ORGANISM O B-GENEPROD 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 of LC3B with without IFNγ in RH , RH + GRA15WT . experiments were done 3 times with each of strains .

B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-ORGANISM O B-ORGANISM O B-GENEPROD 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 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 ) .

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

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 B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD 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 .

O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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 ) .

B-EXP_ASSAY O B-GENEPROD O O O O O O B-CELL_TYPE B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O

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 .

B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 (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 .

B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 .

B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 .

B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 .

B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 .

B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 .

O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O

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 ) .

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

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 ) .

O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY 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 B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O B-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

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-GENEPROD O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-ORGANISM O B-ORGANISM I-ORGANISM O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

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 B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD 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 ) .

B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD 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 ) .

B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD 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 ) .

B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O

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 ) .

B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD 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 ) .

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

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 B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-CELL_TYPE O O O O B-CELL_TYPE 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 B-SUBCELLULAR O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O B-TISSUE O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O B-CELL_TYPE 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 B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O B-CELL_TYPE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O 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 B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE I-CELL_TYPE 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.


E . bar diagram quantifies frequencies of SARS - CoV - 2 - positive in different brain organoid sections derived from two donor iPSC lines ( IMR90 Crx - iPS , see methods ) . Please note that each point represents one organoid section . SARS - CoV - 2 shows an enhanced tropism for Day - 60 organoids . Note , comparative statistics are shown between different groups respective days post infection ( dpi ) of organoids , significance is given as Asterisks in Day - 60 groups . There is no significant difference in SARS - CoV - 2 - positive between 2 - 4 - dpi within each groups . At least twelve organoids sections from four ( n = 4 ) independent batches , from each donor day post infections ( dpi ) were analyzed . One - way ANOVA , followed by Tukey ' s multiple comparisons , * * * P < 0 . 001 . Data presented as mean + .

O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-CELL_TYPE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The bar diagram quantifies frequencies of SARS-CoV-2-positive cells in different brain organoid sections derived from two donor iPSC lines (IMR90 and Crx-iPS, see methods). Please note that each point represents one organoid section. SARS-CoV-2 shows an enhanced tropism for Day-60 organoids. Note, comparative statistics are shown between different age groups and respective days post infection (dpi) of organoids, and the significance is given as Asterisks in Day-60 groups. There is no significant difference in SARS-CoV-2-positive cells between 2- and 4-dpi within each age groups. At least twelve organoids sections from four (n=4) independent batches, from each donor and day post infections (dpi) were analyzed. One-way ANOVA, followed by Tukey's multiple comparisons test, ***P<0.001. Data presented as mean + SEM.


F . Subcellular localization SARS - CoV - 2 in cortical neurons . High - resolution imaging de - convolution show peri - localization of SARS - CoV - 2 . SARS - CoV - 2 ( AB4 , green ) nucleus ( grey ) . Figures display scale bars . Representative from at least 200 are examined . surrounds perinuclear border red encircles nucleus .

O O B-EXP_ASSAY I-EXP_ASSAY B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O

F. Subcellular localization SARS-CoV-2 in cortical neurons. High-resolution imaging and de-convolution show peri-nuclear localization of SARS-CoV-2. SARS-CoV-2 (AB4, green) and nucleus (grey). Figures display scale bars. Representative images from at least 200 cells are examined. White line surrounds perinuclear border and red line encircles the nucleus.


G . Determination of viral progeny . Supernatants of SARS - CoV - 2 exposed Vero , brain organoids were analyzed for viral assessed by qRT - PCR . While an increase in viral was detected in supernatants of Vero , no apparent increase was identified in brain organoid supernatants . Data are obtained from five technical replicates from four ( n = 4 ) independent batches of organoids . Data presented as mean + .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_LINE O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Determination of viral progeny. Supernatants of SARS-CoV-2 exposed Vero cells, and brain organoids were analyzed for viral RNA assessed by qRT-PCR. While an increase in viral RNA was detected in the supernatants of Vero cells, no apparent increase was identified in brain organoid supernatants. Data are obtained from five technical replicates from four (n=4) independent batches of organoids. Data presented as mean + SEM.


A . Tau immunoreactivity ( magenta ) specifies cortical plate ( CP ) surrounding luman ( L ) ( i ) . Selected optic sections at high magnification ( ii iii ) highresolution imaging ( iv ) show Tau localization only in axons of cortical neurons . Note somas of neurons are free from Tau . At least eight organoids from four different batches ( n = 4 ) are tested . Figures display scale bars . B . Tau localization in SARS - CoV - 2 - positive neurons ( AB4 , green ) in selected optic sections ( i ) . Note , in contrast , to control groups , SARS - CoV - 2 - exposed organoids display mislocalized Tau ( magenta ) majorly into somas of neurons ( arrowheads ) . Selected confocal slices are shown to distinguish Tau mislocalization into neuronal soma ( arrowheads ) . At high magnification , neuronal soma is further specified by perinuclear localization of SARS - CoV - 2 ( green ) ( ii iii ) . Bar diagrams at right quantifies of neurons ( Mock SARS - CoV - 2 exposed ) exhibiting mislocalized Tau ( iv ) fraction of SARS - CoV - 2 - positive neurons exhibiting Tau - positive axons spanning different cortical areas ( v ) . For statistics , at least 300 from six organoids from four different batches ( n = 4 ) were tested . Figures display scale bars . Unpaired t with Welch ' s correction , * * * P < 0 . 001 . Data presented as mean + SD .

O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O B-SUBCELLULAR O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O B-GENEPROD O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Tau immunoreactivity (magenta) specifies the cortical plate (CP) surrounding the luman (L) (i). Selected optic sections at high magnification (ii and iii) and highresolution imaging (iv) show Tau localization only in axons of cortical neurons. Note the somas of neurons are free from Tau protein. At least eight organoids from four different batches (n=4) are tested. Figures display scale bars. B. Tau localization in SARS-CoV-2-positive neurons (AB4, green) in selected optic sections (i). Note, in contrast, to control groups, SARS-CoV-2-exposed organoids display mislocalized Tau (magenta) majorly into the somas of neurons (arrowheads). Selected confocal slices are shown to distinguish Tau mislocalization into neuronal soma (arrowheads). At high magnification, neuronal soma is further specified by the perinuclear localization of SARS-CoV-2 (green) (ii and iii). Bar diagrams at right quantifies the percentage of neurons (Mock and SARS-CoV-2 exposed) exhibiting mislocalized Tau (iv) and the fraction of SARS-CoV-2-positive neurons exhibiting Tau-positive axons spanning different cortical areas (v). For statistics, at least 300 cells from six organoids from four different batches (n=4) were tested. Figures display scale bars. Unpaired t test with Welch's correction, ***P<0.001. Data presented as mean + SD.


D . In contrast to controls ( i ) , Tau AT180 antibody ( magenta ) that specifically recognizes phosphorylated Threonine 231 of Tau distinctly localizes at somas of SARS - CoV - 2 - positive neurons ( AB4 , green ) ( ii ) . At least four organoids from two different batches ( n = 2 ) are tested . Figures display scale bars .

O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY B-SMALL_MOLECULE O O B-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O 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. In contrast to controls (i), Tau AT180 antibody (magenta) that specifically recognizes the phosphorylated Threonine 231 of Tau protein distinctly localizes at the somas of SARS-CoV-2-positive neurons (AB4, green) (ii). At least four organoids from two different batches (n=2) are tested. Figures display scale bars.


E . Co - localization of SARS - CoV - 2 ( AB4 , green ) phosphorylated Tau ( magenta ) at somas of cortical neurons revealed by high - resolution imaging de - convolution . Representative from at least 300 examined . Figures display scale bars . F . bar diagram quantifies fraction of Tau AT180 - positive neurons that co - localize with SARS - CoV - 2 - positive neurons . For statistics , at least 250 from four organoids two independent batches ( n = 2 ) were examined . Unpaired t with Welch ' s correction . Data presented as mean + .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY B-GENEPROD O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O 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. Co-localization of SARS-CoV-2 (AB4, green) and phosphorylated Tau protein (magenta) at somas of cortical neurons revealed by high-resolution imaging and de-convolution. Representative images from at least 300 cells examined. Figures display scale bars. F. The bar diagram quantifies the fraction of Tau AT180-positive neurons that co-localize with SARS-CoV-2-positive neurons. For statistics, at least 250 cells from four organoids and two independent batches (n=2) were examined. Unpaired t test with Welch's correction. Data presented as mean + SEM.


A . from mock - organoids display a healthy nucleus labeled by DAPI ( blue ) ( i ) . SARS - CoV - 2 - positive ( green ) display condensed ( middle panel , ii ) fragmented ( bottom panel , iii , arrows ) . At least 75 from two ( n = 2 ) independent batches of organoids were examined . Figures display scale bars .

O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O 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. Cells from mock-organoids display a healthy nucleus labeled by DAPI (blue) (i). SARS-CoV-2-positive cells (green) display condensed (middle panel, ii) and fragmented DNA (bottom panel, iii, arrows). At least 75 cells from two (n=2) independent batches of organoids were examined. Figures display scale bars.


B . Compare to mock organoids ( i ) SARS - CoV - 2 - exposed organoids ( ii ) display increased TUNEL - positive ( displayed as inverted LUT ) at cortical plate that is specified by TUJ - 1 ( magenta ) . At least four organoids from two ( n = 2 ) independent batches of organoids were examined . Figures display scale bars . bar diagram below quantifies frequencies of TUNEL - positive between mock SARS - CoV - 2 - exposed organoids . Four organoids from two ( n = 2 ) independent batches were examined . Unpaired t , * P < 0 . 05 . Data presented as mean + SD .

O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O 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. Compare to mock organoids (i) SARS-CoV-2-exposed organoids (ii) display increased TUNEL-positive cells (displayed as inverted LUT) at the cortical plate that is specified by TUJ-1 (magenta). At least four organoids from two (n=2) independent batches of organoids were examined. Figures display scale bars. The bar diagram below quantifies the frequencies of TUNEL-positive cells between mock and SARS-CoV-2-exposed organoids. Four organoids from two (n=2) independent batches were examined. Unpaired t test, *P<0.05. Data presented as mean + SD.


C . Most of SARS - CoV - 2 - positive ( AB4 , green ) are TUNEL - positive ( i ) some of SARS - CoV - 2 - positive are caspase - positive ( ii ) . Caspase - positive in SARS - CoV - 2 - exposed organoids display pT231Tau localization at soma specified by AT - 180 ( ii ) , which are not observed in in mock organoids ( iii ) . Arrowheads point SARS - CoV - 2 - positive ( AB4 , green ) that are also positive for TUNNEL ( red ) , caspase 3 ( yellow ) Tau AT - 180 ( magenta ) . Figures display scale bars . Bar diagrams at right quantifies proportions of TUNEL caspase - positive among SARS - CoV - 2 - positive . second graph below quantifies proportions of pT231Tau - positive among caspase positive between control exposed groups . At least 400 from 4 organoids two independent ( n = 2 ) batches were examined . Unpaired t , * * P < 0 . 01 . Data presented as mean + . TUNEL - positive in control un - exposed organoids do not contain pT231Tau ( iii ) . Figures display scale bars . Bar diagrams at right quantifies proportions of pT231Tau - positive among TUNNEL - positive between control exposed groups . At least 350 from 4 organoids two independent ( n = 2 ) batches were examined . Unpaired t , * * * P < 0 . 001 . Data presented as mean + .

O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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. Most of the SARS-CoV-2-positive cells (AB4, green) are TUNEL-positive (i) and some of the SARS-CoV-2-positive cells are caspase-positive (ii). Caspase-positive cells in SARS-CoV-2-exposed organoids display pT231Tau localization at the cell soma specified by AT-180 (ii), which are not observed in in mock organoids (iii). Arrowheads point SARS-CoV-2-positive cells (AB4, green) that are also positive for TUNNEL (red), caspase 3 (yellow) and Tau AT-180 (magenta). Figures display scale bars. Bar diagrams at right quantifies proportions of TUNEL and caspase-positive cells among SARS-CoV-2-positive cells. The second graph below quantifies the proportions of pT231Tau-positive cells among caspase positive cells between control and virus exposed groups. At least 400 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, **P<0.01. Data presented as mean + SEM. TUNEL-positive cells in control un-exposed organoids do not contain pT231Tau (iii). Figures display scale bars. Bar diagrams at right quantifies proportions of pT231Tau-positive cells among TUNNEL-positive cells between control and virus exposed groups. At least 350 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, ***P<0.001. Data presented as mean + SEM.


( B ) HA - ATG9A - BirA * was expressed in HCT - 116 ATG13 WT , ATG13 KO or ATG13 KO reconstituted with WT ATG13 or ATG13 ∆ HORMA . were grown in full DMEM media , treated with 50 µM biotin for 12 hrs , followed by detergent lysis incubation with streptavidin resin . graph on right shows of normalized ATG101 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD .

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

(B) HA-ATG9A-BirA* was expressed in HCT-116 ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with WT ATG13 or ATG13 ∆HORMA. Cells were grown in full DMEM media, treated with 50 µM biotin for 12 hrs, followed by detergent lysis and incubation with streptavidin resin. The graph on right shows quantification of normalized ATG101 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests.


( C ) were treated as in panel B but included reconstitution with ATG13 ∆ 2AA mutant . graph on right shows of normalized ATG101 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD . ( right ) .

O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Cells were treated as in panel B but included reconstitution with ATG13 ∆2AA mutant. The graph on right shows quantification of normalized ATG101 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests. (right).


( D ) HA - ATG9A - BirA * was overexpressed in WT ULK1 / 2 Double KO MEFs . were subjected to streptavidin pulldown immunoblotting with indicated antibodies . graph on right shows of normalized ATG13 infrared signal . Mean ± . n = 3 ( biological replicates ) . Significance measured using one - sample t - compared to hypothetical mean of 1 ( right ) . ns = p > 0 . 05 , * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HA-ATG9A-BirA* was overexpressed in WT and ULK1/2 Double KO MEFs. Cells were subjected to streptavidin pulldown and immunoblotting with indicated antibodies. The graph on right shows quantification of normalized ATG13 infrared signal. Mean ± SEM. n=3 (biological replicates). Significance measured using one-sample t-test compared to hypothetical mean of 1 (right). ns=p>0.05, *=p≤0.05, **= p≤0.01, ***=p≤0.001, ****= p≤0.0001.


( A ) - ( G ) Representative of ATG9A colocalization with different organelle autophagy markers . HCT - 116 ATG9A - HA KI - ATG13 WT or ATG13 KO were grown in full DMEM media , fixed labeled with antibodies for HA indicated organelle / markers imaged ( Scaler bar = 10 µm ) . ( H ) of ATG9A colocalization with indicated organelle markers of golgi ( GOLGIN97 ) , mitochondria ( TUFM ) , ER ( PDIA3 ) , endosomal system ( VPS26A ) , early endosome ( EEA1 ) , lysosome ( LAMP1 ) , autophagy adaptor ( p62 ) , autophagosome markers ( ATG16L1 , WIPI4 , ATG2A , LC3II , WIPI2 ) respectively . Mean ± , n = 1 independent experiment with 30 technical replicates . for p62 colocalization ( G ) are from same single independent

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

(A)-(G) Representative images of ATG9A colocalization with different organelle and autophagy markers. HCT-116 ATG9A-HA KI-ATG13 WT or ATG13 KO cells were grown in full DMEM media, fixed and labeled with antibodies for HA and indicated organelle/cellular markers and imaged (Scaler bar=10 µm). (H) Quantification of ATG9A colocalization with indicated organelle markers of golgi (GOLGIN97), mitochondria (TUFM), ER (PDIA3), endosomal system (VPS26A), early endosome (EEA1), lysosome (LAMP1), autophagy adaptor (p62), and autophagosome markers (ATG16L1, WIPI4, ATG2A, LC3II, WIPI2) respectively. Mean ± SEM, n=1 independent experiment with 30 technical replicates. Images for p62 colocalization (G) are from the same single independent experiment


( A ) Endogenous p62 / SQSTM1 level in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO FIP200 KO clones was measured by immunoblotting with indicated antibodies . were grown in full DMEM media , treated with or without 100 nM Bafilomycin for 24 hrs whole lysates were subjected to immunoblotting ( left ) . graph on right shows of normalized p62 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD ( right ) . ( B ) Endogenous p62 / SQSTM1 level in HEK - 293T ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO FIP200 KO clones was measured by immunoblotting with indicated . were grown in full DMEM media , treated with or without 100 nM Bafilomycin for 24 hrs whole lysates were subjected to immunoblotting . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD ( right ) .

O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O 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) Endogenous p62/SQSTM1 level in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated antibodies. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting (left). The graph on right shows quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) Endogenous p62/SQSTM1 level in HEK-293T ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated proteins. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right).


( C ) Confocal of ATG9A colocalization with p62 / SQSTM1 . HCT - 116 ATG9 - HA KI - ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO FIP200 KO were grown in full DMEM media , fixed , labeled with antibodies for HA p62 / SQSTM1 imaged ( Scale bar 10 µm ) . ATG101 KO was stained quantified separately . are from same single independent experiment ( D ) of average surface of p62 / SQSTM1 in C . Mean ± , n = 1 independent experiment with 30 technical replicates . ATG101 KO was stained quantified separately . Mean ± , n = 1 independent experiment with 30 technical replicates . A break was inserted in Y axis to accommodate wide range of p62 / SQSTM1 sizes . ( E ) of ATG9A colocalization with p62 / SQSTM1 . Mean ± , n = 1 independent experiment with 30 technical replicates . ATG101 KO was stained quantified separately . Mean ± , n = 1 independent experiment with 30 technical replicates .

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

(C) Confocal images of ATG9A colocalization with p62/SQSTM1. HCT-116 ATG9-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO cells were grown in full DMEM media, fixed, labeled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar 10 µm). ATG101 KO was stained and quantified separately. Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates.


( A ) Endogenous p62 / SQSTM1 level was measured in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO OR ATG13 KO reconstituted with ATG13 WT , ATG13 ΔHORMA ATG13 Δ2AA by immunoblotting with indicated ( left ) . were grown in full DMEM media whole lysates were subjected to immunoblotting ( left ) . of normalized p62 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD ( right ) . ( B ) HCT - 116 in A were incubated in EBSS for 4 hours then processed analyzed as in A .

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

(A) Endogenous p62/SQSTM1 level was measured in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO OR ATG13 KO cells reconstituted with ATG13 WT, ATG13 ΔHORMA and ATG13 Δ2AA by immunoblotting with indicated proteins (left). Cells were grown in full DMEM media and whole cell lysates were subjected to immunoblotting (left). Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) HCT-116 cells in A were incubated in EBSS for 4 hours then processed and analyzed as in A.


( C ) Confocal of ATG9A colocalization with p62 / SQSTM1 in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO or ATG13 KO reconstituted with ATG13 WT , ATG13 Δ2AA ATG13 ΔHORMA . were grown in full DMEM media , fixed , labelled with antibodies for HA p62 / SQSTM1 imaged ( Scale bar = 10 µm ) . are from same single independent experiment ( D ) of average surface of p62 / SQSTM1 in C . Mean ± , n = 1 independent experiment with 30 technical replicates . A break was inserted in Y axis to accommodate wide range of p62 / SQSTM1 sizes . ( E ) of ATG9A colocalization with p62 / SQSTM1 in C . Mean ± , n = 1 independent experiment with 30 technical replicates .

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

(C) Confocal images of ATG9A colocalization with p62/SQSTM1 in HCT-116 ATG9A-HA KI- ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with ATG13 WT, ATG13 Δ2AA and ATG13 ΔHORMA. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar=10 µm). Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1 in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates.


( B ) Confocal of Ubiquitin colocalization with p62 / SQSTM1 in HCT116 ATG13 WT HCT116 ATG13 KO . were grown in full DMEM media , fixed , labelled with antibodies for Ubiquitin p62 / SQSTM1 imaged ( Scale bar = 10 µm ) ( left ) . of Ubiquitin colocalization with p62 / SQSTM1 ( right ) . Mean ± , n = 1 independent experiment with 30 technical replicates .

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

(B) Confocal images of Ubiquitin colocalization with p62/SQSTM1 in HCT116 ATG13 WT and HCT116 ATG13 KO cells. Cells were grown in full DMEM media, fixed, labelled with antibodies for Ubiquitin and p62/SQSTM1 and imaged (Scale bar=10 µm) (left). Quantification of Ubiquitin colocalization with p62/SQSTM1 (right). Mean ± SEM, n=1 independent experiment with 30 technical replicates.


( C ) Modified chase experiment . HEK293T ATG13 WT HEK293T ATG13 KO stably expressing HA ATG9A - BirA * were labeled with biotin for 24 hours , media was replaced with full DMEM streptavidin pulldown was performed at indicated time points . of normalized p62 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using Student ' s t ( bottom ) . ns = p > 0 . 05 , * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 .

O O O O O O O B-CELL_LINE B-GENEPROD O B-CELL_LINE B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Modified pulse chase experiment. HEK293T ATG13 WT and HEK293T ATG13 KO cells stably expressing HA ATG9A-BirA* were pulse labeled with biotin for 24 hours, media was replaced with full DMEM and streptavidin pulldown was performed at indicated time points. Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using Student's t test (bottom). ns=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****= p≤0.0001.


( C ) Confocal of Venus colocalization with ATG9A ULK1 in HCT116 ATG9A - HA KI ATG13 - ATG101 double KO stably expressing VenusC - ATG101 - 3X FLAG VenusN - ATG13 - Myc or VenusN - ATG13 Δ2AA - Myc . were grown in full DMEM media , fixed , labelled with antibodies for HA ULK1 imaged ( Scale bar = 10 µm ) . of overlap shown as of total Venus ( bottom ) . Mean ± , n = 1 independent experiment with 10 technical replicates .

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

(C) Confocal images of Venus colocalization with ATG9A and ULK1 in HCT116 ATG9A-HA KI ATG13-ATG101 double KO cells stably expressing VenusC-ATG101-3X FLAG and VenusN-ATG13-Myc or VenusN-ATG13 Δ2AA-Myc. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and ULK1 and imaged (Scale bar=10 µm). Quantification of overlap shown as percent of total Venus puncta (bottom). Mean ± SEM, n=1 independent experiment with 10 technical replicates.


( E ) Confocal of EGFP - p62 / SQSTM1 colocalization with HA ATG9A ATG13 in HCT116 ATG9A - HA KI stably expressing EGFP - p62 / SQSTM1 . were grown in full DMEM media with or without 1 μM Wortmannin for time 4 hrs , fixed , labelled with antibodies for HA ATG13 imaged ( Scale bar = 10 µm ) .

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

(E) Confocal images of EGFP-p62/SQSTM1 colocalization with HA ATG9A and ATG13 in HCT116 ATG9A-HA KI cells stably expressing EGFP-p62/SQSTM1. Cells were grown in full DMEM media with or without 1 μM Wortmannin for time 4 hrs, fixed, labelled with antibodies for HA and ATG13 and imaged (Scale bar=10 µm).


A - C . Scanning electron microscopy ( ) of ( A ) nanowires ( VNw ; scale - 5 µm ) ( B ) crude nanosheets ( VSh ; scale - 200 nm ) , ( C ) ultrathin - nanosheets ( Vs ) , scale - 20 µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

A-C. Scanning electron microscopy (SEM) images of (A) nanowires (VNw; scale - 5 µm) and (B) crude nanosheets (VSh; scale - 200 nm), (C) ultrathin-nanosheets (Vs), scale - 20 µm.


C . Michaelis - Menten plot with variation of H2O2 ( 0 - 400 µM ) in presence of Vs ( 20 ng / µL ) , GSH ( 2 mM ) , NADPH ( 0 . 2 mM ) , GR ( 1 . 7 units ) in phosphate buffer ( 100 mM , pH 7 . 4 ) at 25 ° C .

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

C. Michaelis-Menten plot with the variation of H2O2 (0-400 µM) in the presence of Vs (20 ng/µL), GSH (2 mM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C.


D . Michaelis - Menten plot with variation of GSH ( 0 - 7 mM ) in presence of Vs ( 20 ng / μL ) , H2O2 ( 200 µM ) , NADPH ( 0 . 2 mM ) , GR ( 1 . 7 units ) in phosphate buffer ( 100 mM , pH 7 . 4 ) at 25 ° C .

D. Michaelis-Menten plot with the variation of GSH (0-7 mM) in the presence of Vs (20 ng/μL), H2O2 (200 µM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C.


G , H . U1 Grx1 - roGFP2 were supplemented with BSO ( G ) or GSH ( H ) for 16 h to deplete or replenish GSH , respectively . Following this , were treated with Vs for 15 min , exposed to H2O2 , ratiometric response was measured .

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

G, H. U1 Grx1-roGFP2 cells were supplemented with BSO (G) or GSH (H) for 16 h to deplete or replenish GSH, respectively. Following this, cells were treated with Vs for 15 min, exposed to H2O2, and the ratiometric response was measured.


E . U1 - Grx1 - roGFP2 were serum starved for 30 min in presence or absence of Vs sodium selenite ( 0 . 5 nM ) , biosensor response was measured . Data were compared to serum starved control ( C ) .

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

E. U1-Grx1-roGFP2 cells were serum starved for 30 min in the presence or absence of Vs and sodium selenite (0.5 nM), and the biosensor response was measured. Data were compared to serum starved control cells (C).


F . U1 were either serum - starved or supplemented with Se ( 0 . 5 nM ) or Vs ( 0 . 62 ng / μL ) HIV reactivation was measured at 6 h post starvation by gag RT - PCR .

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

F. U1 cells were either serum-starved or supplemented with Se (0.5 nM) or Vs (0.62 ng/μL) and HIV reactivation was measured at 6 h post starvation by gag RT-PCR.


Total isolated from untreated ( UT ) , PMA - treated , Vs - treated Vs + PMA treated U1 was examined by NanoString technology to assess expression of genes responsive to oxidative stress HIV . ( C Heat map showing functional categories of DEGs under PMA / UT , Vs / UT PMA + Vs / UT comparisons . mRNA were normalised using internal control β2 microglobulin ( B2M ) , fold change ( FC ) was calculated using nSolver 4 . 0 software . Genes showing an absolute FC > 1 . 5 , P < 0 . 05 were considered as significantly altered .

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

Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. (C Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered.


Total isolated from untreated ( UT ) , PMA - treated , Vs - treated Vs + PMA treated U1 was examined by NanoString technology to assess expression of genes responsive to oxidative stress HIV . D ) Heat map showing functional categories of DEGs under PMA / UT , Vs / UT PMA + Vs / UT comparisons . mRNA were normalised using internal control β2 microglobulin ( B2M ) , fold change ( FC ) was calculated using nSolver 4 . 0 software . Genes showing an absolute FC > 1 . 5 , P < 0 . 05 were considered as significantly altered .

Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. D) Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered.


B - D . A similar was performed using Jurkat ( CD4 + T ) viral replication was assessed by ( B ) gag RT PCR , ( C ) p24 ELISA in culture supernatant , ( D ) immunoblotting for p24 ( viral capsid ) in whole lysate .

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

B-D. A similar assay was performed using Jurkat (CD4+ T cell line) and viral replication was assessed by (B) gag RT PCR, (C) p24 ELISA in the culture supernatant, and (D) immunoblotting for p24 (viral capsid protein) in the whole cell lysate.


F . CD4 + T purified from human PBMCs ( 3 healthy donors ) were activated , pre - treated with 25 ng / μL Vs for 15 min , infected with 0 . 05 moi of HIV - 1 NL - 4 . 3 . released in supernatant was quantified by p24 ELISA . Vs treatment was repeated every 48 h .

O O B-GENEPROD O B-CELL_TYPE O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O

F. Primary CD4+ T cells purified from human PBMCs (3 healthy donors) were activated, pre-treated with 25 ng/μL Vs for 15 min, and infected with 0.05 moi of HIV-1 NL-4.3. Virus released in supernatant was quantified by p24 ELISA. Vs treatment was repeated every 48 h.


B . Expanded CD4 + T from 3 were cultured in presence of IL - 2 ARVs , with without 25 ng / µL Vs for 21 days . Vs treatment was given for 15 min every 3rd day . HIV transcripts were quantified by RT - qPCR at day 14 , day 21 , at 24 h post - stimulation of cultured for 21 days by prostratin . Limit of detection for RT - qPCR was 3 viral transcripts per million .

O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

B. Expanded CD4+ T cells from 3 patients were cultured in presence of IL-2 and ARVs, with and without 25 ng/µL Vs for 21 days. Vs treatment was given for 15 min every 3rd day. HIV transcripts were quantified by RT-qPCR at day 14, day 21, and at 24 h post-stimulation of cells cultured for 21 days by prostratin. Limit of detection for RT-qPCR was 3 viral transcripts per million cells.


C . At day 21 , were stimulated with 1 µM prostratin for 24 h HIV transcripts were quantified by RT - qPCR . Reduction in viral stimulation in Vs treated samples are represented as values . ND - non determined . D . Aggregate plot for 3 from data ( C ) .

O O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

C. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT-qPCR. Reduction in viral stimulation in Vs treated samples are represented as percentage values. ND - non determined. D. Aggregate plot for 3 patients from data (C).


Mean ± CB2 mRNA in cerebellar extracts of WT ASM - KO mice ( n = 5 mice per group ) .

O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O O O O

Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM-KO mice (n = 5 mice per group).


Western blot against CB2 PSD - 95 ( used as loading control ) graph showing mean ± CB2 in cerebellar extracts of WT ASM - KO mice ( n = 6 mice per group ) .

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

Western blot against CB2 and PSD-95 (used as loading control) and graph showing mean ± SEM CB2 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 6 mice per group).


Mean ± CB1 mRNA in cerebellar extracts of WT ASM - KO mice ( * * P = 0 . 0013 , n = 6 mice per group , Student ' s t - ) .

O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O

Mean ± SEM CB1 mRNA levels in cerebellar extracts of WT and ASM-KO mice (**P = 0.0013, n = 6 mice per group, Student's t-test).


Western blot against CB1 GAPDH ( used as loading control ) graph showing mean ± CB1 in cerebellar extracts of WT ASM - KO mice ( n = 3 mice per group ) .

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

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 3 mice per group).