EMBO/SourceData huggingface dataset:
Figure 6(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments.(B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.
Figure 7(A) Relative values (RV) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected (UN) monkeys, n = 7 for SIVE, n = 12 for SIV and n = 9 for UN, P<0.05.B) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects (NNDS), n = 8 for HAD and n = 10 for NNDS, P<0.0001.(C) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys, as well as those with HAD in brain sections from hippocampus. p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues. Higher magnification views are shown in green insets. Black arrows indicate p62- or ubiquitin-immunoreactive structures within the neuronal cell body or proximal processes, and red arrowheads indicate a dot-like immunoreactivity in the neuropil. Scale bar, 20 µm.
Figure 1IFNγ enhances macrophage host cell death after type I (RH) and type II (Pru) Toxoplasma gondii (Tg) infection. Graphs show cell death as measured by LDH release assay (left) and viability measured by XTT assay (right) in untreated or IFNγ-primed THP-1 cells infected with type I or type II Tg for 24 h.LDH release assays from THP-1 cells left untreated or primed with IFNγ, transfected with siRNA against indicated GBPs or non-targeting control (CTRL) and infected with indicated strains of Tg for 24 h.LDH release assay from primary monocyte-derived macrophages (MDM) left untreated or treated with IFNγ, transfected with siRNA against GBP1 or non-targeting control (CTRL) and infected with indicated strain of Tg for 24 hours. Mean ± SEM of n = 4 donors shown.LDH release assay from indicated wildtype, GBP1 knockout (∆GBP1) or GBP1 reconstituted (∆GBP1+Tet-GBP1) cells infected with type I or type II Tg for 24 hours. Cells were untreated or treated with IFNγ or additionally treated with doxycycline (Dox) as indicated.Realtime propidium iodide (PI) uptake assay from indicated THP-1 cells infected with type I or type II Tg, and untreated or treated with IFNγ and/or Dox as labelled. AU, arbitrary units.LDH release assay from wildtype THP-1 or ∆GBP1 cells stably reconstituted with Dox-inducible expression plasmids of the indicated mutants of GBP1. Cells were pre-treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h.
Figure 2Propidium iodide (PI) uptake in indicated wildtype THP-1 or ∆CASP-1, ∆CASP-4, ∆ASC cells, or THP-1 stably expressing GSDMD-targeting miRNA (GSDMDmiR) infected with the indicated strains of Toxoplasma gondii (Tg) for 24 h. Cells were untreated or treated with IFNγ as indicated. Mean area under the curve (AUC) ± SEM from n = 3 experiments are shown. ns, not significant, from two-way ANOVA following adjustment for multiple comparisons.Real-time apoptosis assay (Annexin V (AnnV)-Glo luminescence) from IFNγ-primed primary human MDMs transfected with non-targeting control (siCTRL; left) or GBP1 siRNA (siGBP1; right) and left uninfected or infected with type I or type II Tg. Depicted is the mean ± SEM of n = 4 donors. AU, arbitrary units.Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h. Cells were untreated or pre-treated with IFNγ before infection. Treatment with TNFα and cycloheximide (CHX) served as positive control for apoptosis. Images are representative of n = 4 biologically independent experiments.Live time-lapse imaging to visualize apoptotic cell death and measure caspase-3/7 activities using a fluorescent caspase substrate. THP-1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg. Plot in (D) shows cumulative caspase-3/7 activity from an 18-hour time-course (mean ± SEM from n = 3 independent experiments). Representative phase-contrast images overlaid with fluorescence signal for caspase-3/7 activity (green) images are shown in (E) from IFNγ-primed THP-1 cells infected with type I Tg captured at indicated times post-infection. Scale bar 20 μm.AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human caspases and infected with type I or type II Tg. Area under the curve (AUC) from a real-time AnnV-Glo luminescence assays similar to those in (B) are plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against CASP8, CASP10 and non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays similar to those in (B) are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.
Figure 3AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human NLR/ALR sensors and infected with type I or type II Toxoplasma gondii (Tg). Area under the curve (AUC) from real-time assays are plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against AIM2 or non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.Representative images (top) from IFNγ-primed THP-1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase-8. Blue, Nuclei; Red, ASC; Green, active caspase-8 (IETD-FITC) and Grey, Tg. Scale bar, 5 μm or 2 μm in the magnified images. Quantification of ASC specks (bottom) in Tg-infected THP-1 WT or ∆CASP1 treated with IFNγ or left untreated at 8 hours post infection, plotted as mean ± SEM from n = 3 independent experiment.Immunoblots from primary MDMs infected with Tg strains for indicated times. Cells were untreated or pre-treated with IFNγ before infection. Images are representative of n = 4 biologically independent experiments.
Figure 4Representative images (A) and quantification (B) from IFNγ-primed THP-1 ∆GBP1 stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or monocyte derived macrophages (MDM) stained for GBP1. Cells were infected with type I or type II Toxoplasma gondii (Tg) for 2 h. Blue, Nuclei; Grey, Tg; Red, mCH-GBP1 or Magenta, GBP1. Scale bar, 5 μm. Graph in (B) shows mean percentage of Tg vacuoles targeted by GBP1 from n = 4 experiments.
Figure 5LDH release assay from indicated THP-1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium (STm) WT or a SPI-1 mutant ΔinvA for 4 h at indicated multiplicities of infection (MOI). Mean ± SEM of n = 3 experiments are shown.Propidium iodide (PI) uptake assay from IFNγ-primed primary MDMs transfected with non-targeting control siRNA (CTRL) or siRNA against GBP1 (left) or indicated lines of THP-1 (right) infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.PI uptake assay from IFNγ-primed primary MDMs transfected with indicated siRNA or non-targeting control (CTRL) and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 4 independent experiments.PI uptake assay from IFNγ-primed or untreated THP-1 ΔCASP-1 cells transfected with indicated siRNA and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows overview of the pathway leading to caspase-1 and caspase-4 activation.LDH release assay from indicated THP-1 cells treated with IFNγ and Dox and infected with STm-GFP for 4 h. Shown is the mean ± SEM of n = 3 experiments.Representative images from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm.quantification (G) from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm. Graph in (G) shows percentage of STm vacuoles targeted by GBP1.Quantification of GBP1 recruitment to STm-GFP containing vacuoles (SCV) in MDMs pre-treated with IFNγ or left untreated (UT). Mean ± 95% CI of GBP1 fluorescence intensity measured on the indicated number (n) of SCVs for each condition from three independent experiments are shown.Representative images from THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S. Cells were infected with STm for 2 h. Blue, Nuclei & STm (Hoechst dye); Red, mCH-GBP1; Green, YFP-CASP4C258S. Scale bar, 5 μm.Quantification of THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S and infected with STm for 2 h. Graph shows percentage of STm vacuoles targeted by GBP1, CASP4 or both from n = 3 experiments. n.d. not detected.
Figure 6Cell death measured using Annexin V (AnnV)-Glo assay of THP-1 ∆CASP1 cells transfected with non-targeting control (CTRL) or GBP1 siRNA and treated as indicated. Cells were primed with IFNγ, transfected with soluble Tg antigen (STAg) or both, or left untreated (UT). Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with soluble Tg antigen (STAg) that was untreated (UT) or treated as indicated. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.Immunoblots from IFNγ-primed THP-1∆CASP-1 or THP-1 ∆ASC cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (-) or transfected with STAg for 12 h. Images are representative of n = 2 independent experiments.AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (UT) or transfected with STAg as indicated for 18 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows an overview of pathways leading to caspase-8 activation.Propidium iodide (PI) uptake assay from MDMs transfected with non-targeting control (CTRL) or GBP1 siRNA and then left untreated (UT), treated with LPS or transfection reagent (Lipofectamine) only or transfected with LPS for 4 h. Area under the curve (AUC) from real-time assays plotted as mean ± SEM from n = 4 independent experiments. Schematic on right shows an overview of pathways leading to caspase-4 activation.
Figure 1a, Diagram of the cell cycle composed of G1, S, G2, and M phases (not to scale). Phase durations were quantified by time-lapse fluorescence microscopy using a PCNA-mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min.b, Mean phase durations in RPE, U2OS, and H9 cell lines. Error bars represent standard deviations.c, Coefficient of variation (CV) of phase durations.d, Percentage of the total variation in cell cycle duration contributed by individual phases.e, Correlations between individual cell cycle phase durations. Sample sizes were adequate to detect correlations n = 125 (RPE), 130 (U2OS), 113 (H9). R2, square of Pearson correlation coefficient.
Figure 3b, Shift in phase durations of RPE cells overexpressing Myc. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: Myc, n = 116c, Pairwise correlation between cell cycle phase durations of RPE cells overexpressing Myc. Number of cells: Myc, n = 116e, Shift in phase durations of RPE cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 115f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 115h, Shift in phase durations of RPE cells incubated at 34°C. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: 34°C, n = 122i, Pairwise correlation between phase durations for cells incubated at 34°C. Number of cells: 34°C, n = 122k, Shift in phase durations of RPE cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 119.l, Pairwise correlation between phase durations for cells treated with NCS. Number of cells: NCS, n = 119.
Figure 4g, Shifts in phase durations for RPE cells treated with 2 μM CVT-313. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 117 cells).h, Pairwise correlation between cell cycle phase durations upon treatment with CVT-313.j, Shifts in phase durations for RPE cells overexpressing cyclin D. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 113 cells).k, Pairwise correlation between cell cycle phase durations upon overexpression of cyclin D. P indicates P-value.
Figure 5b, Shift in phase durations of U2OS cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 114c, Pairwise correlation between phase durations for U2OS cells treated with NCS. Number of cells: NCS, n = 114e, Shift in phase durations of U2OS cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 153.f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 153.
Figure 1B. Comparison of ΔPrP expression levels. Representative immunoblot is shown for detection of (3F4)MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4. ΔPrPs are similar in expression level glycosylation appearance, except for Δ159-167 which has an extra fragment larger than the diglycoform (lane 7, arrow).C. ΔPrPs are not converted into PK-resistant PrP in prion-infected cells. 22L-ScN2a cells were transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, and cell lysates digested with PK or not. Immunoblot was done using mAb 3F4.. D. Pilot study for dominant-negative inhibition (DNI) of Δ159 and Δ159-175. DNI was assessed by co-transfecting 22L-ScN2a cells with (3F4)MoPrP and Δ159 or Δ159-175, respectively, and testing for PK-resistant (3F4)MoPrP. Left panel shows representative immunoblot (mAb 3F4) and right panel the statistical analysis of quantified PK-res levels of a triplicate experiment. Empty pcDNA3.1 plasmid was used as control for co-transfection in lane 1. The error bars indicate standard deviation. *, p<0.05.
Figure 2A. N-linked glycans of ΔPrPs are not complex-type. Immunoblot comparing EndoH- and non-digested lysates of ΔPrP159 and Δ159-175 transfected cells. Left panel was probed with mAb 3F4 mAb, detecting transiently expressed ΔPrPs. On the right panel, the membrane was re-probed with mAb 4H11, detecting total PrP. Arrowheads depict deglycosylated ΔPrPs.B. The additional band of Δ159-167 represents a third N-glycan sensitive to EndoH. Upon EndoH digestion, all bands converge into a non-glycoform band (lane 3 and 4). In Δ159-167(169) the third glycosylation site is not present anymore.C. ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface. Confocal microscopy images of N2a cells transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, either permeabilized (right) or non-permeabilized (left) with 0.2% TX100 after fixation. Cells were immuno-labeled with mAb 3F4. (3F4)MoPrP-expressing cells show bright immunopositivity on the cell surface, whereas ΔPrPs are not observed on the cell surface. Also intracellular staining differs strongly between (3F4)MoPrP and ΔPrP expressing cells Scale bars, 25 µm.
Figure 3A. ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159-175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non-infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30.B. ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159-175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.
Figure 4A. ΔPrPs are substantially soluble in detergent without GdnHCl denaturation. Immunoblots probed with mAbs 3F4 (left) or 4H11 (right, membrane re-probed) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159-175. Det denotes detergent phase, Aq aqueous phase.B. ΔPrPs are highly hydrophobic after denaturation with GdnHCl. Representative immunoblot of TX114-treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159-175 almost exclusively in detergent phase (lanes 1-4). PIPLC treatment (+, even lanes) was done after denaturation with GdnHCl or not (−, uneven lanes). PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions.C. Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion. After further diluting out GdnHCl by phase-separation and removal of the aqueous phase containing GdnHCl, PIPLC was able to digest ΔPrPs. Left two panels (upper mAb 3F4, lower 4H11) are shorter expositions, right panels longer expositions to show also difference in PrP levels in the detergent phase. Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion (e.g. upper-left panel, lanes 1, 3 vs. 2, 4). Migration of ΔPrPs in the aqueous phase of PIPLC-digested samples was slightly slower than that of PIPLC-undigested samples (−) (upper-right panel, lane 9 vs. 10).
Figure 5A. Degradation of ΔPrPs by lysosomal and proteasomal systems. (Left) N2a cells transiently transfected with Δ159 or Δ159-175 were treated for 7 hours with inhibitors either for lysosomal degradation (bafilomycin A1, Baf, 120 nM), autophagy (3-methyladenine, 3MA, 10 mM), or the proteasome (MG132, 10 µM) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159-175 (mAb 3F4). Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs, respectively. An increment in diglycoforms is observed in Baf- and 3MA-treated cells, whereas in MG132-treated cells also the non-glycoforms are increased. (Right) A graph showing quantification of all PrP bands by densitometric analysis. Data from 3 independent (one in duplicate) experiments for Δ159 and 2 independent (one in duplicate) for Δ159-175 were statistically analyzed for mean and standard deviation (error bars).B. ΔPrPs can be localized in LAMP1-immunopositive vesicles. Immunofluorescence analysis shows distribution of ΔPrP (mAb 3F4) and LAMP1 in cells with or without 6M GdnHCl antigen-retrieval treatment. N2a cells transfected with ΔPrP159 were fixed, permeabilized and incubated without (upper panel) or with 6M GdnHCl (lower panel). With GndHCl treatment ΔPrP is diffusely distributed (arrowheads) and co-localizes poorly with LAMP1. After GdnHCl treatment (lower panel), bright 3F4-immunopositive puncta (arrows) are observed which partly co-localize with LAMP1-positive structures (merge). Scale bars, 10 µm.
Figure 6A. DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrPSc moiety of (3F4)MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrPSc levels were calculated as proportion of PK-resistant (3F4)MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159-165, Δ159-167 and Δ159-167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars).B. DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171-175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171-175 exerted only inefficient DNI, similar to Δ159-175 (right panel).