Fig 1: Exosomes inhibit the occurrence of PE. (a) The exosomes in HTR-8/Svneo cells were observed by an inverted microscope. (b) Cell proliferation in HTR-8/Svneo cells treated with PBS and exosomes detected by EdU assay (unpaired t-test, **P < 0.01). (c) Cell migration in HTR-8/Svneo cells treated with PBS and exosomes detected by wound healing assay (two-way ANOVA, **P < 0.01). (d) Cell invasion in HTR-8/Svneo cells treated with PBS and exosomes detected by Transwell assay (unpaired t-test, **P < 0.01). , PBS. , exo. (e) Activity of c-caspase 3 detected by c-caspase 3 Kit (unpaired t-test, **P < 0.01). (f) Cell apoptosis rate detected by TUNEL staining (unpaired t-test, **P < 0.01).
Fig 2: ABT263 abrogates anti-PD1/exomiR-4315-induced resistance to chemotherapy in an in vivo model of lung cancer.A Cisplatin-induced cell death measure and PARP and Caspase-3 cleavage studies were applied to show that the phenotype of cisplatin resistance induced by exosomes derived from T cells exposed to aPD1 (Exo/aPD1) was abrogated by the use of ABT263.A Cell. B Schematic representation of our in vivo experimentations. C Graph represents the impact of treatment on tumor volume, Bim expression at mRNA (RT-qPCR experiments) and protein (ELIZA, Bim ELIZA Kit MyBioSOURCE#MBS9500064, USA) levels and on the level of serum cytochrome c (Cytochrom C ELIZA kit, Biovision#E4286-100, France). Each treatment included four mice. D Correlation between the impact of treatment on tumor volume and the level of serum cytochrome c in mice.
Fig 3: Exosomes derived from T cells exposed to aPD1 (Exo/aPD1) promote a phenotype of cisplatin-induced apoptosis in A549 cells via miR-4315.Cisplatin-induced cell death measure, PARP and Caspase-3 cleavages were applied to show that exosomes derived from T cells exposed to aPD1 (Exo/aPD1) promote a phenotype of cisplatin-induced apoptosis. RT-qPCR and in-cell ELIZA were applied to show that this phenomenon is associated with the miR-4315-mediated down-regulation of Bim.
Fig 4: Caspase-3/IL-16 and GSDME as potential markers of COVID-19 severity(A) Measure of the presence of various inflammatory mediators in plasmas from hospitalized patients presenting COVID-19 disease and analyzed according to their disease severity degree (n = 60 patients, including 15 with moderate COVID-19, 15 with moderate COVID-19 on admission, 15 with severe COVID-19 on admission, and 15 IFN alterations with severe COVID-19). Samples were prepared at day 0 posthospitalization.Data information: data shown as means from n = 12 different donors per category (moderate/moderate ? severe/severe/IFN alterations); each category is represented with a colored circle; * p = 0.05, **p = 0.01, ***p = 0.001 for the indicated comparisons using t test with Bonferroni correction.
Fig 5: NLRP1 engages a caspase-3/Gasdermin E-dependent pyroptosis pathway upon SARS-CoV-2 infection(A) Western blot examination of Gasdermin E, caspases-3, and caspases-8 processing in A549NLRP1+ and A549NLRP1- cells after 24 h of infection with SARS-CoV-2 (MOI 0.05) in the presence or absence of the pan-caspase inhibitor Z-VAD (25 µM). Immunoblots were performed against full-length and processed forms of Gasdermin E (p55 and p30), caspase-8 (p54 and p15), caspase-3 (p35 and p17/19), SARS-CoV-2 nucleocapsid (p40), NLRP1 N-terminal (p130/110), and ACTIN (p40).(B) Western blot examination of Gasdermin E and caspases-3 processing in NHBEWT and NHBENLRP1-/- cells after 36 h of infection with SARS-CoV-2 (MOI 1). Immunoblots were performed against full-length and processed forms of Gasdermin E (p55 and p30), caspase-3 (p35 and p17/19), SARS-CoV-2 nucleocapsid (p40), NLRP1 N-terminal (p130/110), and ACTIN (p40).(C and D) Measure of caspase-1 (C) and caspase-3/-7 (D) activities in SARS-CoV-2-infected (MOI 0.5) NHBEWT or A549NLRP1+ cells for 36 h in the presence or absence of inhibitors of caspase-1 (Z-YVAD, 40 µM) or caspase-3/-7 (Z-DEVD, 30µM). Val-boro (5 µM) was used a positive control of NLRP1-driven caspase activity for 10 H.(E) Measure of cell lysis (LDH release) in A549NLRP1+ or NHBE-infected cells with SARS-CoV-2 (MOI 0.05 and 1, respectively) for 24 h in the presence/absence of the pan-caspase inhibitor Z-VAD (25 µM), the caspase-1 inhibitor Z-YVAD (40 µM), the caspase-8 inhibitor Z-IETD (40 µM), or the caspase-3 inhibitor Z-DEVD (30 µM).(F) Western blot characterization of genetic invalidation of CASP3 in A549 population cells using CRISPR-Cas9 approaches and measure of cell lysis (LDH release) in A549NLRP1+ or A549NLRP1+/CASP3--infected cells with SARS-CoV-2 (MOI 0.05) for 24 h in the presence/absence of the pan-caspase inhibitor Z-VAD (25 µM), the caspase-1 inhibitor Z-YVAD (40 µM), or the caspase-3 inhibitor Z-DEVD (30 µM). Efficiency of genetic invalidation by single-guide RNAs (sgRNAs) targeting GFP or caspase-3 was evaluated at the whole cell population.(G) Western blot characterization of genetic invalidation of GSDME in A549 population cells using CRISPR-Cas9 approaches and measure of cell lysis (LDH release) in A549NLRP1+, A549NLRP1+/GSDMD-, or A549NLRP1+/GSDME--infected cells with SARS-CoV-2 (MOIs 0.001, 0.01, and 0.1) for 24 h. Efficiency of genetic invalidation by single-guide RNAs (sgRNAs) targeting GFP or GSDME was evaluated at the whole cell population.(H) Western blot characterization of genetic invalidation of GSDME in NHBE population cells using CRISPR-Cas9 approaches and measure of cell lysis (LDH release) in NHBEWT or NHBEGSDME-/--infected cells with SARS-CoV-2 (MOIs 0.1, 0.5, and 1) for 36 h. Efficiency of genetic invalidation by single-guide RNAs (sgRNAs) targeting GFP or GSDME was evaluated at the whole cell population.Data information: western blot (A, B, and E–G) images are from one experiment performed 3 times. Graphs (C–G) show data presented as means ± SEM from n = 3 independent pooled experiments; ***p = 0.001 for the indicated comparisons with t test.
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