Journal: Cellular & molecular immunology

Article Title: Plasmacytoid dendritic cells promote acute kidney injury by producing interferon-α.

doi: 10.1038/s41423-019-0343-9

Figure Lengend Snippet: Fig. 4 pDCs induce apoptosis of TECs through IFN-α. Cleaved caspase 3 expression in mouse kidneys after adoptive transfer (a), pDC depletion (b), IFN-α administration (c), and IFN-α neutralization (d) was examined. Densitometric analysis of western blots of cleaved caspase 3 was performed by ratioing the observed value to that of the average control mouse titer (arbitrary units [a.u.]), and the results are shown in the lower panel. ** P < 0.01 vs. WT Cis, DTRBDCA2 Veh, and Veh. ***P < 0.001 vs. control. e Purified TECs were cultured with the supernatant from pDC cultures in the presence or absence of an anti-IFN-α antibody for 24 h, followed by evaluation of the caspase 3 activity level. *** P < 0.001 vs. control. The data are expressed as the mean ± s.e.m. The pictures shown are one representative experiment of at least two independent experiments. The densitometric analysis results shown are representative of the analysis of two western blots. Statistical comparisons among groups were calculated by ANOVA followed by Tukey’s test.

Article Snippet: Supernatants were collected for TNF-α detection using an ELISA kit (#88–7324, eBioscience).52 Stimulation of pDCs TECs were placed in serum-free medium for 24 h and further stimulated with 100 μM cisplatin with or without a 50 μM concentration of the caspase 3 inhibitor Z-DEVD-FMK (MedChemExpress, Monmouth Junction, USA).

Techniques: Expressing, Adoptive Transfer Assay, Neutralization, Western Blot, Control, Cell Culture, Activity Assay

Journal: Cellular & molecular immunology

Article Title: Plasmacytoid dendritic cells promote acute kidney injury by producing interferon-α.

doi: 10.1038/s41423-019-0343-9

Figure Lengend Snippet: Fig. 5 pDCs are activated by factors released by apoptotic TECs. a TECs were stimulated with 100 μm cisplatin with or without a 50 μm concentration of the caspase 3 inhibitor Z-DEVD-FMK. The supernatants containing cisplatin and inhibitor were replaced and then collected after another 24 h of culture. pDCs were isolated and incubated with TEC supernatant for 24 h. The representative histograms of the flow cytometric analysis of CD80, CD86, CD40, and MHC class II expression on ex vivo-stimulated pDCs are shown in the left panel. The mean fluorescence intensity (MFI) values are given in the right panel. **P < 0.01 and ***P < 0.001 vs. Veh. b The IFN-α concentration in the supernatant of ex vivo-stimulated pDCs was analyzed with ELISA. ***P < 0.001 vs. Veh. The results shown are from one representative experiment of at least two independent experiments in triplicate cultures with 4–5 samples per group. The data are expressed as the mean ± s.e.m. Statistical comparisons among groups were calculated by ANOVA followed by Tukey’s test.

Article Snippet: Supernatants were collected for TNF-α detection using an ELISA kit (#88–7324, eBioscience).52 Stimulation of pDCs TECs were placed in serum-free medium for 24 h and further stimulated with 100 μM cisplatin with or without a 50 μM concentration of the caspase 3 inhibitor Z-DEVD-FMK (MedChemExpress, Monmouth Junction, USA).

Techniques: Concentration Assay, Isolation, Incubation, Expressing, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Cellular & molecular immunology

Article Title: Plasmacytoid dendritic cells promote acute kidney injury by producing interferon-α.

doi: 10.1038/s41423-019-0343-9

Figure Lengend Snippet: Fig. 6 Depletion of pDCs prevents IR-induced AKI. a The percentage of pDCs in kidney CD45+ cells 24 h after IR was measured by flow cytometry (left panel). A representative flow cytometry plot for pDCs in injured kidneys is shown in the right panel. b The MFI of cell surface phenotype markers on intrarenal pDCs after IR is shown. c DTRBDCA2 mice and control mice were treated with DT 24 h before IR. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels and kidney expression of KIM-1 were measured 24 h after IR. d The kidney pathology was evaluated and is shown at a magnification of ×400. Scale bar: black 100 μm. The histopathological score of the kidneys was evaluated and is shown in the right panel. e Flow cytometric analysis of kidney immune cells after IR-induced AKI was performed, and the results are shown. f The kidney expression of TNF-α and IL-1β after IR-induced AKI was analyzed and is shown. g The kidney IFN-α concentration was measured by ELISA. The results are expressed in pg/mg total protein. h Cleaved caspase 3 expression in mouse kidneys was examined and is shown in the upper panel. Densitometric analysis of western blots is shown in the lower panel. i Schematic representation showing that pDCs exacerbate AKI via IFN-α. In this study, TECs were shown to contribute to pDC recruitment and activation after AKI. Then, kidney-infiltrating pDCs lead to the release of IFN-α, which exacerbates interstitial inflammation and tubular epithelial cell damage. The data are expressed as the mean ± s.e.m. The results shown are from one representative experiment of at least two independent experiments with 4–6 mice per group or triplicate cultures with 4–5 samples per group. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical comparisons among groups were calculated by ANOVA followed by Tukey’s test.

Article Snippet: Supernatants were collected for TNF-α detection using an ELISA kit (#88–7324, eBioscience).52 Stimulation of pDCs TECs were placed in serum-free medium for 24 h and further stimulated with 100 μM cisplatin with or without a 50 μM concentration of the caspase 3 inhibitor Z-DEVD-FMK (MedChemExpress, Monmouth Junction, USA).

Techniques: Cytometry, Control, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay

Journal: Oncology Reports

Article Title: Expression level of Bcl-XL critically affects sensitivity of hepatocellular carcinoma cells to LIGHT-enhanced and interferon-γ-induced apoptosis

doi: 10.3892/or.17.5.1067

Figure Lengend Snippet: Figure 3. Expression levels of caspase-3, caspase-8, caspase-9, Bcl-XL, Bak and Bid in Hep3B cells treated with IFN-Á/LIGHT. (A) Hep3B cells were treated with 100 ng/ml IFN-Á and various concentrations of LIGHT for 24 h or 72 h as indicated. Cell lysates containing 30 μg of protein were subjected to 12% Tris-glycine gel electrophoresis followed by Western blotting with rabbit polyclonal antibodies against caspase-3, caspase-8, caspase-9, Bcl- XL, Bak, Bid, respectively. Cells treated with 100 ng/ml IFN-Á or 100 ng/ml LIGHT alone and untreated cells were used as the controls. ß-actin was used as an internal control for total protein loading. (B) Hep3B cells (2x105) treated with 100 ng/ml sLIGHT and 100 ng/ml of IFN-Á were also incubated with 100 μM of caspase-3 inhibitor Z-DEVD-fmk, caspase-9 inhibitor Z- IEHD-fmk, caspase-8 inhibitor Z-IEHD-fmk, or normal saline for 96 h. Cell viability was then measured. The viability of untreated (without any cytokine and inhibitor) cells was set at 100%, and the relative viability of other samples was calculated accordingly. The experiments were performed in triplicate, and in at least two separated experiments. The bar indicates the standard error. **p<0.01, versus group treated with normal saline.

Article Snippet: A recombinant LIGHT soluble protein that only contained the extracellular region of human LIGHT and caspase inhibitors Z-DEVD-fmk, Z-IETD-fmk and Z-LEHD-fmk was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot, Control, Incubation, Saline

Journal: Molecular cancer research : MCR

Article Title: Empty spiracles homeobox 2 (EMX2) transcription factor functions as a tumor suppressor in renal cell carcinoma by targeting CADM1

doi: 10.1158/1541-7786.MCR-24-0496

Figure Lengend Snippet: A-B: Annexin V/PI staining combined with flow cytometry analysis to assess cell death in EMX2-stably expressed ACHN cells and control cells in both adhered and suspension conditions. Representative images (A) and statistics (B) were shown. C-D: Illustrative stacked histograms and quantified mean fluorescence intensity (MFI) of CMXRos staining were presented for EMX2 overexpressing ACHN cells and control cells in both adherent and suspension states. The results were expressed as fold change relative to the control. Representative images (C) and statistics (D) were shown. E-F: Confocal imaging displays the fluorescence intensity ratio of green fluorescent monomers (depolarization)/red fluorescent aggregates (polarization) in mitochondria of EMX2-overexpressing cells compared to control cells. Representative images (E) and statistics (F) were shown. G. Heat map showing the expression of mitochondrial ETC (electron-transport chain) regulated genes in EMX2-overexpressing ACHN cells compared to control cells under suspension condition. H. Annexin V/PI staining followed by flow cytometry analysis was conducted to detect the cell death of EMX2 overexpressing ACHN cells and control cells in suspension condition aided by an introduction of Matrigel (80 μg/ml). The results were expressed as fold change relative to the control. I-J: Western blot analyzed the protein level of CASPASE-3, cleaved CASPASE-3, PARP1, cleaved PARP1 and BCL-2 in EMX2 overexpressing and control cells in both adhered and suspension conditions. Representative images (I) and statistics (J) were shown. K. Cell death of EMX2 overexpressing ACHN cells and control cells under both adherent and suspension conditions, with or without Talazoparib (15 μM), was analyzed by Annexin V/PI staining followed by flow cytometry. The results were expressed as fold change relative to the control. L. Annexin V/PI staining followed by flow cytometry analysis was performed to check cell death of EMX2 overexpressing ACHN cells and control cells in both adhered and suspension conditions with or without Z-DEVD-FMK (10 μM). The results were expressed as fold change relative to the control. Error bars represent the mean ± SD of three biological replicates. Two-sided Student’s t test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns: not significant.

Article Snippet: Cells were pretreated with the Caspase-3 inhibitor Z-DEVD-FMK (Selleck, Houston, TX) at 10 μM and PARP inhibitor Talazoparib (Selleck, Houston, TX) at 15 μM for 24 h.

Techniques: Staining, Flow Cytometry, Stable Transfection, Control, Suspension, Fluorescence, Imaging, Expressing, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Empty spiracles homeobox 2 (EMX2) transcription factor functions as a tumor suppressor in renal cell carcinoma by targeting CADM1

doi: 10.1158/1541-7786.MCR-24-0496

Figure Lengend Snippet: EMX2-mediated transcriptional regulation orchestrates multifaceted suppression of RCC progression. EMX2 directly binds to the CADM1 promoter and triggers transcriptional expression. Upregulation of EMX2-CADM1 enhances PARP1 activity and disrupts mitochondrial membrane potential (MMP), leading to parthanatos in movable and migrating RCC cells. EMX2-CADM1 also promotes RCC cell apoptosis under adherent conditions through the activation of the Caspase-3 pathway. Concurrently, increased EMX2-CADM1 expression inhibits PI3K-AKT signaling, impairing RCC cell proliferation and migration. These orchestrated effects ultimately converge to the suppression of renal tumor growth and invasion.

Article Snippet: Cells were pretreated with the Caspase-3 inhibitor Z-DEVD-FMK (Selleck, Houston, TX) at 10 μM and PARP inhibitor Talazoparib (Selleck, Houston, TX) at 15 μM for 24 h.

Techniques: Expressing, Activity Assay, Membrane, Activation Assay, Migration

Journal: Neurotoxicology

Article Title: p73 gene in Dopaminergic Neurons is Highly Susceptible to Manganese Neurotoxicity

doi: 10.1016/j.neuro.2016.04.012

Figure Lengend Snippet: N27 dopaminergic cells were pre-treated with either 20 or 50 μM of the caspase-3 inhibitor zDEVD-fmk before exposure to 300 μM of Mn for 24 h in reduced sera. Expression of ΔNp73 protein was measured by Western blot assays (A). Densitometric analysis was performed (B). Data are expressed as percent of control and represented as mean ± S.E.M. from three independent experiments. Asterisks (*, p < 0.05; **, p<0.01) indicate significant differences between treatment and control groups.

Article Snippet: We purchased the tetrapeptide caspase-3 inhibitor zDEVD-fmk from ApexBio (Houston, TX).

Techniques: Expressing, Western Blot