Fig 2: Loss of Kindlin-2 inhibits breast cancer progression in vivo.A Representative images of breast tumor sections from PyMT;WT and PyMT; cKO littermates at 8-week-age stained with anti-Kindlin-2, anti-cyclin D1 or anti-AR Tyr-534 phosphorylation antibodies, respectively. Scale bar: 100 µm. The right panels of A showed higher magnification images of the areas outlined with black squares in the left panels. B Quantification of staining density of Kindlin-2, cyclin D1 or AR Tyr-534 phosphorylation level in tumor sections was shown. ***p < 0.001 vs. PyMT;WT, n = 4 mice for PyMT; WT mice; n = 3 mice for PyMT; cKO mice; for each mice, the quantification was performed from at least fifteen images. C Immunoblotting analysis of primary tumor cells isolated from the PyMT;WT and PyMT; cKO mice with antibodies as indicated. D Mammary gland whole-mounts were prepared from PyMT;WT and PyMT; cKO littermates with indicated ages (left panel). Arrows indicate the hyperplastic area. Quantification of hyperplastic areas in mammary glands was shown in the right panel. *p < 0.05, **p < 0.01 vs. PyMT;WT, n = 8 mice at 2 weeks of age; n = 9 mice at 4 and 8 weeks of age; n = 3 mice at 6 weeks of age. Original magnification, ×30 (2 weeks); ×6.3 (6 and 8 weeks). Quantification of the largest tumor volume (E) and the total tumor volume (F) per mouse. ***p < 0.001 vs. PyMT;WT. n = 9 mice per group. G Kaplan–Merier tumor-free curves of PyMT;WT and PyMT; cKO mice. ***p < 0.001 vs. PyMT;WT, n = 13 mice per group. wk weeks, WT wild-type, cKO conditional knockout.

Fig 3: 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-ß1 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

Fig 4: Depletion of kindlin-2 increases ROS production and apoptosis. a–c A549 cells were infected with K2 shRNA (Sh-K2) lentivirus or control lentivirus (Sh-NC). Two days after the infection, the cells were infected with lentiviral expression vectors encoding 3xFLAG-tagged kindlin-2 (3fl-K2) or 3xFLAG empty vector (3fl). Three days after the lentiviral infection, the cells were analyzed by western blotting with antibodies recognizing K2 or GAPDH (as a loading control) (a). The levels of ROS were analyzed using DHE fluorescence probe as described in the Methods (b). Scale bar, 75 µm. The mean fluorescence intensities (MFI) were calculated using the Image-pro plus software from two different experiments (c, at least n = 15 field per group were counted from at least three independent experiments). d The cells (as specified in the figure) were immunofluorescently stained with DAPI and anti-cleaved caspase-3 antibody. Scale bar = 25 µm. The percentages of cleaved caspase-3-positive cells were quantified (e) as described in the Methods. Replicates (n = 5) from one representative experiment are shown. Data in c and e are presented as mean ± SEM using one-way ANOVA with Tukey–Kramer post-hoc analysis, *P < 0.05; ***p < 0.001

Fig 5: Kindlin-2 interacts with PYCR1 and colocalizes with PYCR1 in mitochondria. a, b Co-IP of PYCR1 with kindlin-2. Human A549 (a) or HCI-H358 (b) cells were analyzed by IP with monoclonal anti-kindlin-2 antibody or irrelevant mouse IgG (as a control) as described in the Methods. The cell lysates (lane 1), control IgG (lane 2), and anti-kindlin-2 immunoprecipitates (lane 3) were analyzed by western blotting with antibodies as indicated. c Co-IP of PYCR1 with FLAG-kindlin-2. Kindlin-2 KO A549 cells were infected with lentiviral vector encoding 3xFLAG-tagged kindlin-2 (3fl-K2) or control lentiviral vector lacking kindlin-2 sequence (3fl). The 3fl-K2 and control 3fl infectants were analyzed by IP with anti-FLAG antibody. The 3fl-K2 cell lysates (lane 1) and IP samples from 3fl (lane 2) or 3fl-K2 (lane 3) cells were analyzed by western blotting with kindlin-2 or PYCR1 antibodies. d GST-kindlin-2 bound to glutathione-Sepharose beads were incubated with increased concentrations (lane 2, 0 µg ml-1; lane 3, 10 µg ml-1; lane 4, 20 µg ml-1; lane 5, 40 µg ml-1) of purified His-tagged PYCR1. GST-kindlin-2 fusion protein pulldown was analyzed as described in the Methods. The sample in lane 1 was prepared as that in lane 4, except GST-kindlin-2 was replaced with GST. e–g GST-tagged full-length or mutant forms of kindlin-2 (illustrated in e) or GST alone (as a negative control) were used to pull down His-tagged PYCR1 as described in the Methods. The inputs, GST and GST-kindlin-2 fusion protein pulldowns, were analyzed by western blotting with antibodies against His and GST, respectively. h A549 cells were plated on fibronectin-coated coverslips and dually stained with mouse monoclonal anti-kindlin-2 and rabbit polyclonal anti-PYCR1 antibodies. The primary antibodies were detected with Alexa Fluor 488-conjugated anti-mouse IgG or Alexa Fluor 647-conjugated anti-rabbit IgG secondary antibodies. Scale bar = 10 µm. i The cytosolic fraction (Cyto, lane 1), mitochondrial fraction (Mito, lane 2), and total cell lysates (Total, lane 3) were analyzed by western blotting with antibodies to kindlin-2, PYCR1, tubulin, and prohibitin-2 (PHB2) as indicated