il 5rα  (Bioss)

Journal: Frontiers in Immunology

Article Title: Gαi1/3 signaling mediates IL-5-induced eosinophil activation and type 2 inflammation in eosinophilic chronic rhinosinusitis

doi: 10.3389/fimmu.2024.1460104

Figure Lengend Snippet: IL-5 induced Gαi1/3 association with activated IL-5 receptor and Gab1. WT and Gαi1/3 DKO MEFs were treated with IL-5 (25 ng/mL) for 5 min, the association between IL-5Rα, Gab1, and Gαi1/3 was tested by co-ip assay (A) . EoL-1 cells were treated with GM-CSF (50 ng/mL) and IL-5 (50 ng/mL) for 5 min, the association between IL-5Rα, Gab1, and Gαi1/3 was tested by co-ip (B) , and processed for confocal microscopy to detect the presence of Gαi1, Gαi3, and IL-5Rα (C) . Bar = 1 μm.

Article Snippet: Antibodies against Gαi1 (sc-13533), Gαi2 (sc-13534), Gαi3 (sc-365422), Gab1 (sc-133191), Akt (sc-81434), Erk1/2 (sc-514302), S6K (sc-8418), p-Gab1 (AP0256), p-Akt s473 (sc-101629), p-Erk1/2 (sc-136521), p-S6K (sc-8416), STAT5 (sc-74442), p-STAT5 (AP-0887), IL-5Rα (bs-2601R-100ul), IgE (ab75673), and tryptase (ab2378) were purchased from Bioss (Woburn, MA, USA), ABcolnal (Wuhan, China), Abcam (Cambridge, MA, USA), and Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Co-Immunoprecipitation Assay, Confocal Microscopy

Journal: eBioMedicine

Article Title: The impact of Charcot-Leyden Crystal protein on mesothelioma chemotherapy: targeting eosinophils for enhanced chemosensitivity

doi: 10.1016/j.ebiom.2024.105418

Figure Lengend Snippet: Eosinophils differentiated from EOL1 cells inhibit the response to cisplatin and pemetrexed . (a) Valproate-differentiated EOL1 (Dif-EOL1) supernatant (SN) was added to M14K mesothelioma cells at 25% v/v for 48 h. M14K were then treated with 10 μM cisplatin and 10 μM pemetrexed (C + P) for 48 h. After fluorescent labelling of IL-5Rα and CCR3, EOL1 progenitors and Dif-EOL1 were analysed by flow cytometry. The relative Median Fluorescence Intensity (rMFI) corresponds to the ratio of fluorescence intensities associated with IL-5Rα (b) and CCR3 (c) with control isotypes. Bars represent mean ± standard deviation (SD) from 6 independent experiments. (d) Dif-EOL1 were labelled for CCR3 and actin, stained with DAPI and analysed by confocal microscopy (magnification 40×). (e) Flow cytometry was used to discriminate EOL1 progenitors from Dif-EOL1 based on size (forward scatter; FSC) and granulometry (side scatter; SSC). (f) Eosinophil peroxidase activity in EOL1 and Dif-EOL1 cells (stimulated with mock or IL-5; 100 ng/mL). Absorbance of the chromogenic substrate (OPD) was measured at 492 nm with a spectrophotometer. (g) CD63-positive cells (number/mm 2 ) were recorded by time-lapse microscopy (Incucyte imaging S3 Live-Cell system equipped with a 20X objective) in Dif-EOL1 cultures in presence or not of IL-5 (100 ng/mL). (h) Percentages of apoptotic M14K cells (2D) were determined by flow cytometry after Annexin V/propidium iodide (PI) staining. Early (Annexin V + PI − ) and late (Annexin V + PI + ) apoptotic cells were counted for each condition. (i) After ethanol permeabilization and PI staining, the cell cycle profiles (2D) were analysed by flow cytometry. The percentages of cells with fragmented genomic DNA ( i.e., Sub-G1) were evaluated. (j) M14K spheroids were generated by the liquid overlay method for 72 h in presence or absence of Dif-EOL1 supernatant. After treatment with C + P for 48 h, the proportion of M14K cells in Sub-G1 was determined by flow cytometry after spheroid dissociation, cell permeabilization and PI staining. (k) After transfer of the spheroids into an adherent 24-well plate, cell migration was monitored with an Olympus CKX41 microscope. (l) The surface occupied by the cell culture in mm 2 was measured after 24 h. Data are expressed as means ± SD, each point representing an independent test. Normality was checked by Shapiro–Wilk and equality of the variances was checked by Brown–Forsythe. Variance of the means was compared by t-test (panels b and c) or by one-way ANOVA followed by Tukey's multiple comparison test (panels f, h, i, j and l).

Article Snippet: After 2 washes with PBS-FBS 2%, Dif-EOL1 cells were labelled for 1 h on ice with 1 μg/mL of anti-IL-5Rα antibody (Invitrogen Cat# PA525159, RRID: AB_2542659 ).

Techniques: Flow Cytometry, Fluorescence, Control, Standard Deviation, Staining, Confocal Microscopy, Activity Assay, Spectrophotometry, Time-lapse Microscopy, Imaging, Generated, Migration, Microscopy, Cell Culture, Comparison