EMBO/SourceData huggingface dataset:
Figure 3Left upper and right panel: HEp-2 cells re­mained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 4 h. Cells were lysed and the de­amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels; left lower panel: HEp-2 cells were lysed and the cell extracts were incubated with full-length CNFY or the N-ter­minally deleted toxin variants for 4 h. The de­amidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE after detection with anti-RhoA antibodies.HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was ob­served by fluorescence micro­­scopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNFY-treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (ΔcnfY) harboring the empty expression vector were used as negative controls.
Figure 6Nitrocefin (2 mM) was added to the supernatant from 25°C overnight Yersinia cultures expressing the indicated CNFY derivatives to determine β-lactamase activity by measuring changes in absorbance at 390 nm (yellow) and 486 nm (red). The data represent the mean ± SD of three independent experiments, carried out in triplicates.The viability of Y. pseudotuberculosis YPIII expressing the indicated CNFY derivatives was assessed in equalized bacterial cultures using the BacTiter-Glo Microbial Cell Viability Assay kit (Promega). The data represent the mean ± SD of three independent experiments, carried out in triplicates.
Figure 7Comparative analysis of RhoA activation in HEp-2 cell lysate by CNFY and the recombinant D4-5 protein. Purified CNFY or the D4-5 fragment (1 µM) was added to extracts of HEp-2 cells and incubated for 10, 20, 30 or 60 min. De­amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels after detection with anti-RhoA antibodies.Comparative analysis of recombinant RhoA deamidation by CNFY and the D4-5 fragment. Recombinant RhoA was incubated with purified CNFY or the D4-5 fragment and samples were separated by SDS-PAGE after the indicated times before subjecting to trypsin digestion and quantification of deamidation of Q63 by mass spectrometry. Error bars represent standard deviations of triplicate measurements.
Figure 2(A) Tumor volume of MDA-MB-468 mammary fat-pad xenografts treated with vehicle, AZD8186 (50 mg/kg, og twice/day), erlotinib (50 mg/kg IP once/day) alone or in combination (5-6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test **P=0.0028 and ***P<0.0001. (B) Tumor volume of HCC-70 mammary fat-pad xenografts treated with vehicle, AZD8186 (150 mg/kg, og once/day), erlotinib (50 mg/kg IP once/day) alone or in combination (6-7 mice per group, mean±SEM). Growth curves were compared using two-way ANOVA statistical test. Statistical significance of two-way ANOVA statistical test ***P=0.0006 and ****P<0.0001.(C) Waterfall representation of changes in the volume of individual HCC-70 tumors during the treatment. Statistical comparison between different treatment groups by Mann-Whitney test **P=0.0023 and ***P=0.0006.(D) Change in the body weight of mice harbouring HCC70 xenograft tumors (6-7 mice per group, mean±SEM) during 21 days of treatment.(E) A cell line derived from a mammary tumor spontaneously developed in a Wap-cre:Ptenfl/fl:Tp53fl/fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6/J recipient mice. Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure. When tumours reached an average volume of 100mm3, they were treated with vehicle or a combination of AZD8186 (150mg/kg, og once/day) and erlotinib (50mg/kg IP once/day). Tumours were then measured during the treatment (6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test ****P<0.0001.
Figure 3(A) MDA-MB-468 cells were treated for 24h with vehicle, PI3Kβi (AZD8186 250 nM), EGFRi (gefitinib 3 μM) or cetuximab (100 μg/ml), alone or in combination. The cell lysates were probed with the indicated antibodies.(B) MDA-MB-468 cells were treated for 24h with vehicle, AKTi (MK2206 450 nM), gefitinib (3 μM) or cetuximab (100 μg/ml), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.(C, D) HCC70 (C) and ZR-75-1 (D) parental cells or PI3Kβi-Res (derivative cells with acquired resistance to AZD8186), or AKTi-Res (acquired resistance models to MK2206) were treated with vehicle, PI3Kβi (AZD8186 250 nM) or AKTi (MK2206 1 μM) for 24 hours. The whole cell lysates were then probed with the indicated antibodies. Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P-S6 and S6 tot in (D) have been cut and reassembled for figure purposes.(E) HCC70 parental, PI3Kβi-Res or AKTi-Res cells were treated with vehicle, AKTi (MK2206 250 nM) or gefitinib (3 μM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.(F) HCC70 MK res (acquired resistance models to MK2206) were treated with serial dilutions of gefitinib, alone or in combination with MK2206 (810 nM), as indicated, and viability measured after 4 days of treatment. Average ± SD of triplicates and representative of two independent experiments.(G) HCC70 AZD res (acquired resistance models to AZD8186) were treated with serial dilutions of gefitinib, alone or in combination with AZD8186 (270 nM), as indicated, and viability measured after 4 days. Average ± SD of triplicates and representative of two independent experiments.(H,I) p110β co-immunoprecipitates with EGFR in MDA-MB-468 (H) and in HCC70 (I). Cells were pre-treated with different inhibitors, including vehicle, pan-PI3Ki GDC0941 (1 μM for MDA-MB-468 or 0.5 μM for HCC70), EGFRi (gefitinib 3 μM), PI3Kβi (AZD8186 250 nM) or a combination of EGFRi and PI3Kβi. Cell lysates were incubated with IgG control or anti-EGFR antibody and the immuno-complexes or the total lysates were immune-blotted with the indicated antibodies.(J) EGF-induced increase in phospho-AKT is dependent on p110β kinase activity. MDA-MB-468 were starved in 0% FBS and pre-treated with vehicle or PI3Kβi (AZD8186 250 nM) for 1h. Cell lysates were probed with the indicated antibodies. Phospho-AKT and pan-AKT bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT normalised to the control (left hand lane) is shown.
Figure 4(A Results of CRISPR-Cas9 screening in combination with AZD8186 100nM (A) The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions (AZD8186 and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A). <0.05>B) Results of CRISPR-Cas9 screening in combination with GDC0941 400nM (B). The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions GDC0941, and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. <0.05>(C) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941-treated conditions for MDA-MB-468 cells transduced with non-target Control, GNB2 or GNG5 sgRNAs (N=2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1.5 times the inter-quartile range. Statistical significance of unpaired t-test ****P<0.0001.(D) Biochemical analysis of GNB2 KO cells. Cell lysates of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software.
Figure 5(A) MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were treated with vehicle, GDC0941 1 μM, gefitinib 3 μM or lapatinib 1 μM for 24 hours. The cell lysates were probed with the indicated antibodies.(B-D) Viability assays of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones treated with serial dilutions of the indicated drugs for six days. Mean ± SD of triplicates and representative of two or three independent experiments.(E) Patient samples from METABRIC dataset classified as TNBCs (N=299) were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN-WT groups. Box & Whisker plot (Median/IQR/1.5*IQR Whiskers). P value calculated by unpaired t-test.
Figure 6(A) Schematic of the results from the drug screening with compounds targeting GPCR signaling. MDA-MB-468 cells were treated with the compounds of the GPCR-targeted library at three different concentrations (0.1, 1 or 10 μM) in combination with vehicle, GDC0941 450 nM or lapatinib 1 μM. The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI. Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle, GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot, respectively. Readings acquired following treatment with the three different concentrations of the GPCR-targeted drugs are reported in 3 different colours. Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot. The values reported are mean of a biological triplicate of the experiment.(B) MDA-MB-468 were treated for 24 h with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.(C) MDA-MB-468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes, alone or in combination with vorapaxar. The cell lysates were probed with the indicated antibodies. Phospho-AKT, pan-AKT, phospho-ERK1/2 and pan-ERK1/2 bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT and phospho-ERK1/2 to pan-ERK1/2 normalised to the control peptide-treated conditions for WT and GNB2 KO cells (first and forth lanes, respectively) is shown.(D) p-S6/loading control signal from western blots experiments in which a panel of six TNBC PTEN-null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The values were normalised to vehicle treatment for all cell lines. Mean of 2 independent experiments ± SD. P values calculated by two-tailed paired student t-test *P=0.0235 and **P=0.002. Cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395, BT-549, HCC70 AZD8186-resistant and HCC70 MK2206-resistant.(E) Long-term proliferation assay of MDA-MB-468 cells treated with vorapaxar (5 or 2.5 μM), GDC0941 (1 μM) or lapatinib (1 μM), alone or in the indicated combinations. Cells were treated for two weeks and stained by crystal violet. One representative experiment of three is shown.
f1b, Carboxy-terminal V5immunoblot of full-length (68 kDa) and cleaved (36 kDa) ATG16L1β. Silver stain (bottom gels) depicts total protein. Data represent 3 independent experiments. IB, immunoblot.c, TNF-α-mediated processing of humanATG16L1 in monocyte-derived macrophages. Scatter plot of immunoblot densitometry depicts a ratio of cleaved:full-length ATG16L1 (Extended Data Fig. 2). n = 7 donors.d, TNF-α-mediated processing of murineATG16L1 in macrophages. Immunoblot represents 5 independent experiments. Scatterplot represents data pooled from 5 independent experiments. WT, wild type.
f2a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 μM staurosporine or 20 ng ml−1 TNF + 10 μg ml−1 CHX.b, HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a.c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &amp;amp;mgr;g ml−1 CHX.d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 µM staurosporine or 20 ng ml−1 TNF + 10 µg ml−1 CHX. Data in a-c represent 2 independent experiments; data in d represent 4 independent experiments.
f3a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose.c, Immunoblot of phosphorylated (p-) AMPK&amp;amp;agr; (Thr 172) and lipidated LC3 (LC3-II).d, Quantification of mean LC3-II area in murine macrophages, n = 4 mice.e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units.f, Quantification of mean LC3-II area in wild-type (Casp3+/+) or Casp3-knockout (Casp3−/−) macrophages, n = 3 mice. Data in a-f represent 2 independent experiments.
f4a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments.d, Visualization of early autophagosomes by transmission electron micrographs (TEM). Red arrows, early autophagosomes, characterized by vesicular structures with a double-membrane. Blue arrows, multivesicular lysosomal structures, identified by degraded intravesicular cargo. Micrographs representative of 10-12 images per donor, n = 4 (WT), n = 3 (T300A). A total of 29 (WT) and 26 (T300A) images containing autophagosomes was analysed. Histogram quantifies autophagic vesicles as a ratio of autophagosome:cytosolic area.
f5a-c, Y. enterocolitica colony forming units (c.f.u.) in human and murine macrophages infected for 6 h (20 m.o.i.). Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype, total n = 16 (non-risk, WT), 15 (T300A, risk). Data in b represent 3 independent experiments, n = 4 mice. Data in c represent 3 independent experiments, n = 6.d, TNF-α, IL-1β and IL-6 levels in culture media of WT and T316A macrophages infected with 20 m.o.i. Y. enterocolitica. Data represent 2 independent experiments, n = 4 mice.e, Ileum and colon histology (haematoxylin and eosin staining) of WT mice. Images are representative of 3 (PBS) and 8 (Y. enterocolitica) treated male mice.f, Measurement of cytokine mRNA transcripts in mesenteric lymph nodes. Data are pooled from 2 independent experiments of 3 mice (PBS) and 7 mice (Y. enterocolitica) per experiment. Transcripts are normalized to Gapdh (2−Dgr;Ct).g, Y. enterocolitica c.f.u. in mesenteric lymph nodes of WT and T316A knock-in mice 48 h following oral gavage. Data represent 3 independent experiments, n = 9 male mice.
Figure 1D. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11), gasdermin D (GSDMD) and GAPDH (loading control) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Data are from one three independent experiments (DE. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in D. Data information: Each symbol represents an independent biological replicate (E). **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (E). Data are from three independent experiments E; mean and s.e.m. in EG. IncuCyte live-imaging analysis of WT and Casp11-/- BMDM viability after infection with M. catarrhalis as in D, or transfection with LPS. Data information: Data are pooled from two (G) independent experiments mean and s.e.m. in G).
Figure 2A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Nlrp3-/-, Casp11-/- and Aim2-/- BMDMs left untreated (Med.) or 5 h after transfection with 5 µg of LOS from M. catarrhalis (O35E) or 5 µg of LPS from E. coli. Data information: Data are from three independent experiments (A,B. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B, NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B, Data are pooled from three independent experiments (B, mean and s.e.m. in B,
Figure 3F. Transcript abundance for genes encoding GTPases in WT and Ifnar1-/- BMDMs infected with M. catarrhalis as in C. Transcript abundance is provided in counts per million (CPM). Data information: Data are pooled from three independent experimentsG. qRT-PCR analysis of genes encoding the family of guanylate binding proteins (GBP1-11) in WT and Ifnar1-/- BMDMs left untreated (Med.) or following treated as in C. Data information: Each symbol represents an independent biological replicate G). NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test G)). Data are from one experiment representative of four (G) independent experiments (mean and s.e.m. in G).
Figure 4A. Confocal microscopy analysis of intracellular M. catarrhalis (green) and GBP1, GBP2, GBP3, GBP5 or GBP7 (red) in WT BMDMs left untreated (Med.) or assessed 12 h after infection with M. catarrhalis (Ne11, MOI 20). B. Quantitation of GBP1-, GBP2-, GBP3-, GBP5- and GBP7-postive M. catarrhalis in WT BMDMs as treated in A. Data information: Arrowheads indicate bacteria colocalised with GBPs. To quantify the proportion of GBP-positive bacteria in a field of view, we used the single red channel (anti-GBP staining) and compared this to the green channel (anti-M. catarrhalis staining). The number of GBP-positive M. catarrhalis was divided by the total number of M. catarrhalis counted to obtain the proportion of GBP-positive bacteria (B). A total of 100 BMDMs were analysed to quantify the proportion of GBP-positive bacteria per cell using confocal microscopy (B) Data are from three independent experiments (A, or are pooled from two (B) independent experiments Scale bars, 5 µm (A,F. Recovery of M. catarrhalis (as colony-forming units (CFU) from primary WT, Gbpchr3-KO and Ifnar1-/- lung fibroblasts 4, 8 and 12 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Each symbol represents an independent biological replicate F). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test Data are from three independent experiments F; mean and s.e.m.G. Quantitation of intracellular bacteria by Transmission electron microscopy (TEM) in WT, Gbpchr3-KO and Ifnar1-/- BMDMs infected with M. catarrhalis (Ne11, MOI 50) for 12 h. Each symbol represents an independent experiment (E and F). Data information: A total of 100 BMDMs were analysed to quantify the intracellular bacterial burden using electron microscopy (G). Data are from one experiment representative of two (G) independent experiments mean and s.e.m.
Figure 5A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Gbp1-/-, Gbp2-/-, Gbp3-/-, Gbp5-/-, Gbp7-/-, Gbp4/8/9-/-, Gbp11­­-/- and Casp11-/- BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 µg LPS from E. coli.B. Release of IL-1β, IL-18, LDH, TNF, IL-6 and KC from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B)). Data are are pooled from three independent experiments (B; mean and s.e.m. in B).
Figure 6B. Quantification of OD600 of M. catarrhalis in BHI media over 6 h in the presence of solvent control (Sol. Ctrl.), 80 µg/mL recombinant GBP2 or 50 µg/mL kanamycin. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B Data are from one experiment representative of two (A) or three (B, Each independent experiment consists of three (B, technical replicates.C. Quantification of ATP from M. catarrhalis in BHI media over 6 h in the presence of Sol. Ctrl., 30 µg/mL recombinant GBP2 or 50 µg/mL kanamycin. Data information: Data are pooled from two (C) independent experiments Each independent experiment consists of two (C) technical replicates.D. Viability of M. catarrhalis (as a percentage of CFU in relation to Sol. Ctrl.) assessed 6 h after incubation with recombinant GBP2 at 3, 30 or 300 µg/mL. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test D) Data are from one experiment representative of three D, independent experiments Each independent experiment consists of three D, technical replicates.
Figure 7A, B. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Casp11-/- mice (n=10) after M. catarrhalis infection. C, D. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Nlrp3-/- mice (n=11) after M. catarrhalis infection. E, F. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp1-/- mice (n=9) after M. catarrhalis infection. G, H. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp2-/- mice (n=8) after M. catarrhalis infection. I, J. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp3-/- mice (n=8) after M. catarrhalis infection. K, L. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=5) and Gbp5-/- mice (n=8) after M. catarrhalis infection. Data information: A mix of male and female mice 6-8 weeks old were used in each experiment and were injected i.p. with 2×107 CFU of M. catarrhalis and analysed after 6 h. Each symbol represents an individual mouse (A-L). Each panel represents data from a single experiment (mean and s.e.m. in A-L). Each experiment was performed at least two times. * P<0.05; ** P<0.01; *** P<0.001 (two-tailed t-test (A-L)).
Figure 1C Frequency of C179+ or V5-tag+ 293T cells 24h after transfection of AAV-vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE (n = 3).D Immunoblot of 293T cells 72 h after transduction with AAV-vectors at a MOI of 106. Antigen expression was detected with an anti-V5-tag antibody. Equal loading was controlled with a GAPDH antibody (n = 3).