Figure 2

Soft matrix downregulates β1 integrin via lysosome-mediated protein degradation. (a) Representative western blot analysis results of NMuMG cells grown on matrices of varying stiffness at the indicated times. The protein levels of active β1 integrin and total β1 integrin were analyzed. β actin was used as an internal control. Also see Supplementary Fig. S3. (b) Quantification results of total β1 integrin were from (a) and two other experiments. β-actin-normalized data in each condition was compared with those of cells cultured on dishes at 0 h. All data are expressed as relative mean ± SEM from three independent experiments. (c) The β1 integrin mRNA levels of NMuMG cells grown on matrices of varying stiffness for 4 h were assessed and the 18 S rRNA is used a loading control. (d) Quantification results of β1 integrin mRNA levels from (c). (e) Representative western blot analysis results of cycloheximide (CHX)-chase assay of β1 integrin turnover in NMuMG cells grown on matrices of varying stiffness. NMuMG cells were pre-treated with or without 50 μg/ml CHX for 30 min before detached from tissue culture plastics. Then, cells were detached (time = 0) with low dose of trypsin and replated on different matrices with or without the sustained CHX treatment for the indicated time. Also see Supplementary Fig. S3. (f) Quantitative results of β1 integrin turnover were from (e). β-actin-normalized data in each condition was compared with those of cells in suspension at 0 h. (f) Top: Representative western blot analysis results of NMuMG cells grown on 0.2 kPa gel and treated with different concentrations of NH₄Cl. The protein level of integrin β1 was analyzed. β actin was used as an internal control. Bottom: Quantification results of β1 integrin were from three independent experiments. β-actin-normalized data in each condition was compared with those of cells cultured on dishes. Also see Supplementary Fig. S3. (g) Representative confocal immunofluorescence images of NMuMG cells grown on the indicated conditions for 4 h. Cells were stained for β1 integrin (blue), active β1 integrin (green), and lysosomal-associated membrane protein-1 (LAMP-1) (red). Scale bar = 10 μm. All data are expressed as relative mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.