Journal: Cell Death Discovery

Article Title: Trifluoperazine causes mast cell apoptosis through a secretory granule-mediated pathway

doi: 10.1038/s41420-026-03122-x

Figure Lengend Snippet: A Human lung smooth muscle cells (HLSMCs), lung fibroblasts (HLFs), and small airway epithelial cells (HSAECs) were treated with TFP at the indicated concentrations for 24 h. Human peripheral blood eosinophils and neutrophils were treated with TFP for 2 h. Cell viability was assessed by staining the cells with Annexin V (AnnV) and DRAQ7. Viable cells, AnnV − DRAQ7 − ; apoptotic cells, AnnV + DRAQ7 − ; necrotic/late apoptotic cells, AnnV + DRAQ7 + . HSAECs, n = 6 from three independent experiments; HLSMCs, eosinophils, neutrophils, n = 4 from four independent experiments; HLFs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA for HLSMCs, HLFs; Kruskal-Wallis for HSAECs; Friedman test for eosinophils, neutrophils). B – C Bone marrow-derived MCs (BMMCs) and peritoneal cell-derived MCs (PCMCs) treated under the same conditions as in ( A ) for 24 h. BMMCs, n = 5 from two independent experiments; PCMCs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA). Untreated (control) cells were used for statistical comparisons to all other groups in all figures. The bar charts show mean values + SEM or median + interquartile range. * P < 0.05; ** P < 0 .01; **** P < 0.0001. D Effect of TFP on DNA degradation. MCs were preincubated with bafilomycin A1 (Baf A1) (20 nM) for 2 h followed by treatment with TFP (10 μΜ) for 2 h. DNA was extracted from MCs and fragmentation was assessed by agarose gel electrophoresis. St standard marker.

Article Snippet: Primary human lung fibroblasts (HLFs) (PCS-201-013) and primary human lung smooth muscle cells (HLSMCs)(PCS-130-010) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Control, Agarose Gel Electrophoresis, Marker

Journal: PLoS ONE

Article Title: C/EBPβ-Thr217 Phosphorylation Signaling Contributes to the Development of Lung Injury and Fibrosis in Mice

doi: 10.1371/journal.pone.0025497

Figure Lengend Snippet: A. Immunoblots for RSK, C/EBPβ-phospho-Thr217, procaspase 8 and C/EBPβ were performed on C/EBPβ immunoprecipitates from activated primary human LMF lysates as described in . RSK and phosphorylated C/EBPβ were induced in activated LMF but decreased in activated LMF treated with the ERK1/2 inhibitor (10 µg for 24 hr) or with the C/EBPβ peptide (200 µg for 24 hr). Inactive procaspase 8 was associated with phosphorylated C/EBPβ in untreated, activated LMF, while active caspase 8 was associated with unphosphorylated C/EBPβ in activated LMF treated with the ERK1/2 inhibitor or with the C/EBPβ peptide. Human LMF expressed full-length C/EBPβ from the second AUG . β-Actin was used to correct for lung lysate input. We performed single analysis of the samples. Results from triplicate samples of two independent experiments are shown. B. The ERK1/2 inhibitor decreased the fold association of RSK with C/EBPβ (n: 6 per group; 0.07+/−0.01, P <0.0001), and the fold expression of C/EBPβ (n: 6 per group; 0.05+/−0.006, P <0.0001) and C/EBPβ-PhosphoThr266 (n: 6 per group; 0.33+/−0.078, P <0.0001) in activated, human lung fibroblasts. There was also an increased fold association between unphosphorylated human C/EBPβ and active caspase 8 (n: 6 per group; active caspase 8; 5.70+/−0.59, P <0.0001). The cell permeant Ac-KAla217VD-CHO peptide also inhibited C/EBPβ expression (n: 6 per group; 0.12+/−0.01, P <0.0001), the phosphorylation of C/EBPβ-Thr266 (n: 6 per group; 0.17+/−0.03, P <0.0001), the association of RSK with C/EBPβ (n: 6 per group; 0.24+/−0.08, P <0.0001) and increased the association of RSK with C/EBPβ (n: 6 per group; 0.24+/−0.08, P <0.0001). We performed single analysis of the samples. C. α-SMA and TGF-β were induced in activated LMF but the expression of these fibrogenic genes was inhibited by treatment with the ERK1/2 inhibitor (10 µg for 24 hr) or with the peptide (200 µg for 24 hr). β-Actin was used to correct for lung lysate input. Representative results from two independent studies. D. The ERK1/2 inhibitor decreased the fold expression of α-SMA (n: 6 per group; 0.24+/−0.11, P <0.0001) and TGF-β1 (n: 6 per group; 0.14+/−0.02, P <0.0001). The cell permeant Ac-KAla217VD-CHO peptide also inhibited the fold expression of α-SMA (n: 6 per group; 0.24+/−0.11, P <0.0001) and TGF-β1 (n: 6 per group; 0.14+/−0.02, P <0.0001). We performed single analysis of the samples. E. Annexin-V-PE binding in vivo in activated LMF was increased after treatment with the ERK1/2 inhibitor (20 µg for 8 hr) or with the peptide (200 µg for 24 hr). Values are the percentage of cells expressing annexin-V-PE binding as described in . Activated, human lung fibroblasts treated with the ERK1/2 inhibitor (n: 6; 66.33+/−5.68%, P <0.0001) or with the Ac-KAla217VD-CHO peptide (n: 6; 61.00+/−9.27%, P <0.0001) displayed increased percent annexin-V binding compared to control (n: 6; 4.15+/−0.94%). We performed single analysis of the samples. Results from triplicate samples of three independent experiments are shown.

Article Snippet: Primary human lung fibroblasts isolated from normal human lung parenchyma (Cell Applications; San Diego, CA) were cryopreserved at first passage and can be cultured and propagated at least 12 population doublings.

Techniques: Western Blot, Expressing, Phospho-proteomics, Binding Assay, In Vivo, Control

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: Exhaled breath condensates from healthy children induce cell death of in vitro cultured cells by activation of apoptosis

doi: 10.5114/ada.2019.87087

Figure Lengend Snippet: A – The culture of human lung fibroblasts (HLF) in the phase contrast microscope after 4-hour incubation with double distilled water (DDW), used as a test control (upper picture) or with a representative EBC sample (bottom picture). B – The membranes of Human Apoptosis Array exposed to HLF lysates incubated for 4 h with DDW (left picture) or a representative EBC sample (right picture). Each pair of spots in white frames represents a respective target molecule. The numbers at the frames correspond to respective molecules on graph C: 1 – CD95/Fas, 2 – pro-caspase-3, 3 – cleaved caspase-3, 4 – SMAC/Diablo, 5 – Hsp27, 6 – Hsp70. The brightest spots correspond to reference markers (dashed line frames). C – Relative expressions of selected apoptosis pathway-related proteins with the highest signal density, calculated as the percent of reference spots

Article Snippet: The normal human lung fibroblasts (HLF), purchased from Cell Applications Inc. (San Diego, CA), passages 4 th to 8 th , and murine endothelial cell line C166 (ATCC ® CRL-2581TM) from American Type Culture Collection (ATCC, Manassas, VA), were used for in vitro studies.

Techniques: Microscopy, Incubation, Control