EMBO/SourceData huggingface dataset:
Figure 1B. Representative 2D projection of confocal images showing caspase activation at the dissection time point (red channel) and in the recent past (green channel, arrows) within putative escort somatic cells via DBS-S-QF sensor; TUNEL staining indicates apoptosis (grey, arrowhead); DAPI labels the nuclei in the entire Figure. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection. Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; ;QUAS-Gal4 /+ (BL83123). C. Representative 2D projection of confocal images showing escort and follicular somatic cells permanently labelled with DBS-S-QF sensor (green channel, arrows); the arrowhead indicates the presence of germline cells positive for TUNEL staining (red, arrowhead). Notice the lack of TUNEL signal and therefore apoptosis in somatic cells permanently labelled with DBS-S-QF sensor (green). Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection. Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; QUAS-flippase (BL30126)/+; Actin5C FRT-stop-FRT lacZ-nls/+ (BL6355) Data information: Scale bars represents 10 µm in the entire Figure.D. Graph showing the percentage of germaria permanently labelled with DBS-S-QF overtime at 25 °C (N=4 biological replicates; n=520 and n=536 germaria were inspected at 7 and 14 days post adult eclosion, respectively) and 29 °C (N=7, n=799 at 7 days and n=588 at 14 days). A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; QUAS-flippase (BL30126)/+; Actin5C FRT-stop-FRT lacZ-nls/+ (BL6355). Data information: The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets.E. Percentage of germaria showing permanently labelled with DBS-S-QF the ECD over time at 25 °C and 29 °C. FasIII immunostaining was used to locate the DBS-S-QF labelling in the inspected germaria. The N and n numbers of these experiments are indicated in D. A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotypes are shown in D. F. Percentage of germaria showing permanently labelled with DBS-S-QF the ECD over time at 25 °C and 29 °C. FasIII immunostaining was used to locate the DBS-S-QF labelling in the inspected germaria. The N and n numbers of these experiments are indicated in D. A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotypes are shown in D. Data information: The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets.
Figure 5B. Relative number of Ptc-positive puncta per germaria of the following genotypes, from left to right: DroncKO Tub-G80ts /+ (n=10). ptc-Gal4 /+; Tub-G80ts/+ (n=9). ptc-Gal4 /+; DroncKO Tub-G80ts /+ (n=10). A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (***p≤0.001 Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.C. Western blot showing Ptc (upper lane) and actin (bottom lane, loading control) expression in ovaries of the genotypes shown in A. Notice the Ptc accumulation in double heterozygous germaria (ptc-Gal4 /+; DroncKO Tub-G80ts /+).E. Pearson colocalization index between Ptc and Atg8 in germaria of the following genotypes: ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749) (n=12). ptc-Gal4 (BL2017)/UAS-GFP-mCherry-Atg8 (BL37749); DroncKOTub-G80ts (BL7019)/+ (n=18). An unpaired parametric Welch's t-test was used to establish the statistical significance (** p≤0.01). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.F. Fraction of Ptc positive that overlaps with Atg8 in germaria of the genotypes shown in E. n number is indicated in E. An unpaired parametric Welch's t-test was used to establish the statistical significance (*** p≤0.001). The Y axis in the graph shows the Manders' Coefficient M1-M2. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.G. Fraction of Atg8+ autophagosomes that overlaps with Ptc in germaria of the genotypes shown in E. n number is indicated in E. An unpaired parametric Welch's t-test was used to establish the statistical significance (** p≤0.01). The Y axis in the graph shows the Manders' Coefficient M2-M1. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.
Figure 6B. Total number of Atg8+ autophagosomes in germaria of the following genotypes ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749) (n=21). ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749); DroncKOTub-G80ts (BL7019)/+ (n=28). An unpaired parametric Welch's t-test was used to establish the statistical significance (* p≤0.05). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.C. Volume of Atg8+ autophagosomes in germaria of the genotypes described in B. n number is indicated in B. A non-parametric Mann-Whitney t-test was used to determine statistical significance (*** p<0.001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.H. Western blot showing the expression levels of the autophagy marker p62 (upper lane), Caspase-9 (middle lane) and Actin (bottom lane, loading control) in either scrambled or Caspase-9 deficient OVCAR-3 cells; the protein levels of the different read outs were measured at 72h after siRNA treatment in cells grown during the last 4 h before sample processing in our standard cell culture conditions, in cell culture media containing EtOH (0.2%), and in cell culture media containing EtOH (0.2%) + bafilomycin A1 (400nM).I. Quantification of p62 protein levels in the experimental conditions described in H. one sample T Wilcoxon test was used to calculate statistical significance, * p≤0.05, N≥3. Bars indicate value of the mean while error bars represent the standard deviation.K. Quantification of the total number of Castor-expressing cells within the FasIII cellular domain in the genotypes described in J. n number is indicated also in J. An ordinary one-way ANOVA and Dunnett's multiple comparison post-test were used to determine statistical significance (n.s.= not significant, ** p≤0.01, **** p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.
Figure 1(C, D Confocal micrographs of HeLa cells expressing GFP-TECPR1 and treated with osmotic shock assay or LLOMe (C), or infected with the bacteria indicated (D) and fixed at 1 h (S. Typhimurium and L. monocytogenes) or 0.5 h (S. flexneri) post-infection. Cells were stained for Galectin-8 and DNA (DAPI). Scale bar, 20 μm.(F) Percentage of bacteria positive for mCherry-TECPR1 in ATG5 knockout (KO) or ATG5-complemented MEF cells at 1 h (S. Typhimurium) or 0.5 h (S. flexneri) post-infection. Mean +/- SD of four (S. Typhimurium) or two (S. flexneri) independent experiments. n > 100 bacteria per coverslip. (G) Percentage of S. Typhimurium positive for GFP-TECPR1 at 1 h post-infection in HeLa cells pretreated or not with 100 nM Wortmannin. Mean +/- SD of two independent experiments. n > 100 bacteria per coverslip.H) Confocal micrographs of HeLa cells expressing GFP-TECPR1 either alone or together with FLAG-tagged neutral sphingomyelinase 2 (nSMase2; (H)) Cells were stained for Galectin-8 and DNA (DAPI). Scale bar, 20 μm. (I) Percentage of S. Typhimurium positive for GFP-TECPR1 or endogenous Galectin-8 at 1 h post-infection in HeLa cells expressing FLAG-nSMase2 or not. Mean +/- SD of two (TECPR1) or three (Gal8) independent experiments. n > 100 bacteria per coverslip.
Figure 2(C) Percentage of S. Typhimurium positive for the indicated constructs in HeLa cells, fixed at the indicated timepoints post-infection. Mean +/- SEM of 3-4 independent experiments performed in duplicate. n > 100 bacteria per coverslip.(E) Confocal micrographs of HeLa cells expressing TECPR1 N' DysF-GFP with or without FLAG-nSMase2, infected with S. Typhimurium, fixed at 30 min post-infection and stained with anti-Galectin 8 antibody. DNA stained with DAPI. Scale bar, 20 μm.(F) Percentage of S. Typhimurium positive for N' DysF-GFP in Hela cells, expressing FLAG-nSMase or not, fixed at 30 min post-infection. Mean +/- SEM of three independent experiments performed in duplicate.(G and H) Confocal micrographs of HeLa cells expressing the indicated GFP-tagged DysF domains, infected with mCherry-S. Typhimurium and fixed at 30 min post-infection (G) or treated with osmotic shock assay, fixed and stained with anti-Galectin 8 antibody (H). The bottom row in H depicts an enlarged version of the boxed region shown above. Scale bar, 20 μm.
Figure 3(B Percentage of S. Typhimurium positive for the indicated GFP-tagged N-terminal DysF constructs at 30 min post-infection. Mean +/- SD of two (EK134,135AA) or three independent experiments. n > 100 bacteria per coverslip. p value from one-way ANOVA with Dunnett's multiple comparisons test.C) Percentage of S. Typhimurium positive for the indicated GFP-tagged N-terminal DysF constructs at 30 min post-infection. Mean +/- SD of three independent experiments. n > 100 bacteria per coverslip. p value from one-way ANOVA with Dunnett's multiple comparisons test.(F) Confocal micrographs of HeLa cells expressing either GFP-TECPR1 WT or W154A and treated with LLOMe or DMSO vehicle control for 10 min and fixed. Scale bar, 20 μm. (G) Number of GFP-positive puncta per cell were enumerated in samples depicted in (F). Data from 50 cells from a single experiment, representative of two. p value from one-way ANOVA with Tukey's multiple comparisons test.
Figure 4(C-E) Liposome sedimentation assay. The indicated proteins were incubated with either liposomes (containing sphingomyelin or not) or buffer only, pelleted by centrifugation and equal portions of supernatant and pellet fractions analysed by SDS-PAGE and Coomassie staining. Representative gels depicted (C). Fraction of TECPR1 (D) or DysF WT or W154A (E) proteins present in pellet under the indicated conditions, expressed as % binding. Note pelleting in the absence of liposomes likely due to affinity of proteins for tube walls. Mean +/- SD of three independent experiments. p value from one-way ANOVA with Tukey's multiple comparisons test.(F) In vitro LC3 lipid conjugation assay. Liposomes containing sphingomyelin or not were incubated with LC3 lipid conjugation machinery: ATG7 (E1), ATG3 (E2), ATG5-ATG12 (E3), LC3B, MgCl2, ATP and the indicated TECPR1 proteins. Control reactions without ATG3 or ATP were included. Mixes were incubated at 37 C for 3 h, centrifuged and equal fractions of both supernatant and pellet analysed by SDS-PAGE and Coomassie staining. Lipidated LC3B-II appears in pellet fraction whereas both soluble LC3B-I and AMPylated LC3B are in the supernatant, the latter of the same apparent molecular weight as LC3B-II.(G) Fraction of LC3B protein present in pellet from complete in vitro LC3 lipid conjugation assay with the indicated TECPR1 proteins in either absence or presence of sphingomyelin. Mean +/- SD from three independent experiments. p value from one-way ANOVA with Tukey's multiple comparisons test.
Figure 5(A Confocal micrographs of indicated MEF cell lines infected with mCherry-expressing S. Typhimurium, fixed at 30 min post-infection and stained with anti-ATG5 antibody (A) Scale bar, 20 μm.C) Confocal micrographs of indicated MEF cell lines infected with mCherry-expressing S. Typhimurium, fixed at 30 min post-infection and stained with anti-LC3 antibody (C). Scale bar, 20 μm.(E) Percentage of S. Typhimurium positive for endogenous LC3 in indicated MEF cells treated with 100 nM Wortmannin or DMSO vehicle control, fixed at 1 h post-infection. Mean +/- SEM of three independent experiments performed in duplicate. n > 100 bacteria per coverslip. p value from one-way ANOVA with Tukey's multiple comparisons test.(F) Confocal micrographs of either control of ATG16L1-deficient MEF cells, transfected with either control or TECPR1 siRNA (siRNA no. 66 depicted), infected with mCherry-expressing S. Typhimurium for 30 min, fixed and stained with anti-LC3 antibody. Scale bar, 20 μm.
Figure 6(A) Percentage of S. Typhimurium positive for Galectin-8 and/or GFP-TECPR1 in either control Hela cells or those expressing GFP-TECPR1 at 1 h post-infection. Mean +/- SEM of three independent experiments performed in duplicate. n > 100 bacteria per coverslip. (B) Confocal micrographs of HeLa cells expressing the indicated GFP-TECPR1 constructs or not (control), infected with S. Typhimurium for 30 min, fixed and stained with anti-Galectin-8 and DAPI. Scale bar, 20 μm.­­ Arrowheads indicate bacteria positive for Galectin 8, of which more are present in cells expressing GFP-TECPR1 WT but not W154A. Arrow indicates bacterium to which GFP-TECPR1 but not Galectin 8 has been recruited.(C) Percentage of S. Typhimurium positive for Galectin-8 in Hela cells expressing the indicated GFP-TECPR1 constructs or not (control) and infected with S. Typhimurium for 1 h. Mean +/- SEM of five independent experiments performed in triplicate. p value from one-way ANOVA with Tukey's multiple comparisons test.(D) Fold intracellular proliferation of S. Typhimurium in HeLa cells expressing the indicated GFP-TECPR1 constructs or not (control) as assessed by colony forming unit assay. Mean +/- SEM of four (ΔPH) or five (control, WT, W154A and ΔAIR) independent experiments. p value from one-way ANOVA with Dunnett's multiple comparisons test.
Figure 1(A-B) Capsids for WT, K158A, K158A/T8I and T8I were classified into the indicated categories. Representative tomograms of K158A mutant (A) and the percentage (B) for each category are shown. The margin of error was calculated Images and quantification are from the same samples.(C) Virion stability assay using TIRF microscopy to quantify the number of viral particles in presence of SLO and IP6 for the indicated mutant after 1 hour. Mean data represent n=2 technical replicates. Scale bar 10 μm.(F) Quantification of CXCL10 and IFIT1 transcripts by qPCR of the indicated THP-1 cell lines stimulated for 24 hours with LPS (1 ng/ml) normalised to mock. Mean data n=2 biological replicates.(H) Quantification of the indicated chemokine transcripts by qPCR from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants normalised to mock infected. Mean data represent n=3 biological replicates.(I-J) Protein quantification of CXCL10 (I) and TNFa (J) by Luminex™ from supernatant of PMA-treated THP-1/SAMHD1 KO cells infected with the indicated HIV-1 mutants. Data were normalised to mock. Mean data represent n=2 biological replicates.
Figure 2(D) qPCR quantification of the indicated chemokine transcripts from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with the indicated VLPs mutants. (E) qPCR quantification of the indicated chemokine transcripts level from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants.(F) Protein quantification of IL-6 by Luminex™ from PMA-treated THP-1/SAMHD1 KO cells infected with the indicated HIV-1 mutants.
Figure 3IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection with either poly I:C (0.5 μg/ml) (B) mean data represent n=2 biological replicatesIFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection with HT-DNA (0.1 μg/ml) (C) mean data represent n=2 biological replicatesIFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection infected for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants (D).IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO infected with for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants in the presence of DMSO or 0.5 μg/ml H151 (E)
Figure 4(B-D) Protein quantification of CXCL10 (B), IL-6 (C) and TNFa (D) by Luminex™ from HMDMs infected with the indicated HIV-1 mutants.(F-H) Protein quantification from SupT1 cells as in (B-D)).Protein quantification from infected Jurkat T cells as in (B-D).
Figure 1CSF p3-Alcβ37 values in subjects who were categorized along the AD continuum (n = 131). Cut-off values are 359.6 pg/mL for Aβ42 (A+ indicates <359.6 pg/mL), 30.6 pg/mL for p-tau (T+ indicates >30.6 pg/mL), and 105.3 pg/mL for t-tau (N+ indicates >105.3 pg/mL) respectively. Statistical significance was determined by a one-way ANOVA followed by Tukey's multiple comparisons test (means ± SEM; A-: n = 36, A+T-N-: n = 51, A+T+N-: n = 30, A+T+N+: n = 14), and significant p-values (p < 0.01, p < 0.0001) are indicated on the graph.
Figure 2(C) Effect of p3-Alcβ37 and its partial peptides on the viability of mouse primary cultured neurons. Wild-type (WT) neurons (div 15-20) were incubated for 24 h in the presence (10 μM) or absence (-) of p3-Alcβ1-11, p3-Alcβ9-19, p3-Alcβ20-37, and p3-Alcβ1-37. Neuronal viability was evaluated with MTT assays and expressed relative to that of cells cultured in the absence of peptides (assigned a value of 1.0). Statistical analysis was performed using a one-way ANOVA, followed by Dunnett's multiple comparisons test (means ± SEM; n = 10-12), and significant p-values (p < 0.01, p < 0.0001) are indicated on the graph.(D, E, F) Effect of p3-Alcβ on the neuronal toxicity of Aβ42 oligomers. WT neurons (div 15-20) were incubated for 24 h in the presence (10 μM) or absence (-) of p3-Alcβ9-19 and p3-Alcβ1−37 with (+) or without (-) Aβ42 oligomers (Aβo, 2.5 μM). Neuronal viability was evaluated by MTT (D), ATP generation (E), and LDH release (F) assays and expressed relative to neurons cultured in the absence of p3-Alcβ and Aβo (assigned a value of 1.0). Statistical significance was determined by Dunnett's multiple comparisons tests (means ± SEM; MTT: n = 36, ATP: n = 40, LDH: n = 60). The significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) versus cells incubated in the absence (-) of p3-Alcβ and Aβo are indicated in the graphs. The significant p-values (#p < 0.05, #p < 0.01) versus cells incubated in the presence (+) of Aβo and in the absence (-) of p3-Alcβ) are indicated in the graphs.(G) Effect of Aβ42 oligomers (Aβo) and p3-Alcβ peptides on the generation of reactive oxygen species (ROS). WT neurons (div 15-17) were incubated for 24 h with (+) or without (-) Aβo (2.5 μM) and in the presence (+) or absence (-) of p3-Alcβ (10 μM). ROS generation was assessed and expressed relative to that of neurons incubated in the absence (-) of Αβo, which was assigned a value of 1.0. Statistical significance between samples with or without Aβo was evaluated with a Student's t-test (means ± SEM; n = 10), and the significant p-value (p < 0.05) is indicated on the graph.
Figure 3(A, B) Suppression of Ca2+ influx induced by Aβo in neurons treated with p3-Alcβ9-19 (A) and p3-Alcβ37 (B). Mouse neurons (div 11-13) pretreated with Fluo 4-AM were stimulated at 5 min (arrow) with (+) or without (-) Aβo (5.2μM) in the presence (+) or absence (-) of p3-Alcβ (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 18 min (5-23 min at time points) is shown (right) as the AUC expressed relative to that of cells cultured in the absence (-) of p3-Alcβ and Aβo (assigned a value of 1.0).(C, D) Suppression of Ca2+ influx into neurons followed by Ca2+ administration in the presence of p3-Alcβ9-19 (C) and p3-Alcβ37 (D). Mouse neurons (div 11-13) pretreated with Fluo 4-AM in calcium-depleted medium were administered Ca2+ (2 mM) at 5 min (arrow) in the presence or absence of p3-Alcβ9-19 (C) and p3-Alcβ37 (D). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 7.5 or 9 min (5 to 12.5 min in panel C and 5 to 14 min in panel D at the indicated time points) is shown (right). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparison test (means ± SE; n = 14 (A), n = 15 (B), n = 10 (C)). The significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) are indicated on the graphs. Statistical significance was determined with a Student's t-test (means ± SEM; n = 11 (D)), and the significant p-value (p < 0.001) is indicated on the graph.