Journal: Protoplasma

Article Title: The migration and fusion events related to ROCK activity strongly influence the morphology of chicken embryo intestinal organoids

doi: 10.1007/s00709-018-1312-3

Figure Lengend Snippet: The dynamic of organoids growth—the migration and fusion events in various culture conditions. The formation of organoids was recorded under an inverted microscope equipped with an incubation chamber, by collecting one image every half an hour in the first 3 days of culture. a The location of organoids moving through the Matrigel parallel to the insert surface was marked by numbers (1–6); the position of given organoid center at time 0 is shown with a white number; and at 6 h—yellow, 12 h—orange, 18 h—red, and 23 h—blue. The black dashed lines on the photograph in the lower right corner of the panel represent the distances covered by moving organoids in the first whole day of culture (see also Suppl. video ). b The organoids moving towards one another and the fusion events between them were also recorded. The boundaries of three individual organoids that fused into one structure were contoured in red, green, and yellow. The lumen formation is marked by a white cross (see also Suppl. video ). c The presence of ultrapure lipopolysaccharides from Escherichia coli 0111:B4 (LPS, 1 μg/ml) in the medium caused the formation of larger organoids on days 2 and 3 from seeding, as compared to the organoids cultured without such stimulation (C, red arrows). The addition of Y-27632 (10 μM) resulted in the formation of more organoids but of smaller size, regardless of the LPS presence (C, green arrows, see also Suppl. video ). d The epithelial lining obtained by incubation of the intestines of 19-day-old chicken embryos in PBS contacting EGTA and glucose for 2 h was used for total protein isolation and subsequent immunoblotting (time 0). The tissue fragments isolated in the same way were used to start the organoid cultures. Organoids were cultured for 3 days without LPS and Y-27632 or with LPS (1 μg/ml) or with Y-27632 (10 μM) or both. Upon completion of the time-lapse video recording, the media were exchanged and cultures were transferred to the incubator. After 96 h of culture, the total cellular proteins were isolated and the presence of enterocyte markers (sucrase-isomaltase and villin), myofibroblasts marker (α-smooth muscle actin—α-sma), and loading control (growth factor receptor-bound protein 2—grb2) was detected by immunoblotting. The protein samples from organoid cultures treated with Y-27632 inhibitor contain much more α-sma. e The projection area of organoids and number of organoids in one field of view were calculated from digitized images of cultures kept with the absence (open bars) or presence (closed bars) of ROCK inhibitor, which were taken at 72 h from seeding. All fusion events occurring in the first 48 h were calculated by a time-lapse video analysis (the mean of five independent measurements is presented). An ANOVA test was performed to confirm the significance of the difference between organoids cultured with and without Y-27632: p value below 0.05 was considered significant and marked by asterisks (see also Suppl. video )

Article Snippet: The immunodetection was undertaken as previously described (Panek et al. ) using the following antibodies: polyclonal anti-sucrase-isomaltase (1:200, SAB2102141, Sigma-Aldrich, St. Louis, MO, USA), monoclonal anti-α-smooth muscle actin (1:1000, 1A4, Sigma-Aldrich, St. Louis, MO, USA), monoclonal antibody against chicken villin (1:2000, MCA292, AbD Serotec, Raleigh, NC, USA).

Techniques: Migration, Inverted Microscopy, Incubation, Cell Culture, Isolation, Western Blot, Marker, Control

Journal: Frontiers in Immunology

Article Title: The role of gasdermin-mediated mitochondrial RNA release in amplifying secondary immune response during microbial infection

doi: 10.3389/fimmu.2025.1668763

Figure Lengend Snippet: GSDMD cleavage mediates mtRNA release and secondary inflammatory responses via the VISA pathway. (A, B) THP-1 cells primed with LPS and treated with Ng, and cells pretreated with VX-765 and LPS + Ng had their cytoplasmic RNA extracted and 2 μg/mL transfected into fresh THP-1 cells. qPCR analysis showed significantly higher IFN-β (A) and IL-6 (B) expression in fresh THP1 cells transfected from treated with LPS + Ng alone compared to VX-765-pretreated cells. Data are presented as mean ± SEM (****p < 0.0001, ***p < 0.001). (C, D) PM cells primed with LPS and treated with Ng, and cells pretreated with VX-765, then treated with LPS + Ng, had their cytoplasmic RNA extracted, and 2 μg/mL transfected into fresh PM cells. qPCR analysis showed significantly higher IFN-β (C) and IL-6 (D) expression in fresh PM cells transfected from treated with LPS + Ng alone compared to VX-765-pretreated cells. Data are presented as mean ± SEM (***p < 0.001, ****p < 0.0001). (E, F) WT PM cells and VISA -/- PM cells, primed with LPS and then treated with Ng, had their cytoplasmic RNA extracted and 2 μg/mL transfected into fresh WT PM cells and VISA − / − PM cells. qPCR analysis showed significantly higher IFN-β (E) and IL-6 (F) expression in fresh WT PM cells than in fresh VISA -/- PM cells. Data are presented as mean ± SEM (***p < 0.001, **p < 0.01). (G) Six WT mice (three peritoneal injected with 10 mg/kg LPS and three untreated) and three VISA -/- mice peritoneal injected with 10 mg/kg LPS. Western blot analysis of lung tissues showed no GSDMD cleavage or pTBK1 expression in untreated WT mice. In contrast, LPS-injected WT mice exhibited both GSDMD cleavage and pTBK1 expression, whereas LPS-injected VISA -/- mice displayed GSDMD cleavage but no pTBK1 expression. (H) THP-1 cells were primed with LPS alone, cells primed with LPS were then treated with Ng, and cells pretreated with VX-765, followed by LPS + Ng treatment, and their culture media was collected. RNA extraction and qPCR analysis revealed higher expression of mtRNA (ND5, ND6, and CYTB) in the extracellular space of cells treated with LPS + Ng alone compared to VX-765-pretreated cells. Data are presented as mean ± SEM (**p < 0.01, **p < 0.01, *p < 0.05). (I) PM cells primed with LPS only, primed with LPS, then treated with Ng, and cells pretreated with VX-765, followed by LPS + Ng treatment, had their culture media collected. RNA extraction and qPCR analysis revealed higher expression of mtRNA (ND1, COX1, and CYTB) in the extracellular space of cells treated with LPS + Ng alone compared to VX-765-pretreated cells. Data are presented as mean ± SEM (**p < 0.01, ****p < 0.0001, *p < 0.05), all experiments were repeated three times, and representative experiments are shown.

Article Snippet: Monoclonal rabbit antibodies against GSDME (ab215191) were purchased from Abcam at a dilution of 1:2000, and monoclonal rabbit antibodies against GSDMD (97558) from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibodies against phospho-TBK1 (ab109272) from Abcam at a dilution of 1:2000, monoclonal rabbit antibody against Caspase-1 (3866) purchased from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibody against NLRP3 (15101) sourced from Cell Signaling Technology at a dilution of 1:1000 and monoclonal antibody against VDAC1 (55259-1-AP) from Proteintech.

Techniques: Transfection, Expressing, Injection, Western Blot, RNA Extraction

Journal: Frontiers in Immunology

Article Title: The role of gasdermin-mediated mitochondrial RNA release in amplifying secondary immune response during microbial infection

doi: 10.3389/fimmu.2025.1668763

Figure Lengend Snippet: Gasdermin D and E mediated mtRNA release and VISA pathway activation induce secondary inflammatory response. Cells treated with LPS and Ng trigger the activation of the NLRP3 pathway, leading to the recruitment of caspase-1, which in turn cleaves GSDMD . In contrast, cells infected with VSV activate the PKR pathway and caspase-3, resulting in the cleavage of GSDME ( , ). Our findings indicate that the N-terminal fragments of both GSDMD and GSDME facilitate mitochondrial membrane permeabilization and the release of mtRNA into the cytoplasm. This mtRNA is subsequently recognized by the double-stranded RNA sensors RIG-I and MDA5, prompting their activation . Activated RIG-I interacts with VISA (MAVS) on the mitochondrial membrane , initiating a signaling cascade that involves various kinases, including TBK-1, IKKϵ, IKKα, and IKKβ ( , ). This cascade culminates in the phosphorylation and activation of key transcription factors, notably IRF-3/7 and NF-κB. These activated transcription factors then translocate to the nucleus to drive the expression of type I interferons (IFNs) and an array of pro-inflammatory cytokines ( , ). Furthermore, the N-terminal fragments of Gasdermin D and E contribute to the formation of pores in the plasma membrane, facilitating the release of mtRNA into the extracellular space due to the permeabilization induced by GSDMD and GSDME.

Article Snippet: Monoclonal rabbit antibodies against GSDME (ab215191) were purchased from Abcam at a dilution of 1:2000, and monoclonal rabbit antibodies against GSDMD (97558) from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibodies against phospho-TBK1 (ab109272) from Abcam at a dilution of 1:2000, monoclonal rabbit antibody against Caspase-1 (3866) purchased from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibody against NLRP3 (15101) sourced from Cell Signaling Technology at a dilution of 1:1000 and monoclonal antibody against VDAC1 (55259-1-AP) from Proteintech.

Techniques: Activation Assay, Infection, Membrane, Phospho-proteomics, Expressing, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: The role of gasdermin-mediated mitochondrial RNA release in amplifying secondary immune response during microbial infection

doi: 10.3389/fimmu.2025.1668763

Figure Lengend Snippet: GSDMD inhibition modulates cytokine production in LPS and Ng responses. (A, B) THP1 cells were primed with 100 ng/mL LPS for 2 hours and activated with 5 μM/mL nigericin (Ng) for one hour, with qPCR analysis demonstrating elevated expression of IFN-β (A) and IL-6 (B) . An unpaired t-test determined statistical significance. Data represent ± SEM (**p < 0.01, ***p < 0.001). (C, D) mouse peritoneal macrophages (PM cells) primed with 100 ng/mL LPS for 2 hours, treated with 5 μM/mL Ng for one hour, qPCR analysis demonstrating elevated expression of IFN-β (C) and IL-6 (D) , an unpaired t-test determined statistical significance. Data represent ± SEM (**p < 0.01, **p < 0.01). (E, F) THP-1 and PM cells primed with 100 ng/mL LPS alone exhibited reduced NLRP3 activation, caspase-1, and GSDMD cleavage. In contrast, cells primed with LPS and activated with 5 μM Ng showed enhanced NLRP3 activation, caspase-1, and GSDMD cleavage, leading to TBK1 phosphorylation. Cleaved caspase-1 protein (p20) was isolated from the supernatant (SN), and pTBK1 protein expression levels were quantified using ImageJ; an unpaired t-test determined statistical significance. Data represent ± SEM (***p < 0.001, ****p < 0.0001). (G, H) THP1 cells primed with 100 ng/mL LPS alone, cells treated with LPS + Ng, cells pretreated with 20 μM VX-765 for 2 hours and then treated with LPS + Ng, qPCR analysis showing decreased IFN-β (E) and IL-6 (F) expression in VX-765 pretreated THP-1 cells. Data represent mean ± SEM (*p < 0.05, ***p < 0.001). (I, J) PM cells primed with LPS alone, cells treated with LPS + Ng, cells pretreated with 20 μM VX-765 for 2 hours and then treated with LPS + Ng, qPCR analysis showing decreased IFN-β (G) and IL-6 (H) expression in VX-765 pretreated PM cells. Data represent mean ± SEM (**p < 0.01, *p < 0.05). (K, L) Western blot analysis of THP1 and PM cells showed no GSDMD cleavage and TBK1 phosphorylation in LPS-primed cells alone and cells pretreated with 20 μM VX-765 for 2 hours followed by LPS + Ng treatment. In contrast, LPS + Ng treatment induced GSDMD cleavage and elevated TBK1 phosphorylation, pTBK1 protein expression levels were quantified using ImageJ; an unpaired t-test determined statistical significance. Data represent ± SEM (****p < 0.0001, ***p < 0.001). (M, N) THP1 cells primed with LPS alone, cells primed with LPS, then treated with Ng, cells primed with LPS, then treated with 100 μM/mL dimethyl fumarate (DMF) for 2 hours and then treated with Ng, qPCR analysis showing decreased IFN-β (K) and IL-6 (L) expression in DMF treated THP1 cells. Data represent mean ± SEM (***p < 0.001, ****p < 0.0001). (O, P) PM cells primed with LPS alone, cells primed with LPS and then treated with Ng, cells primed with LPS, then treated with 100 μM/mL DMF for 2 hours and then treated with Ng, qPCR analysis showing decreased IFN-β (M) and IL-6 (N) expression in DMF treated PM cells. Data represent mean ± SEM (**p < 0.01, **p < 0.01). (Q, R) Western blot analysis showed that LPS priming, followed by Ng treatment, induced GSDMD cleavage and elevated TBK1 phosphorylation in THP-1 and PM cells. However, cells primed with LPS, treated with DMF, and treated with Ng inhibited GSDMD cleavage and reduced TBK1 phosphorylation, pTBK1 protein expression levels were quantified using ImageJ; an unpaired t-test determined statistical significance. Data represent ± SEM (****p < 0.0001, ***p < 0.001), all experiments were repeated three times, and representative experiments are shown.

Article Snippet: Monoclonal rabbit antibodies against GSDME (ab215191) were purchased from Abcam at a dilution of 1:2000, and monoclonal rabbit antibodies against GSDMD (97558) from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibodies against phospho-TBK1 (ab109272) from Abcam at a dilution of 1:2000, monoclonal rabbit antibody against Caspase-1 (3866) purchased from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibody against NLRP3 (15101) sourced from Cell Signaling Technology at a dilution of 1:1000 and monoclonal antibody against VDAC1 (55259-1-AP) from Proteintech.

Techniques: Inhibition, Expressing, Activation Assay, Phospho-proteomics, Isolation, Western Blot

Journal: Frontiers in Immunology

Article Title: The role of gasdermin-mediated mitochondrial RNA release in amplifying secondary immune response during microbial infection

doi: 10.3389/fimmu.2025.1668763

Figure Lengend Snippet: GSDMD as key drivers of mitochondrial dysfunction: (A) THP1 cells primed with LPS alone, cells primed with LPS, then treated with Ng, cells pretreated with VX-765, then treated with LPS + Ng. Subjected to mitochondrial Isolation, Western blot analysis of GSDMD cleavage was observed in both cytoplasmic and mitochondrial fractions of LPS + Ng-treated cells; no GSDMD cleavage was observed in cells pretreated with VX-765 or those primed with LPS alone. (B) PM cells primed with LPS, cells primed with LPS, then treated with Ng, cells pretreated with VX-765, then treated with LPS + Ng. Subjected to mitochondrial Isolation, Western blot analysis of GSDMD cleavage was observed in both cytoplasmic and mitochondrial fractions of LPS + Ng-treated cells; no GSDMD cleavage was observed in cells pretreated with VX-765 or those primed with LPS alone. (C) THP1 cells primed with LPS alone, cells primed with LPS, then treated with Ng, cells pretreated with VX-765, then treated with LPS + Ng. Cells stained with JC-1 dye, LPS + Ng alone displayed a higher proportion of JC-1 monomers and fewer aggregates, in contrast to THP-1 cells primed with LPS alone and cells pretreated with VX-765. (D, E) THP1 cells were primed with LPS alone, primed with LPS, then treated with Ng, pretreated with VX-765, then treated with LPS + Ng treatment, and mitochondrial damage was assessed using JC-1 staining. Flow cytometry analysis confirmed the results observed by inverted microscopy; the histogram shows the percentage. (F) PM cells primed with LPS alone, cells primed with LPS, then treated with Ng, cells pretreated with VX-765, then treated with LPS + Ng. Cells stained with JC-1 dye, LPS + Ng alone displayed a higher proportion of JC-1 monomers and fewer aggregates, in contrast to PM cells primed with LPS alone and cells pretreated with VX-765. (G, H) PM cells were primed with LPS alone, primed with LPS, then treated with Ng, pretreated with VX-765, then treated with LPS + Ng treatment, and mitochondrial damage was assessed using JC-1 staining. Flow cytometry analysis confirmed the results observed by inverted microscopy; the histogram shows the percentage, all experiments were repeated three times, and representative experiments are shown. The histogram shows the percentage of Flow Cytometry analysis, with statistical significance determined by an unpaired t-test. Data represent ± SEM (***p < 0.001, ****p < 0.0001).

Article Snippet: Monoclonal rabbit antibodies against GSDME (ab215191) were purchased from Abcam at a dilution of 1:2000, and monoclonal rabbit antibodies against GSDMD (97558) from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibodies against phospho-TBK1 (ab109272) from Abcam at a dilution of 1:2000, monoclonal rabbit antibody against Caspase-1 (3866) purchased from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibody against NLRP3 (15101) sourced from Cell Signaling Technology at a dilution of 1:1000 and monoclonal antibody against VDAC1 (55259-1-AP) from Proteintech.

Techniques: Isolation, Western Blot, Staining, Flow Cytometry, Inverted Microscopy