EMBO/SourceData huggingface dataset:
Figure 8(A) Early stage Drosophila egg chambers showing giant germ-line nurse cells surrounded by E-cadherin-positive follicle cell epithelium. A follicle cell undergoing mitotic rounding is highlighted with an asterisk (*). Scale bar ~3µm. n > 3 independent biological replicates.(B) High resolution imaging of the F-actin cortex, E-cadherin, and phospho-MyoII in a mitotically rounded follicle cell. Note maintenance of adherens junctions including both E-cad and the contractile actomyosin ring during mitotic rounding. Asterisks or pH3 staining indicate a single mitotic cell. Scale bar ~3µm. n > 4 independent biological replicates.
Figure 1(A) DNA damage induces H2AX ADP-ribosylation. 293T cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes). ADP-ribosylated proteins were IPed with anti-ADP-ribose (anti-ADPR) antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.(B) H2AX is ADP-ribosylated at glutamate 141 (E141). shPARG HCT116 cells were treated with H2O2 (2 mM in PBS, 5 minutes) and ADP-ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15.0109 Da, an increment that can be readily distinguished by mass spectrometry. Fragmentation of the NH2OH-derivatized peptides yielded typical b- and y-ion series, allowing easy localization of ADP-ribosylation sites. Glutamate 141 (E141) is indicated by an asterisk.(C) The E141A mutation abolishes the ADP-ribosylation of H2AX. Empty vector (EV), wild type (WT) or the E141A mutant (E141A) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2. ADP-ribosylated proteins were IPed with anti-ADPR antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.(D) ADP-ribosylation on H2AX is suppressed by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. The samples were examined by IP and Western blotting.(E) PARP1, but not PARP2, mediates the ADP-ribosylation on H2AX. Wild type (WT) cells, PARP1-null cells or PARP2-null cells were treated with H2O2.
Figure 2(A) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 or MMS. Cell viability was measured after 24 hours using MTT assay. Values are mean ± SD of three assays.(B) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant (E141A) H2AX were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes), subsequently; the cells were cultured in fresh DMEM medium for 2 weeks. The numbers of colony formation were counted. Values are mean ± SD of three assays.(C) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant (E141A). U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes) and harvested at the indicated recovery time points. Then FPG-modified comet assay was performed. NT: no treatment. Representative comet tails were shown. The olive tail moments (OTM) were summarized from at least 50 cells in each experiment. Values are mean ± SD of three assays.(D) DNA replication in U2OS cells expressing the E141A mutant (E141A) is suppressed. Cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes), followed by BrdU incorporation (30 μM in medium, 30 minutes). Representative images show BrdU-positive cells in cells expressing WT H2AX or E141A following genotoxic stress. Nuclei were stained with DAPI (blue). The bottom panel histogram shows the percentage of BrdU incorporation in each sample, Values are mean ± SD of three assays.(E) Generation of AP sites is suppressed in cells expressing E141A. Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes. AP sites were calculated according to the standard curve. Values are mean ± SD of three assays.
Figure 3(A) ADP-ribosylation mediates the recruitment of Neil3. GFP-Neil3 was expressed in U2OS cells, and cells were treated with olaparib (1 µM, 1 hour) or mock (0.1 % DMSO in medium, 1 hour). Following laser microirradiation treatment, the recruitment of Neil3 was examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.(B) The GRFs of Neil3 alone is sufficient to be recruited to the sites of DNA damage. Truncated mutants of Neil3 were generated and fused with a GFP tag. Following laser microirradiation treatment, the recruitment of truncated mutants Neil3 were examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.(C) PARP inhibitor treatment (olaparib: 1 µM, 1 hour) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions. The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.(D) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. Oxidative damage was induced by KillerRed (KR) system with the exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 minutes. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of Neil3 foci co-localized with KR was quantified.
Figure 4(A) The GRFs of Neil3 recognizes PAR. The recombinant GST fusion proteins were incubated with PAR and the protein-PAR complex was pulled down by glutathione agarose beads. Samples were serially diluted three-fold, spotted onto the nitrocellulose membrane, and subjected to dot blotting using anti-PAR antibody (top panel). FL represents full length Neil3, N-ter represents N-terminal Neil3 (1-300 aa), Mid represents middle Neil3 (300-404 aa), and GRFs represents C-terminal Neil3 GRFs domain (480-606 aa). Recombinant GST protein was used as the negative control (NC). The GST fusion proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (middle panel). The relative PAR-binding in the pull down assay was summarized in the graph (bottom panel). Values are mean ± SD of three assays.(B) Tandem co-immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP-ribosylated H2AX but not the E141A mutant H2AX.(C,D) The GRFs of Neil3 is sufficient and required for the interaction with ADP-ribosylated H2AX. Tandem co-immunoprecipitation and Western blotting assays were used(E,F) The relocation kinetic of full-length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed. GFP-tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells, and relocation kinetics was monitored in a time course following laser microirradiation (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.(G,H) The recruitment of full-length Neil3 or the GRFs of Neil3 to the KR-induced oxidative lesions is mediated by H2AX ADP-ribosylation (left panel). Scale bars, 2 μm. The foci were indicated with white arrowheads. Scale bars, 0.5 μm. Right panel: the percentage of Neil3 or GRFs Neil3 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.
Figure 5(A) PARP inhibition facilitates γH2AX following oxidative stress. The cells were pre-treated with olaparib (1 µM, 1 hour) prior to H2O2 stimulation. 0.1 % DMSO in medium for 1 hour as mock. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.(B) The effect of H2AX E141 ADP-ribosylation on γH2AX following oxidative stress. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.(C) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed-induced oxidative DNA lesions (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Scale bar, 0.5 μm. Right panel: the relative intensity of γH2AX at the site of tetR (intensity of γH2AX at the site of tetR/intensity of γH2AX area away from tetR) was quantified. Mean value with SD is the relative intensity of γH2AX in 20 cells.(D) The level of MDC1 at KillerRed-induced oxidative DNA lesions is increased in the cells expressing the E141A mutant (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of MDC1 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.(E) The recruitment of Neil3/MDC1 chimera but not wild type Neil3 to the KR-induced oxidative lesions in cells expressing the E141A mutant (left panel). Neil3/MDC1 chimera: replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: The percentage of detected protein foci co-localized with KR was quantified. Mean value with SD is from 20 cells.(F) AP sites were measured in cells expressing the E141A mutant or the Neil3/MDC1 chimera, according to the standard curve. Values are mean ± SD of three assays.(G) DNA replication of U2OS cells expression with individual wild type (WT), the H2AX KO + WT, the E141A mutant (E141A) or the Neil3/MDC1 Chimera (Chimera) were analyzed by BrdU incorporation following treatment with H2O2, MMS or mock (PBS). Represented images are shown in the left panel. The graph shows percentages of BrdU-positive cells expressing WT, E141A or Chimera (right panel).
Figure 1A) HFFs were pre-stimulated with IFNγ (10U/mL) for 24 h. Plaque assays were performed for each strain and each condition. Plaque number and area loss were calculated 4 days p.i. for RH and 6 days p.i. for Pru. Assays were performed with RH (n=4), Pru (n=4).B) Relative parasite growth was measured 24 h p.i. in IFNγ-stimulated and unstimulated HFFs by luciferase assay. Growth of each strain in IFNγ-stimulated HFFs is expressed relative to growth in unstimulated HFFs. Experiments were performed with RH (n=4), Pru (n=4).C) Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U/mL IFNγ for 24 h. Following stimulation, HFFs were infected with either RH (MOI=1) or Pru (MOI=3) for another 24 h. Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei. The number of PVs in 5-6 fields of the coverslips were counted for each condition and normalized with the number of host cells in each field. Experiments were performed with RH (n=3), Pru (n=3). Representative images of percentage infected cells for each strain and condition are provided. Scale bar is 100 µm.D) Parasites per vacuole were determined 24 h p.i. with similar conditions and staining as described in (C) except MOIs used were 0.5 and 1 for RH (n=3) and Pru (n=3), respectively.E) Plaque assays were performed similarly as described for (a) with Pru WT (n=20 for plaque number and n=7 for plaque area), PruΔgra15 (n=20 for plaque number and n=7 for plaque area), PruΔgra15+GRA15 complemented (n=12 for plaque number and n=7 for plaque area).F) Plaque assays were performed similarly as described for (A) with RH (n=5 for plaque number and n=3 for plaque area) and RH+GRA15II (n=5 for plaque number and n=3 for plaque area).G) Relative parasite growth was measured as described in (B) with Pru WT (n=12), PruΔgra15 (n=12), PruΔgra15+GRA15 complemented (n=8).H) Number of PVs was calculated as described in (C) with Pru WT (n=3), PruΔgra15 (n=3), RH (n=3) and RH+GRA15II (n=3) and normalized with the number of host cells in each field.
Figure 3A) HFFs were stimulated with IFNγ (10 U/mL for 24 h) or left unstimulated and subsequently treated with PYR41 (1 µM) for 2 h prior to infection. Cells were washed and subsequently infected with Pru parasites (MOI=3) for 3 h. The percentage of vacuoles that stained positive for total ubiquitin, p62 or LC3B was determined (n=3).For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, p62 was calculated.For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, LC3B was calculated.For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, GABARAP was calculated.For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for p62 LC3B was calculated.For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for p62 GABARAP was calculated.
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 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.
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.