EMBO/SourceData huggingface dataset:
Figure 5A-B. Total fresh weight of 21 days old non-colonized- (Mock) and SA190-colonized (SA190) Col-0, tip2;1 and qpip plants grown for 16 days under normal (½ MS) or drought stress proxy conditions (½ MS + 25% PEG). Data information: All plots represent the mean of 3 biological replicates. Error bars represent SE. Boxplot shows data from three independent biological replicates (technical replicates n = (A) 16-20, (B) 8-20 where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001). Asterisks indicate a statistical difference based on the Student's t-test *** p < 0.001).G. Transpiration rate of mock- or SA190- colonized 21 days old plants after transfer of 5-day-old seedlings from ½ MS to ½ MS +/- 25 % PEG. Transpiration was quantified as rate of water loss by calculating the changes in fresh weight for 30 min with reading at every 5 min interval.H-I. Comparison of stomatal aperture of SA190- and non-colonized 21-days old plants grown for 16 days under non-stress (½ MS) or drought stress proxy (½ MS + 25% PEG) conditions. Scale bar = 20 µm. Data information: All plots represent the mean of 3 biological replicates. Error bars represent SE. Boxplot shows data from three independent biological replicates (technical replicates n = (I) 94-104, where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001). Asterisks indicate a statistical difference based on the Student's t-test *** p < 0.001).
Figure 2C-E, boxplots showing the serum level of phenylacetylglutamine, mandelic acid and homovanillic acid in different CHF subgroups in the REM-HF cohort. NGT: n = 23; NGT+CHF: n = 48; Prediabetes+CHF: n = 83; T2D+CHF: n = 56; Prediabetes+CHF+CKD: n = 34; T2D+CHF+CKD: n = 16 (biological replicates). Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.F, the boxplot showing the serum level of ImP in the Swedish prediabetes versus the Chinese REM-HF cohorts. For Swedish prediabetes cohort, NGT: n = 125; prediabetes: n = 262; T2D: n = 17 (biological replicates). For Chinese REM-HF cohort: NGT: n = 23; NGT+CHF: n = 48; Prediabetes+CHF: n = 83; T2D+CHF: n = 56; Prediabetes+CHF+CKD: n = 34; T2D+CHF+CKD: n = 16. Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.G, differences in ImP when stratified by left ventricular ejection fraction (LVEF; two-tailed Wilcoxon rank-sum tests). NGT: n = 23; HFpEF: LVEF ≥50, n = 66; HFmEF: 40<LVEF<50, n = 68; HFrEF: LVEF ≤40, n = 103 participants. Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.
Figure 3B, representative JC-1 staining images showing red fluorescence of JC-1 aggregates and green signal of monomers. Scale bars: 20 μm.
Figure 5A, the Kaplan-Meier survival curve based on the Framingham and GWTG-HF risk scores. 240 participants with clinical variables for Framingham and GWTG-HF risk scores calculation were included in survival analysis (biological replicates). P values were calculated based on the log-rank test.B, the Kaplan-Meier survival curve based on each microbially associated metabolite (grey colors except for imidazole propionic acid) and a combined metabolite risk score. 244 participants who completed the follow-up study were included in survival analysis (biological replicates). P values were calculated based on the log-rank test.C, the time-dependent receiver operating characteristic (ROC) curves for the performance of GWTG-HF scores, metabolite-based risk score and the combination of both scores in CHF prognosis. The AUC values were determined by bootstrap resampling with 1,000 iterations.
Figure 1(A, B) CMH2DCFDA staining (A) and data quantification (B) showing H2O2 level in different aged fly brains with and without treatment with the indicated RET inhibitors (n=5 per group).(I) Dose-dependent effect of CPT on RET-ROS in mitochondria isolated from 45 days old flies and respiring under RET condition (n=4).(K) CMH2DCFDA staining showing H2O2 level in young flies treated with DES or DES + CPT (n=5 per group).
Figure 2(E-H) Survival curves comparing control flies and flies fed with food containing 5 µM CPT (E), 10 µM CPT (**p<0.01 logrank, **p<0.01 Wang Allison) (F), or 20 µM CPT (**p<0.01 logrank, **p<0.01 Wang Allison) (G) from 35 days onwards. Bar graphs (H) show quantification of mean and maximal lifespan (n=3 groups, 20-25 flies per group), using single factor ANOVA with Scheffe's analysis as a post hoc test. : Data are representative of at least three repeats. H) Data are shown as mean ± Survival curves were analyzed with logrank test along with Wang Allison test for maximum lifespan. Asterisks indicate statistical significance (**p< 0.01, *p< 0.05). Dashed lines mark time points of 50% survival.
Figure 3(C) Images and data quantification showing accumulation of protein aggregates in the brains of aged flies with or without CPT treatment (n=5).(D) Images and data quantification showing DA neuron mitochondrial morphology in the brains of aged flies with or without CPT treatment (n=5).Images (E) showing 4-HNE levels within or near TH-positive DA neurons in young flies, old flies, and old flies treated with CPT (n=4 sets, 5 samples per set). Arrows point to HNE signals co-labelled with TH- positive DA neurons.
Figure 4(D) CMH2DCFDA staining of H2O2 and quantification of signal intensity in the muscle tissues of 50-day old control flies and flies with NDUFS2 or NDUFS3 knockdown (n=5 per group).(F) Survival curves of wild type flies with or without NMN treatment from 35 days onwards (**p<0.01 logrank, **P<0.01 Wang Allison) (n=3 groups, 20-25 flies per group).(H) Images and data quantification showing accumulation of protein aggregates in the muscle of young flies, old flies, and old flies treated with CPT (n=5 per group).
Figure 5(A, B) Survival curves showing effect of neuronal dSirt2 knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group). (C, D) Survival curves showing effect of muscle dFoxo knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group). (E, F) Survival curves showing effect of neuronal ATG1 knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group).(H) Images and data quantification in single factor ANOVA with Scheffe's analysis as a post hoc test showing lysotracker staining in the muscle of young flies, old flies, and old flies treated with CPT (n=5 per group).
Figure 6(A, B) Survival curves showing effect of CPT treatment on the lifespan of Mhc>APP.C99 flies (**p<0.01 logrank, **p<0.01 Wang Allison) (A) and elav>APP.BACE flies (**p<0.01 logrank, **p<0.01 Wang Allison) (B) (n=3 groups, 20-25 flies per group).(E) Immunofluorescence images and data quantification showing effect of CPT on the formation of ubiquitin- and 6E10-positive amyloid aggregates in the brain of elav>APP.BACE flies (n=5).(G) Quantification of the effect of CPT on DA neuron number in the PPL1 cluster of elav>APP/BACE fly brain (n=4 sets, 5 brains per set).
Figure 7(A, B) Quantification of effect of CPT on ROS level as detected by staining with the H2O2 dye CMH2DCFDA (A), and on NAD+/NADH ratio (B) in control, FAD, and DS iPSC-derived neurons.(D) Survival curves showing effect of CPT on lifespan of PS19 tauopathy mice (*p<0.05 logrank) (n=9 in vehicle group, 8 in CPT group).
Figure 1B) Kaplan-Meier curve showing % of mice surviving S. pneumoniae infection. Data was analyzed using Kaplan-Meier statistical analysis. N = 5 - 10 mice/group. Data information: Data presented show mean ± SEM of biological replicates.F) Bar graphs comparing bacterial burden (CFU) in the lung and spleen at 48h, 72h and 7d post-S. pneumoniae infection in unvaccinated and BCG vaccinated mice. N = 3 - 5 mice/group. Data information: Data presented in F) show mean ± SEM of biological replicates. Statistical analysis for F) were two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01; ****p < 0.0001.
Figure 2B) Dot plots of gated mononuclear cells from the BAL of control and BCG vaccinated mice. Analysis was performed utilizing default FlowJo V.10 software settings. C) Line graphs comparing absolute neutrophil cell counts in the airways at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (C, were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.D) Dot plots of gated mononuclear cells from the airways of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. Line graphs comparing absolute AM cell counts in the airways at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.G) Line graphs comparing the levels of neutrophilic chemokines in the BAL fluid at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.
Figure 3A) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. B) Line graphs comparing absolute neutrophil cell counts in the lung at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (B, were two-way ANOVA with Sidak's multiple comparisons test.C) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. D - Line graphs comparing absolute AM, cell counts in the lung at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test.Line graphs comparing absolute IM and MDM cell counts in the lung at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test.
Figure 4C) Bar graphs comparing the killing rates of phagocytosed S. pneumoniae by AMs at 1 and 2h post-ex vivo S. pneumoniae infection. N = 3 replicates/group. Data information: Data presented in C, represent mean ± SEM of technical replicates. Statistical analysis for C, were two-tailed unpaired t-tests *p < 0.05.E) Line graphs comparing changes in body weight following S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 4 mice/group. Data information: Data presented represent mean ± SEM of technical replicates. Statistical analysis for (E) was two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05.G) Bar graphs comparing bacterial counts (CFU) in the lung and spleen at 72h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented represent mean ± SEM of technical replicates. Statistical analysis for G) were two-tailed unpaired t-tests, *p < 0.05.
Figure 5B) Line graphs comparing changes in body weight following S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented in (B represent mean ± SEM of biological replicates. Statistical analysis for (B were two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01; ****p < 0.0001.C) Bar graphs comparing bacterial counts (CFU) in the lung and spleen at 72h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented in C) represent mean ± SEM of biological replicates. Statistical analysis for C) were two-way ANOVA with Sidak's multiple comparisons test, (C) was two-tailed unpaired t-test. *p < 0.05; **p < 0.01; ****p < 0.0001.
Figure 6B) Line graphs comparing changes in body weight following S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (B was two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01.D) Bar graphs comparing bacterial counts (CFU) in the lung and spleen at 48h post- S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (D) was one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05; **p < 0.01.
Figure 1A/B Lysates from parental, SMCR8 KO (A) or C9orf72 KO (B) 293T cells were analyzed by immunoblotting. * indicates unspecific protein band detected by C9orf72 antibody. Calnexin served as loading control.C Rescue of C9orf72 protein levels in SMCR8 KO cells after transient overexpression of untagged- or C-terminal tagged SMCR8 analyzed by immunoblotting. * indicates unspecific protein band detected by C9orf72 antibody. Calnexin served as loading control.G Parental and SMCR8 KO cells were subjected to a cycloheximide (CHX) chase in the absence or presence of DMSO or Btz. * indicates unspecific protein band detected by C9orf72 antibody. α‑tubulin served as loading control. NRF2 confirmed proteasomal inhibition by Btz.
Figure 2C SMCR8 KO cells were treated with DMSO or MLN7243 prior to fixation and immunostaining with an anti-C9orf72 antibody. Untreated parental and C9orf72 KO cells served as controls. Scale bar, 10 µm.D Empty or stably C9orf72-HA expressing SMCR8 KO cells were treated with Btz or DMSO followed by lysis and immunoblotting. Untreated parental cells served as control. Calnexin served as loading control. NRF2 confirmed proteasomal inhibition by Btz.E Parental and SMCR8 KO cells stably expressing C9orf72-HA were subjected to CHX chase in absence and presence of DMSO or Btz. NRF2 confirmed proteasomal inhibition by Btz.F Lysates from empty or stably C9orf72-HA expressing SMCR8 KO cells treated with DMSO or Btz were subjected to HA-IP under denaturing conditions in the absence and presence of the catalytic domain of the deubiquitinase USP2.
Figure 3D Lysates from C9orf72-HA expressing parental and SMCR8 KO cells treated with DMSO or Btz were subjected to HA-IP followed by immunoblot analysis. Empty parental and SMCR8 KO served as controls. : NRF2 confirmed proteasomal inhibition by Btz.E SMCR8 KO cells expressing C9orf72-HA were transiently transfected with GFP-UBR5 or GFP and treated with DMSO or Btz. Lysates were incubated with Streptavidin agarose coupled to biotinylated GFP VHH nanobodies and bound proteins were analyzed by immunoblotting.
Figure 4A SMCR8 KO cells stably overexpressing C9orf72-HA were treated with Btz and lysates were subjected to HA-IP under mild conditions in the absence and presence of the catalytic domain of the deubiquitinase USP2. FK2 confirmed deubiquitination by USP2.D SMCR8 KO cells transiently overexpressing GFP, GFP-C9orf72 WT or GFP-C9orf72 mutant K156R, K388R, K414R, K422R, K424R, K425R, K428R (KR7) were treated with Btz followed by streptavidin pulldown with biotinylated GFP VHH nanobodies under denaturing conditions.
Figure 5B SMCR8 KO cells stably expressing GFP-C9orf72 were treated with DMSO or Btz followed by denaturing lysis and differential USP2 treatment. Lysates were incubated with biotinylated GFP VHH nanobodies coupled to Streptavidin agarose and enriched proteins were analyzed by immunoblotting.D SMCR8 KO cells were transiently transfected with siUBR4/5, siCTRL or left untreated but grown in the presence of Btz. Lysates were subjected to immunoblotting. Quantification of C9orf72 levels in SMCR8 KO cells treated with siUBR4/5 or siCTRL. n = 3 biological replicates. Data represent mean ± SD. Statistical analysis of C9orf72/α‑tubulin ratio was performed using one-tailed, unpaired Student's t-test. * p < 0.05. Representative Western blot is shown beside. α‑tubulin served as loading control.
Figure 1(D) Western blot analysis of ADAR1 expression in E13.5 immortalized MEFs of the indicated genotypes +/- interferon-β (IFNβ). Data from two independent cell lines per genotype.(G) Survival analysis of mice of the indicated genotypes; number as indicated for each genotype. Statistical difference determined by Log-rank (Mantel-Cox) test.(H) Total white blood cell counts (WBC) in peripheral blood (PB).(J-L) Peripheral blood (J) red blood cell (RBC), (K) hematocrit (HCT) and (L) platelets.
Figure 2(C) Survival analysis of mice of the indicated genotypes; number as indicated for each genotype. No statistical difference between genotypes by Log-rank (Mantel-Cox) test. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4).(E) Peripheral blood leukocyte populations (by lineage) between genotypes at day 0 and day 28. ; R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM, spleen and thymic analysis at day 28 post tamoxifen treatment(G) Differential analysis of leukocyte populations in the BM. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM analysis at day 28 post tamoxifen treatment(H) BM erythroid cells. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM, analysis at day 28 post tamoxifen treatment(J) Differential analysis of leukocyte populations in the spleen. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). spleen analysis at day 28 post tamoxifen treatment;(K) Thymic cellularity. (L) Differential analysis of thymocyte populations in the thymus. ( R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). thymic analysis at day 28 post tamoxifen treatment(N) qPCR (SYBR green) based analysis of indicated gene expression in BM. Data expressed as mean +/- SEM gene expression related to Ppia expression. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4).
Figure 4, (C) survival analysis of mice of the indicated genotypes.(E) survival of mice from inbreeding to generate P195A/- animals.(H) survival of pups derived from breeding of a Adar1P195A/P195A to a Adar1+/- animal; number as indicated for each genotype.(K) survival of pups derived from breeding an Adar1P195A/- to an Adar1P195A/+ genotype; number indicated for each genotype. Inset photo: 35-day-old Adar1P195A/- male bred from Adar1P195A/P195A x Adar1+/- parents (data in Panel F); sibling 29-day-old Adar1P195A/+ and Adar1P195A/- male bred from an Adar1P195A/- x Adar1P195A/+ (data in Panel I).
Figure 5(B) survival of Adar1P195A/E861AIfih1-/- and littermates (all Ifih1-/-).(D) survival of Adar1P195A/-Ifih1-/- and littermates (all Ifih1-/-).
Figure 6(F) Individual plots of the normalised expression of ISGs and ISR genes in the kidney of the indicated genotypes using Taqman-based qPCR.(G) Western blot of PKR, phospho-eIF2α and total eIF2α levels in whole kidney lysates derived from the indicated genotypes; same mice as used for panel F. Relative phospho-eIF2α expression was calculated against total eIF2α normalised to the average of P195A/+ samples using ImageJ Fiji. All images are from the same gel and re-probed for different antibodies.
Figure 7(E) Viability of cell lines with and without tamoxifen treatment (isogenic pairs) over 14 days of treatment (n=3 independent lines per genotype; biological replicates).(F) qPCR (SYBR green) analysis of Ifit1 (left) and Irf7 (right) expression on day 14 of analysis.(G) Cell viability at IFNβ dosages 0, 50 and 1000U/mL after 24 hours. Statistical analysis was Two-way ANOVA with multiple comparisons correction.(H) qPCR (SYBR green) of Ifit1 (left) and Irf7 (right) expression in the cell lines collected at 3 or 24 hours post-treatment with 50 or 1000 U/mL of IFNβ. No statistical difference between genotypes in Ifit1 (left) and Irf7 (right) expression.
Figure 2BMDMs were treated with cytochalasin D and infected with aggregated or non-aggregated Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (B-E) Examples of macrophages infected with low (B), medium (C), or high (D) doses of non-aggregated bacteria or with bacterial aggregates (E). Scale bars, 20 μm.BMDMs were treated with cytochalasin D and infected with aggregated or non-aggregated Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (F) Percentage survival over time for macrophages in contact with increasing doses of non-aggregated bacteria or with bacterial aggregates. Time 0 for cells "in contact" represents the time when stable contact with the bacteria begins. Each line represents a biological replicate (n ≥ 100 cells per condition).
Figure 3(A) BMDMs expressing a membrane-targeted tdTomato were treated with cytochalasin D, infected with aggregates of Mtb expressing GFP, and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Example of a BMDM interacting with an extracellular Mtb aggregate. Top panels show the macrophage plasma membrane and the bacterial aggregate. Intimate interaction between the macrophage plasma membrane and the bacterial aggregate begins at 4h00 (indicated by the white arrow). Bottom panels show the Annexin V-647 positive plasma membrane domain that appears at 5 hours 20 minutes and co-localizes with the bacterial aggregate. At 8h00, plasma membrane integrity is lost (top panel) and Annexin V accumulates throughout the dead cell (bottom panel).(E-G) Cytochalasin D-treated BMDMs were infected with aggregates of Mtb Erd-tdTomato in the presence of Annexin V-FITC. Selected cells were imaged by time-lapse fluorescence microscopy followed by SEM. Arrows in top panels indicate the cell imaged by SEM. Top left: brightfield image of macrophage. Top left and right: fluorescence image of the Mtb aggregate. Top right: fluorescence image of Annexin V staining. Bottom: correlative SEM image. Scale bars, 20 μm in top panels and 1 μm in bottom panels. (E) Example of a bystander BMDM that is not in contact with a bacterial aggregate. (F) Example of an Annexin V-negative BMDM interacting with a Mtb aggregate. Arrows in bottom panel indicate areas of intact plasma membrane interacting with bacterial aggregates. (G) Example of a BMDM with blebbed Annexin V-positive membrane domains interacting with an Mtb aggregate.