Fig. 7

ECM stiffening regulates kindlin-2-PYCR1 interaction. a The cytosolic (lanes 3 and 5), mitochondrial (lanes 4 and 6), and total (lanes 1 and 2) lysates from A549 cells on stiff (elastic modulus of 40 kPa) or soft (elastic modulus of 0.35 kPa) hydrogels were analyzed by western blotting. b A549 cells plated on stiff or soft hydrogels for 48 h were analyzed by IP and western blotting. Lane 3, the sample was prepared as that of lane 4, except anti-kindlin-2 antibody was substituted with irrelevant mouse IgG. c A549 cells expressing mClover-kindin-2 and mRuby-PYCR1 on stiff or soft hydrogels were analyzed by FLIM-FRET (> 15 cells per group were counted from at least three independent experiments). d, e A549 cells on soft hydrogels were pretreated with or without 10 mM MBCD for 1 h and stained with anti-β1 integrin antibody (d) or analyzed by IP with anti-kindlin-2 antibody (e). Scale bar in d, 25 μm. The lysates (lanes 1 and 2), control IgG (lane 3), and anti-kindlin-2 immunoprecipitates (lanes 4 and 5) were analyzed by western blotting (e). f–l A549 or kindlin-2 KO cells on stiff or soft hydrogels were analyzed by western blotting (f). Protein levels of PYCR1 relative to that of GAPDH were quantified by densitometric analyses and compared with that of A549 cells (normalized to 1) (g, n = 3). The mRNA level of PYCR1 in A549 cells on soft hydrogels was analyzed by RT-PCR and compared with that in A549 cells on stiff hydrogels (normalized to 1) (h, n = 3). The proline levels in A549 or kindlin-2 KO cells on stiff or soft hydrogels were analyzed (i, n = 3). The cells (3.5 × 10⁴/gel) were plated on stiff or soft hydrogels, cultured for 2 days, the number was counted (j, n = 3) and cells were stained with DAPI and anti-Ki67 antibody (k). Scale bar, 75 μm. The percentages of Ki67-positive cells were quantified (l, n = 5). Data in c, g, h, i, j, and l represent means ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis (g, i, j, l) or two-tailed unpaired Student’s t-test (c, h). ***p < 0.001. NS, no significance