Fig 2: Loss of expression of either Kindlin-2, TβRI or ITGB1 inhibits growth and metastasis of TNBC tumors, which can be restored by re-expression of Kindlin-2.Tumor volume (A, C) and weight (B, D) of NSG mice (A, B) or Balb/C mice (C, D) injected with control (Ctrl) MDA-MB-231 or 4T1 cells or their K2-KO, TβRI-KO and ITGB1-KO derivatives up to 40 days post injection. (Representative H&E staining (E, G) of lungs form tumor-bearing mice from A and C, and quantification of metastatic foci (F, H). I Tumor volume of NSG mice injected with control (Ctrl) MDA-MB-231 cells, and their K2-KO or K2-KO rescued with K2-full derivatives up to 36 days post injection. J Representative H&E staining of their corresponding lungs, and quantification of metastatic foci (K). **p < 0.01; ***p < 0.001; ANOVA.

Fig 3: Kindlin-2 is required for the stabilization of the β1-Integrin:Kindlin-1:TβRI protein complex.MDA-MB-231 cells (A) and 4T1 cells (B) were subjected to CRISPR/Cas 9 mediated gene editing to knockout FERMT 2 (K2), ITGB1 (β1-Integrin) or TβRI genes, and their protein expression was assessed by Western Blot analysis. β-Actin is a loading control. qt-RT-PCR of mRNA expression levels of TβRI and β1-Integrin in the K2-KO MDA-MB-231 (C) and 4T1 (D) TNBC cells. E, F Flow cytometry histograms of the cell surface expression levels of β1-Integetrin (E) and its activated form (F) in K2-KO, ITGB1-KO and K2-full rescued K2-KO MDA-MB-231 cells. Western Blot analysis with indicated antibodies showing expressions of TβRI and β1-Integrin proteins in MDA-MB-231 K2-KO after treatment with MG132 (G, H) or after rescue of K2-full length (I, J). The numbers under each WB band represent the fold change in signal intensity with respect to its respective control band in each panel after normalization to the loading control signal. Data shown are representative of 3 replicates. Data are the mean ± SD (n = 3, *p < 0.05, Student’s t-test).

Fig 4: Loss of expression of either Kindlin-2, TβRI or ITGB1 inhibits signaling activities that are specific to β1-Integrin and TβRI.Representative images and average number of adherent control (Ctrl) MDA-MB-231 and 4T1 cells, and their K2-KO, TβRI-KO and ITGB1-KO derivatives on fibronectin (A–D), and on Matrigel (E–H). I–N Representative images and average cell surface area of control (Ctrl) MDA-MB-231 and 4T1 cells, and their K2-KO, TβRI-KO and ITGB1-KO derivatives on fibronectin (I–L), and on Matrigel (M–P) after spreading assay. Actin filaments were stained with Alexa 488 Phalloidin and nuclei were counterstained with DAPI. Q, R, WB results of phosphorylation levels of SMAD2/3 in control (Ctrl) MDA-MB-231 and 4T1 cells, and their K2-KO, TβRI-KO and ITGB1-KO derivatives. Scale bar: 100 µm for adhesion images and 50 µm for spreading images. The numbers under each WB band represent the fold change in signal intensity with respect to its respective control band in each panel after normalization to the loading control signal. Data shown are representative of 3 replicates. Data are the mean ± SD (n = 3, **p < 0.01; ***p < 0.001, Student’s t-test).