Journal: International Journal of Molecular Sciences

Article Title: Mechanical Load and Piezo1 Channel Regulated Myosin II Activity in Mouse Lenses

doi: 10.3390/ijms23094710

Figure Lengend Snippet: Piezo1 activation in ex vivo lenses by Yoda1: Effects on lens clarity and myosin II activity. ( A ) . To determine the effects of Piezo1 activation on lens transparency, lenses derived from P30 mice and maintained under culture conditions were treated with 25 µM Yoda1, an agonist of Piezo1, and changes in lens transparency were monitored for 24 h. Yoda1-treated lenses exhibited a slight haziness starting at the 1 h interval that increased progressively with time, with lenses exhibiting significant increases in swelling and a slight nuclear opacity after 24 h compared to the control lenses. ( B ). In lenses treated with Yoda1 (25 µM) for 1 h, the levels of phospho-MLC were significantly elevated compared to the untreated control lenses. On the other hand, in the 6 and 24 h Yoda1-treated lenses, there was a significant and progressive decrease in the levels of phospho-MLC (pMLC) compared to the control lenses. The levels of total MLC (tMLC), however, were found to be similar between the Yoda1-treated and control lenses throughout the course of drug treatment. Lanes 1 to 3 represent 3 experimental replicates. Bars denote image magnification. *** p < 0.001; **** p < 0.0001. A.U.: Arbitrary Units.

Article Snippet: To determine the levels of total and phospho-MLC in the lens homogenates, the TCA-treated lens samples described above were subjected to immunoblot analysis using anti-phospho-MLC (pMLC) or anti-MLC rabbit polyclonal antibodies (Cat. No. 3674 and 3672, respectively, Cell Signaling Technology) as we described previously [ ].

Techniques: Activation Assay, Ex Vivo, Activity Assay, Derivative Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Microelectromechanical System Measurement of Platelet Contraction: Direct Interrogation of Myosin Light Chain Phosphorylation

doi: 10.3390/ijms22126448

Figure Lengend Snippet: Western Blotting for pMLC detection. Platelet lysate was analyzed for the presence of pMLC after the addition of reagents. The addition of the activators U46619, sildenafil, PMA, okadaic acid resulted in increased MLC phosphorylation which coincided with increased platelet contraction forces. Blots are representative of five independent experiments. The total MLC band used to calibrate the increase in phosphorylation is included.

Article Snippet: The membranes were blocked and incubated with the respective antibodies for MLC and pMLC (1:10,000, cat.# 3674 and #3672, Cell Signaling Technology (Danvers, MA, USA); β-actin loading control antibody (MA5-15738, ThermoFisher), 1:10,000 goat anti-mouse (cat.# 102971-068 LiCOR), and 1:10,000 H+L superclonal goat anti-rabbit (cat.#A27036, Fisher) and developed using chemiluminescent substrate (Licor).

Techniques: Western Blot, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: MASP-1 Increases Endothelial Permeability

doi: 10.3389/fimmu.2019.00991

Figure Lengend Snippet: rMASP-1 treatment reorganizes the actin cytoskeleton of endothelial cells. Confluent layers of HUVECs were seeded onto 18 well ibidi™ slides and were cultured for 2 days. Cells were pretreated with or without PAR antagonists (0.68 μM PAR-1 antagonist; 20 μM PAR-2 antagonist or 0.28 μM PAR-4 antagonist) for 10 min or 2.5 μM ROCK inhibitor (Y-27632) for 15 min, then were treated with 2 μM rMASP-1 or 300 nM thrombin for 20 min. Cells treated with culture medium only served as a negative control. After fixation, cells were stained with anti-pMLC antibody. F-actin cytoskeleton was stained with phalloidin-Alexa488, cell nuclei were labeled with Hoechst 33258 and pictures were taken using an Olympus IX-81 fluorescence microscope and an Olympus XM-10 camera with 40x magnification Olympus lenses (numerical aperture: 0.75). Representative images from three independent experiments are shown. The scale bar applies to all photomicrographs.

Article Snippet: In other experiments, cells were pretreated with 10 μM ML-7 or 2.5 μM Y-27632 or culture medium only for 15 min. After 1% paraformaldehyde-PBS fixation, cells were stained with anti-diphospho MLC (pMLC) antibody (Cell Signaling).

Techniques: Cell Culture, Negative Control, Staining, Labeling, Fluorescence, Microscopy