Mammalian Ste20-like kinase 1 inhibition as a cellular mediator of anoikis in mouse bone marrow mesenchymal stem cells

Figure 4 Upregulated integrinα5β1 expression triggered by Mammalian Ste20-like kinase 1 inhibition protected mouse bone marrow mesenchymal stem cells from anoikis. A: Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL)-positive cells analysed by flow cytometry; B: Representative images and quantification of fluorescence staining of dead cells; C: Reactive oxygen species (ROS) levels were measured by flow cytometry; D: Quantitative analysis of the rate of TUNEL-positive cells; E: Quantification of live cells using a thiazolyl blue tetrazolium bromide assay; F: Quantitative analysis of the intracellular ROS level; G: Cell adhesion was evaluated as the fold change compared back to the control groups; H and I: Western blotting was used to measure the expression levels of Mammalian Ste20-like kinase 1 (Mst1), microtubules associated protein 1 light chain 3 II/I, p62, Beclin1, phospho-focal adhesion kinase, and activated caspase 3. glyceraldehyde-3-phosphate dehydrogenase was used as the loading control. Values are expressed as the mean ± SD. Measurements were performed in three replicates. 3-MA: 3-methyladenine; TUNEL: Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling; ROS: Reactive oxygen species; Mst1: Mammalian Ste20-like kinase 1; LC3-II/I: Microtubules associated protein 1 light chain 3 II/I; p-FAK: Phospho-focal adhesion kinase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase. 1. Control group; 2. Anoikis group; 3. Anoikis + sh-NC group; 4. Anoikis + sh-Mst1 group; 5. Anoikis + sh-Mst1 + si-NC group; 6. Anoikis + sh-Mst1 + si-ITGα5; 7. Anoikis + sh-Mst1 + si-ITGβ1. ^cP < 0.01; ^bP < 0.05; ^aP > 0.05.