Journal: Scientific Reports
Article Title: Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4
Figure Lengend Snippet: The uptake of exogenous CnB occurred via TLR4 receptor-mediated internalization. ( a ) Co-localization of rhodamine-labelled CnB with clathrin-GFP (upper panel) or CnB-GFP with rhodamine-labelled transferrin (lower panel) in SK-HEP-1 cells. The clathrin-GFP transfected cells were co-incubated with 5 μM CnB-rhodamine, or SK-HEP-1 cells were co-incubated with 5 μM CnB-GFP mixed with 5 μM rhodamine-labelled transferrin for 30 min, and visualized using a confocal laser scanning microscope (×63, scale bar 10 μm). ( b ) Free CnB inhibits the uptake of the fluorescently labelled CnB. The cells were co-incubated with excess CnB and DyLight 488-labeled CnB or labelled CnB alone for 30 min and visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×). The fluorescence intensity was quantified using a microplate reader (lower panel). ( c ) Positive correlation between CnB uptake and TLR4 expression. 5 × 10 5 cells from different cell lines were co-incubated with 5 μM CnB for 10 min, subjected to Trizol treatment and RNA extraction. Extracted mRNA was used for qPCR analysis of TLR4. The qPCR results were analyzed and compared with CnB-GFP uptake (lower panel). 5 × 10 6 cells from different cell lines were co-incubated with 5 μM CnB for 10 min and lysed with RIPA buffer, the samples were used for detecting the protein level of TLR4 by western blot analysis (upper panel). ( d ) Co-localization of exogenous CnB-GFP and TLR4-cherry. The TLR4-cherry- or cherry-transfected Hek293 cells were co-incubated with 5 μM CnB-GFP for 30 min, and visualized using a confocal laser scanning microscope (63×, scale bar 20 μm). ( e,f ) Effect of TLR4 knock down on CnB uptake. The influence of TLR4 knock down on CnB uptake was analysed by western blot analysis ( e ) or FI ( f ), (scale bar 50 μm, 20×). ( g ) TAK242 inhibited CnB uptake. The SK-HEP-1 cells were pre-incubated with 10 μM or 5 μM TAK242 or vehicle for 3 h, followed by co-incubation with CnB-GFP for 30 min. The co-incubated cells were washed, acid-stripped and quantified by a microplate reader. Bars represent mean ± s.e.m. from three independent experiments. *P
Article Snippet: The clathrin-GFP plasmid was a gift from the Yu lab (School of Life Science, Tsinghua University).
Techniques: Transfection, Incubation, Laser-Scanning Microscopy, Labeling, Fluorescence, Microscopy, Expressing, RNA Extraction, Real-time Polymerase Chain Reaction, Western Blot