Journal: Cell reports

Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry

doi: 10.1016/j.celrep.2025.116903

Figure Lengend Snippet: (A) Schematic of the STIM1/ORAI1 complex using split Venus as a yellow signal reporter in a bimolecular fluorescence complementation (BiFC) assay. Yellow fluorescence indicates STIM1/ORAI1 interaction. The signal is higher after shear stress compared to the static condition in THP-1 cells. (B) Quantification of the Venus-positive BiFC STIM1/ORAI1 THP-1 cells under shear stress and static conditions. The shear stress condition shows 10.60%-±0.70% Venus-positive cells, while the static condition only has 1.80% ±0.20% Venus-positive cells. (C) STIM1-KD and ORAI1-KD cells have decreased shear stress-mediated IL8 expression as compared to control THP-1 cells, as quantified by qPCR. Replicates, n = 6. (D) SPTAN1-KD and RAF1-KD cells have decreased STIM1 and ORAI1 interaction under shear stress condition based on decreased split Venus STIM1 and ORAI1 signal. Replicates, n = 6. (E) Quantification of flow cytometry data, showing that STIM1-KD and ORAI1-KD cells have decreased shear-mediated cellular necrosis and increased cell survival. Replicates, n = 6. (F) Proposed mode of the cascade molecular reaction induced by shear stress. (G) FR180204 , an ERK inhibitor, decreased shear stress-mediated IL8 expression in CD14 + primary monocytes. (H) Quantification of flow cytometry data, displaying a decrease in shear stress-mediated necrotic cells following treatment with FR180204 in CD14 + primary monocytes.

Article Snippet: Co-detection was performed using IRDye 700 and 800-labeled secondary antibodies (926–68070 and 926–32211, Li-Cor) under the Odyssey infrared laser fluorescence scanner (Li-Cor) and quantified using ImageJ.

Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, Shear, Expressing, Control, Flow Cytometry