proteome profiler human protease inhibitor array kit  (R&D Systems)

Journal: Aging Cell

Article Title: Targeting Hyperoxia‐Induced Cellular Senescence in Developing Human Airway Cells: Senomorphics Versus Senolytics Versus Antioxidants

doi: 10.1111/acel.70538

Figure Lengend Snippet: Hyperoxia‐induced senescent fASM secrete higher levels of SASP. Cells cultured till day 7 in normoxic (21% O 2 ) or hyperoxic (50% O 2 ) environment were incubated with fresh growth media in normoxia for 24 h, and the supernatants were collected. Samples were then analyzed for SASP secretion by Eve Technologies Corporation (Calgary, Alberta, Canada) using Luminex xMAP and the Human Cytokine/Chemokine 96‐Plex Discovery Assay Array (HD96) panel. (A) Out of the 96 proteins, 80 were detected and visualized using a heatmap. (B) A detailed analysis of each detected marker is shown as fold‐change normalized to control. For statistical analysis, 2‐way ANOVA with a two‐stage linear step‐up procedure of Benjamini, Krieger and Yekutieli test, with individual variances computed for each comparison was applied. False discovery rate < 0.05 was considered significant. Data are presented as box plot of n = 7 cell lines per group.

Article Snippet: Supernatants collected at day 8 from normoxia and hyperoxia‐exposed fASM were analyzed for SASP secretion by Eve Technologies Corporation (Calgary, Alberta, Canada) using the Human Cytokine/Chemokine 96‐Plex Discovery Assay Array (HD96) panel.

Techniques: Cell Culture, Incubation, Luminex, Marker, Control, Comparison

Journal: Redox Biology

Article Title: Prophylactic C-terminal occludin–derived peptide attenuates LPS-induced airway inflammation via barrier preservation and mitochondrial ROS regulation

doi: 10.1016/j.redox.2026.104119

Figure Lengend Snippet: Occludin modulates LPS-induced IL-8 secretion, barrier integrity, and cytoskeletal remodeling in human bronchial epithelial cells. (A) The BEAS-2b cells were transfected and were then incubated with LPS in a time-dependent manner before the generation of total cell lysates, and occludin transcripts were assessed by qRT-PCR. ∗ p < 0.05 compared to the control. (B) The cells were treated with LPS in a time-dependent manner. The occludin-specific antibody was assessed by Western blot analysis. β-actin was used as a loading control. (C) A construct expressing wild-type occludin or siRNA-occludin was transiently transfected into BEAS-2b cells. The cells were washed and serum-starved overnight. They were subsequently treated with LPS for 2 h and a cytokine assay was performed with cultured media and cell lysates were harvested for qRT-PCR of IL-8 (D) . The density of the resulting spots of IL-8 was measured using a densitometric analysis. ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT occludin-treated transfectants. (E) The cells were transfected with either the wild-type occludin construct or siRNA-occuldin before incubation with LPS for various times, and then TEER testing was performed. Error bars represent the SEM of at least three independent experiments. (F) Cells were then treated with LPS for 2 h. F-actin staining was performed using ActinRed 555 ReadyProbe reagent (Molecular Probes) following the manufacturer's instructions. Cell nuclei were stained with diluted Deep Red (1:300). The fluorescence intensity was analyzed and statistically evaluated (right panel). ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT occludin-treated transfectants. All data are representative of at least three independent experiments.

Article Snippet: Proteome Profiler Human Cytokine Array kit was purchased from R&D Systems (cat no. ARY005B, USA).

Techniques: Transfection, Incubation, Quantitative RT-PCR, Control, Western Blot, Construct, Expressing, Cytokine Assay, Cell Culture, Staining, Fluorescence

Journal: Redox Biology

Article Title: Prophylactic C-terminal occludin–derived peptide attenuates LPS-induced airway inflammation via barrier preservation and mitochondrial ROS regulation

doi: 10.1016/j.redox.2026.104119

Figure Lengend Snippet: The peptide regulates mitochondrial dysfunction and ROS production by inhibiting LPS-induced p38 activation. (A) The BEAS-2b cells were treated with wild-type occludin peptide (pepWT OCLN) or mutant occludin peptide (pepMut OCLN) and incubated with LPS for 15, 30 min. The phospho-specific and total antibodies were assessed by Western blot analysis. β-actin was used as a loading control. (B) The BEAS-2b cells were transfected with p38 overexpression construct (WT p38) or siRNA-p38 for 24 h and incubated with LPS for 4 h siRNA-scramble was used as a negative control. The proinflammatory cytokine transcripts were assessed by qRT-PCR. ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT p38-transfected cells. (C) The mitochondrial membrane potential of LPS-induced BEAS-2b cells treated with either WT OCLN peptide or mut peptide was stained with JC-1 dye. Images are representative results of 3 independent experiments. (D) The mitochondria fission was stained using phospho-Drp1 antibody and visualized. The fluorescence intensity was analyzed and statistically evaluated (right panel). ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT OCLN peptide-treated transfectants. (E) After the BEAS-2b cells were harvested, cell lysates were used for MTT assay. (F) After mitochondria from the cells was isolated, the mitochondria lysates were used for mtROS measurement. ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS only; ∗∗∗ p < 0.05 compared with LPS- and WT occludin peptide-treated cells. All data shown are representative of three independent experiments.

Article Snippet: Proteome Profiler Human Cytokine Array kit was purchased from R&D Systems (cat no. ARY005B, USA).

Techniques: Activation Assay, Mutagenesis, Incubation, Western Blot, Control, Transfection, Over Expression, Construct, Negative Control, Quantitative RT-PCR, Membrane, Staining, Fluorescence, MTT Assay, Isolation