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

Figure 3 Mammalian Ste20-like kinase 1 inhibition-induced autophagy reduced cell apoptosis in detached mouse bone marrow mesenchymal stem cells. 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 using the ROS probe 2,7-Dichlorodihydrofluorescein diacetate 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: 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 expression were evaluated by Western blot analysis. glyceraldehyde-3-phosphate dehydrogenase served as a control. Values are the mean ± SD, and experiments were completed in triplicate. 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 + 3-MA group. ^cP < 0.01; ^bP < 0.05; ^aP > 0.05.