Figure 5MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( A ) IRGB6 For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times . MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( B ) GBPs , For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times . MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( C ) total ubiquitin , For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times . MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( D ) K63 - linked ubiquitin , For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times . MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( E ) K48 - linked ubiquitin For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times . MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) . IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 or RH for 3 h and subsequently fixed , permeabilized and stained for ( F ) LC3B . For analysis , at least 100 vacuoles were scored . All experiments were performed 3 times . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . All experiments were performed 3 independent times . G ) MEFs were stimulated with IFNγ for 24 h ( 100 U / ml ) or left unstimulated and subsequently infected with Pru , PruΔgra15 and GRA15 complemented parasites for 24 h for measuring parasite growth by luciferase assay . All experiments were performed 3 independent times . H ) Plaque numbers were measured for Pru ( n = 12 ) , PruΔgra15 ( n = 12 ) and GRA15 complemented parasites ( n = 6 ) . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (A) IRGB6 For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (B) GBPs, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (C) total ubiquitin, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (D) K63-linked ubiquitin, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (E) K48-linked ubiquitin For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (F) LC3B. For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.G) MEFs were stimulated with IFNγ for 24 h (100 U/ml) or left unstimulated and subsequently infected with Pru, PruΔgra15 and GRA15 complemented parasites for 24 h for measuring parasite growth by luciferase assay. All experiments were performed 3 independent times.H) Plaque numbers were measured for Pru (n=12), PruΔgra15 (n=12) and GRA15 complemented parasites (n=6).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).


Figure 6A ) Immunoprecipitations and Western blots were performed on MEFs stimulated or not with IFNγ ( 100 U / ml ) and infected with an RH strain expressing type II GRA15 - HA and as a control RH expressing GRA45 - HA . The blots using antibodies against TRAF6 and TRAF2 were made after stripping the first blot . Left panel and right panel were run on a single gel ; vertical white lines indicate excision of irrelevant lanes . B ) IFNγ - stimulated MEFs were infected with Pru , PruΔgra15 and RH for 3 h and subsequently stained for TRAF6 . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as an inset picture with magnification . Experiments were performed 3 times . C ) IRGB6 staining on PVM after infection of wild - type or Traf6 - / - MEFs for 3 h with Pru , PruΔgra15 and RH . On the right - hand side , a representative fluorescent image is shown of IRGB6 coating on the PVM of both wild - type and Traf6 - / - MEF . Scale bar is 10 µm . At least 100 different vacuoles were observed and analyzed for each experiment ( n = 3 ) . D ) Parasite growth was measured 24 h p . i . using luciferase readout from unstimulated and IFNγ - stimulated wild - type , NFκB p65 - / - and Traf6 - / - MEFs . Growth was compared between Pru , PruΔgra15 and PruΔgra15 + GRA15 ( complemented ) strains . Reading from unstimulated cells was considered as 100 % and percentage growth in IFNγ - stimulated cells was expressed relative to unstimulated cells . Experiments were performed three times with each MEF type . E ) Immunoprecipitation and Western blot were performed in MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut and as a control RH expressing GRA45 - HA . Left panel and right panel were run on a single gel ; vertical white lines indicate excision of irrelevant lanes . Full length blots are in the source data for this figure . The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova , respectively . Immunofluorescence analysis of TRAF6 , was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) . Immunofluorescence analysis of IRGB6 , was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) . Immunofluorescence analysis of GBP1 - 5 , was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) . Immunofluorescence analysis of p62 was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) . Immunofluorescence analysis of ubiquitin was done in IFNγ - stimulated MEFs infected with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 3 ) . K ) Plaque assays were performed with RH + GRA15WT , RH + GRA15TRAF2mut or RH + GRA15TRAF2 / 6mut ( n = 4 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Immunoprecipitations and Western blots were performed on MEFs stimulated or not with IFNγ (100 U/ml) and infected with an RH strain expressing type II GRA15-HA and as a control RH expressing GRA45-HA. The blots using antibodies against TRAF6 and TRAF2 were made after stripping the first blot. Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes.B) IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 and RH for 3 h and subsequently stained for TRAF6. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. Experiments were performed 3 times.C) IRGB6 staining on PVM after infection of wild-type or Traf6 -/- MEFs for 3 h with Pru, PruΔgra15 and RH. On the right-hand side, a representative fluorescent image is shown of IRGB6 coating on the PVM of both wild-type and Traf6 -/- MEF. Scale bar is 10 µm. At least 100 different vacuoles were observed and analyzed for each experiment (n=3).D) Parasite growth was measured 24 h p.i. using luciferase readout from unstimulated and IFNγ-stimulated wild-type, NFκB p65-/- and Traf6 -/- MEFs. Growth was compared between Pru, PruΔgra15 and PruΔgra15+GRA15 (complemented) strains. Reading from unstimulated cells was considered as 100% and percentage growth in IFNγ-stimulated cells was expressed relative to unstimulated cells. Experiments were performed three times with each MEF type.E) Immunoprecipitation and Western blot were performed in MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut and as a control RH expressing GRA45-HA. Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes. Full length blots are in the source data for this figure. The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova, respectively.Immunofluorescence analysis of TRAF6, was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).Immunofluorescence analysis of IRGB6, was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).Immunofluorescence analysis of GBP1-5, was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).Immunofluorescence analysis of p62 was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).Immunofluorescence analysis of ubiquitin was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).K) Plaque assays were performed with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=4).


Figure 1A . 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 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 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 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 . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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 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 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 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. 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 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 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.


Figure 2A . 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 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 ) 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 .

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Figure 2A. 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 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) 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.


Figure 3A . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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 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 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.


Figure 2 ( 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 ) 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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) 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 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.


Figure 3 ( 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

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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


Figure 4 ( 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 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 .

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Figure 4(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 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.


Figure 5 ( 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 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 .

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Figure 5(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 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.


Figure 6 ( 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 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 .

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Figure 6(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 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.


Figure 7 ( 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 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 ) .

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Figure 7(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 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).


Figure 1A - 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 .

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Figure 1A-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.


Figure 2C . 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 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 .

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Figure 2C. 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 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.


Figure 3G , 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 .

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Figure 3G, 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.


Figure 4E . 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 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 .

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Figure 4E. 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 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.


Figure 5Total 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 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 .

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Figure 5Total 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 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.


Figure 6B - 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 . 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 .

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Figure 6B-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. 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.


Figure 7B . 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 , 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 ) .

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Figure 7B. 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, 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).


Figure 1Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM - KO mice ( n = 5 mice per group ) . 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 ± 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 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 ) . Immunofluorescence images against CB1 and the following cellular markers : Calbindin for Purkinje cells ( E ) , GFAP for astrocytes ( F ) and F4 / 80 for microglia ( G ) in the cerebellum of WT and ASM - KO mice . DAPI stains cell nuclei . Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells ( shown by white arrows , E ) , astrocytes ( F ) and microglia ( G ) expressed as percentage of the values obtained in WT mice . Graphs to the right show the Mander ' s coefficient that indicates degree of co - localization between CB1 and the cellular markers for Purkinje cells ( E ) , astrocytes ( F ) and microglia ( G ) ( E : * * PFluorecence intensity = 0 . 0082 , * PMander ' s Coefficient = 0 . 0158 ; F : * PMander ' s Coefficient = 0 . 0115 ) ; n = 5 mice per group , Student ' s t - test ) . Scale bar , 100 μm . H . Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age - matched control and ASMD - affected children . Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child ( 16 and 15 replicates in control and ASMD , respectively ) . Scale bar , 10 μm .

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Figure 1Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM-KO mice (n = 5 mice per group).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 ± 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 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).Immunofluorescence images against CB1 and the following cellular markers: Calbindin for Purkinje cells (E), GFAP for astrocytes (F) and F4/80 for microglia (G) in the cerebellum of WT and ASM-KO mice. DAPI stains cell nuclei. Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells (shown by white arrows, E), astrocytes (F) and microglia (G) expressed as percentage of the values obtained in WT mice. Graphs to the right show the Mander's coefficient that indicates degree of co-localization between CB1 and the cellular markers for Purkinje cells (E), astrocytes (F) and microglia (G) (E: **PFluorecence intensity = 0.0082, *PMander's Coefficient = 0.0158; F: *PMander's Coefficient = 0.0115); n = 5 mice per group, Student's t-test). Scale bar, 100 μm.H. Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age-matched control and ASMD-affected children. Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child (16 and 15 replicates in control and ASMD, respectively). Scale bar, 10 μm.


Figure 2Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM - KO mice ( * * * * P < 0 . 0001 , n = 5 independent cultures , Student ' s t - test ) . Western blot against CB1 and GAPDH ( used as loading control ) . Images belong to the same blot but to non - consecutive lanes as indicated by the boxes . Graph showing mean ± SEM CB1 protein levels in cultured neurons from WT and ASM - KO mice ( * * * P = 0 . 0009 , n = 5 independent cultures , Student ' s t - test ) . Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM - KO mice . TOPRO stains cell nuclei . Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units ( left ) or the Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( right ) ( * * * * P < 0 . 0001 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - test ) . Scale bar , 10 μm . Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM - KO mice . Graph shows mean ± SEM Mander ´ s coefficient indicative of the degree of colocalization of CB1 and Lamp1 ( * P = 0 . 0213 , n = 4 mice per group , Student ' s t - test ) . Scale bar , 5 μm . Graphs show mean ± SEM SM levels , expressed as percentage of WT values , in extracts containing the same amount of protein of cultured neurons from WT and ASM - KO mice ( E ) Graphs show mean ± SEM SM levels , expressed as percentage of WT values , in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM ( F ) . Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM ( * * P = 0 . 0021 , n = 5 independent cultures , Student ' s t - test ) . Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM ( n = 2 independent cultures , Student ' s t - test ) Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM . Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units ( left ) or the Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( right ) ( * * * P < 0 . 0001 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - test ) . Scale bar , 10 μm . Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle ( veh ) , with 40μM SM ( SM ) , with 0 , 1μM Bafilomycin ( BafA1 ) or with 40μM SM and 0 , 1μM Bafilomyecin ( SM + BafA1 ) . Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units ( n = 2 independent cultures , one - way ANOVA ) . Mean ± SEM SM levels in cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( * P = 0 . 0157 , n = 3 independent cultures , Student ' s t - test ) . Mean ± SEM CB1 mRNA levels in cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( * * P = 0 . 0015 , n = 5 independent cultures , Student ' s t - test ) . Western blot against CB1 and GAPDH ( used as loading control ) . Images belong to the same blot but to non - consecutive lanes as indicated by the boxes . Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( n = 5 independent cultures , Student ' s t - test ) . Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM - KO mice incubated or not with exogenous SMase . DAPI stains cell nuclei . Graph shows mean ± SEM Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( * P = 0 . 0189 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - test ) . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM-KO mice (****P < 0.0001, n = 5 independent cultures, Student's t-test).Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels in cultured neurons from WT and ASM-KO mice (***P= 0.0009, n = 5 independent cultures, Student's t-test).Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM-KO mice. TOPRO stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (****P < 0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM-KO mice. Graph shows mean ± SEM Mander´s coefficient indicative of the degree of colocalization of CB1 and Lamp1 (*P= 0.0213, n = 4 mice per group, Student's t-test). Scale bar, 5 μm.Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT and ASM-KO mice (E)Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM (F).Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM (**P = 0.0021, n = 5 independent cultures, Student's t-test).Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM (n = 2 independent cultures, Student's t-test)Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (***P <0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle (veh), with 40μM SM (SM), with 0,1μM Bafilomycin (BafA1) or with 40μM SM and 0,1μM Bafilomyecin (SM+BafA1). Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units (n = 2 independent cultures, one - way ANOVA).Mean ± SEM SM levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (*P = 0.0157, n = 3 independent cultures, Student's t-test).Mean ± SEM CB1 mRNA levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (**P= 0.0015, n = 5 independent cultures, Student's t-test).Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM-KO mice incubated or not with exogenous SMase (n = 5 independent cultures, Student's t-test).Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM-KO mice incubated or not with exogenous SMase. DAPI stains cell nuclei. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (*P = 0.0189, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Figure 3Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle or with the indicated concentrations of AEA ( * * * P < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) . Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , the inhibitor of NSM GW , AEA or the combination of GW and AEA ( * * * P < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) . Western blot against NSM and GAPDH ( used as loading control ) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA ( * P = 0 . 0230 , n = 6 independent cultures , Student ' s t - test ) . Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , or with JNJ , PF or URB in the presence or absence of AEA ( * * PJNJ = 0 . 0013 , * PPF = 0 . 0143 , * * * PURB = 0 . 0002 , * * PJNJ + AEA = 0 . 0069 , * * PPF + AEA = 0 . 0077 , * * * PURB + AEA < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) . Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , with PF or with SR141716 + PF ( * PPF = 0 . 0109 , * PSR + PF = 0 . 0343 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) . Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM - KO mice treated with vehicle , AEA , JNJ , PF or URB . Scale bar , 50 μm . Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM - KO mice treated with H2O2 ( as positive control ) , vehicle , AEA , JNJ , PF or URB ( n = 3 independent cultures ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle or with the indicated concentrations of AEA (***P<0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, the inhibitor of NSM GW, AEA or the combination of GW and AEA (***P <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA (*P = 0.0230, n = 6 independent cultures, Student's t-test).Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, or with JNJ, PF or URB in the presence or absence of AEA (**PJNJ = 0.0013, *PPF = 0.0143, ***PURB = 0.0002, **PJNJ+AEA = 0.0069, **PPF+AEA = 0.0077, ***PURB+AEA <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, with PF or with SR141716 + PF (*PPF = 0.0109, *PSR+PF = 0.0343, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM-KO mice treated with vehicle, AEA, JNJ, PF or URB. Scale bar, 50 μm.Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM-KO mice treated with H2O2 (as positive control), vehicle, AEA, JNJ, PF or URB (n = 3 independent cultures).


Figure 4Weekly mean ± SEM body weight of WT and ASM - KO mice treated or not with PF ( * * * P < 0 . 0001 , n = 7 mice per group , statistics reflect the significant difference between the slope of the data in the PF - treated ASM - KO with respect to the other three groups , Linear Regression test ) . Mean ± SEM time spent on the rod in the 4 trials of the rotarod test by WT and ASM - KO mice after two months of vehicle or PF treatment ( * PT3 = 0 . 0488 , * PT4 = 0 . 361 , n = 4 mice per group , two - way ANOVA , Bonferroni post hoc ) . Mean ± SEM time spent in the new arm of the Y maze test by WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWTVeh Vs KO Veh = 0 . 0024 , * * PWT PF Vs KO Veh = 0 . 0016 , * PKO PF Vs KO Veh = 0 . 0391 , n = 9 mice per group , two - way ANOVA , Bonferroni post hoc ) . Mean ± SEM time of immobility in the Tail suspension test of WT and ASM - KO mice after two months of vehicle or PF treatment ( * PWTVeh Vs KO Veh = 0 . 0198 , * * PWT PF Vs KO Veh = 0 . 0067 , n = 6 mice per group , two - way ANOVA , Bonferroni post hoc ) . Mean ± SEM time spent in the open arms of the Elevated Plus Maze by WT and ASM - KO mice after two months of vehicle or PF treatment ( * PWTVeh Vs KO Veh = 0 . 0342 , n = 7 mice per group , two - way ANOVA , Bonferroni post hoc ) . Percentage of survival with respect to weeks of age in WT and ASM - KO mice treated with vehicle or PF ( * * P = 0 . 0031 , n = 5 mice in WT vehicle , WT PF treated and KO vehicle , 6 mice in KO PF treated , Mantel - Cox )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Weekly mean ± SEM body weight of WT and ASM-KO mice treated or not with PF (***P <0.0001, n = 7 mice per group, statistics reflect the significant difference between the slope of the data in the PF-treated ASM-KO with respect to the other three groups, Linear Regression test).Mean ± SEM time spent on the rod in the 4 trials of the rotarod test by WT and ASM-KO mice after two months of vehicle or PF treatment (*PT3 = 0.0488, *PT4 = 0.361, n = 4 mice per group, two - way ANOVA, Bonferroni post hoc).Mean ± SEM time spent in the new arm of the Y maze test by WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0024, **PWT PF Vs KO Veh = 0.0016, *PKO PF Vs KO Veh = 0.0391, n = 9 mice per group, two - way ANOVA, Bonferroni post hoc).Mean ± SEM time of immobility in the Tail suspension test of WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0198, **PWT PF Vs KO Veh = 0.0067, n = 6 mice per group, two - way ANOVA, Bonferroni post hoc).Mean ± SEM time spent in the open arms of the Elevated Plus Maze by WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0342, n = 7 mice per group, two - way ANOVA, Bonferroni post hoc).Percentage of survival with respect to weeks of age in WT and ASM-KO mice treated with vehicle or PF (**P = 0.0031, n = 5 mice in WT vehicle, WT PF treated and KO vehicle, 6 mice in KO PF treated, Mantel-Cox)


Figure 5Mean ± SEM AEA levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWT Veh vs KO Veh = 0 . 0063 ; * * * * PWT Veh vs KO PF < 0 . 0001 ; * * * * PWT PF vs KO PF < 0 . 0001 ; * * * * PKO Veh vs KO PF < 0 . 0001 , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Mean ± SEM SM levels , expressed as percentage of vehicle values , in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * PKO Veh Vs KO PF = 0 . 0465 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Western blot against NSM and GAPDH ( used as loading control ) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( n = 3 mice per group , one - way ANOVA , Bonferroni post hoc ) . Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWTVeh Vs KO Veh = 0 . 0026 , * PKO Veh Vs KO PF = 0 . 0345 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM intensity associated to CB1 , expressed as percentage of values in vehicle - treated mice ( n = 4 mice per group , > 20 Purkinje cells per mouseImmunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graphs show Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( left ) and mean ± SEM lysosomal size ( right ) ( * * * PWT Veh vs . KO Veh = 0 . 0002 ; * * PKO Veh vs . KO PF = 0 . 0052 , n = 4 mice per group , > 20 lysosomes per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 5 μm . Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM number of calbindin positive cells per area ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 00642 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 100 μm . Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM intensity associated to GFAP in arbitrary units ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 0083 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm . Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM number ( left ) and area ( right ) of Iba1 - positive cells ( Left * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 0034 ; Right * * * PWTVeh Vs KO Veh < 0 . 0001 , PKO PF Vs KO Veh = 0 . 00123 ) , n = 4 mice per group , > 20 cells analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Mean ± SEM AEA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWT Veh vs KO Veh = 0.0063; ****PWT Veh vs KO PF < 0.0001; ****PWT PF vs KO PF < 0.0001; ****PKO Veh vs KO PF < 0.0001, n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (***PWTVeh Vs KO Veh <0.0001, *PKO Veh Vs KO PF = 0.0465), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (n = 3 mice per group, one - way ANOVA, Bonferroni post hoc).Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0026, *PKO Veh Vs KO PF = 0.0345), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to CB1, expressed as percentage of values in vehicle-treated mice (n = 4 mice per group, > 20 Purkinje cells per mouseImmunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graphs show Mander´s coefficient for the colocalization of CB1 with Lamp1 (left) and mean ± SEM lysosomal size (right) (***PWT Veh vs. KO Veh = 0.0002; **PKO Veh vs. KO PF = 0.0052, n = 4 mice per group, >20 lysosomes per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 5 μm.Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number of calbindin positive cells per area (***PWTVeh Vs KO Veh <0.0001, **PKO PF Vs KO Veh = 0.00642), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 100 μm. Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to GFAP in arbitrary units (***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0083), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm. Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number (left) and area (right) of Iba1-positive cells (Left ***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0034; Right ***PWTVeh Vs KO Veh < 0.0001, PKO PF Vs KO Veh = 0.00123), n = 4 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


Figure 6H & E staining of cerebellum , hippocampus , cortex and liver from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . Scale bar , 100 μm . Mean ± SEM SM levels , expressed as percentage of vehicle values , in cerebellar , hippocampal , cortical and liver extracts from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . ( * PKOVeh Vs KO 5 = 0 . 0304 , n = 3 mice per group , one - way ANOVA , Bonferroni post hoc ) Immunofluorescences against the microglia marker Iba1 in the cerebellum , hippocampus , cortex or against the macrophage marker F4 / 80 in the liver from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . Graphs show mean ± SEM number of microglia per area unit in cerebellum , hippocampus and cortex or mean ± SEM area of macrophages in the liver ( Hippocampus * * PKOVeh Vs KO 5 = 0 . 0015 , Cortex * PKOVeh Vs KO 5 = 0 . 0364 , Liver * PKOVeh Vs KO 5 = 0 . 0152 , n = 6 mice per group , > 20 cells analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6H&E staining of cerebellum, hippocampus, cortex and liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Scale bar, 100 μm.Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar, hippocampal, cortical and liver extracts from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. (*PKOVeh Vs KO 5 = 0.0304, n = 3 mice per group, one - way ANOVA, Bonferroni post hoc)Immunofluorescences against the microglia marker Iba1 in the cerebellum, hippocampus, cortex or against the macrophage marker F4/80 in the liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Graphs show mean ± SEM number of microglia per area unit in cerebellum, hippocampus and cortex or mean ± SEM area of macrophages in the liver (Hippocampus **PKOVeh Vs KO 5 = 0.0015, Cortex *PKOVeh Vs KO 5 = 0.0364, Liver *PKOVeh Vs KO 5 = 0.0152, n = 6 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.