Fig 2: ILK loss enhances bosutinib and eCF506 treatment in vivo. A, Tumor growth rates for PX459v2 and ILK gRNA 2 tumors growing in immunodeficient Rag2-Il2rg double knockout mice (5 mice per group, 2 tumors per mouse) following treatment with vehicle, bosutinib (75 mg/kg), or eCF506 (40 mg/kg) given once daily via oral gavage. B, Final tumor volumes at day 35. C, Bosutinib and eCF506 inhibit pSrc in vivo as shown via Western blot analysis. D, Quantification of Western blots shown in C. E, Ki67 analysis using QuPath. ****, P < 0.0001, as determined by a two-way ANOVA with Bonferroni multiple comparisons correction. All error bars are SEM.

Fig 3: Heart imaging studies in mini pig MI. (A-D) LVEDD, IVSD, EF and LVPWd improved in the ad-ILK group compared with the ad-null group. n=6, *P<0.05. (E and F) Ad-ILK improves heart function four weeks post-MI. The myocardial perfusion image by ECT and the myocardial metabolic image by PET. LVEDD, LV end-diastolic diameter; IVSD, interventricular septal thickness in diastole; MI, myocardial infarction; PET, positron emission tomography.

Fig 4: Overexpression of ILK prevents apoptosis and increases microvessel density in the infarct zone. (A and B) Triple-staining with TUNEL (green), anti-α-sarcomeric actin antibody (red) and DAPI (blue) in the infarction zone of (A) ad-ILK and (B) ad-null. Scale bars, 50 µm. (C and D) Microvessel density is measured by frozen sections stained with vWF in the infarct zone of (C) ad-ILK and (D) ad-null group; scale bars, 25 µm. (E) Quantification of TUNEL-positive cardiomyocytes in the infarct zones of ad-ILK and ad-null animals. n=6 per group. *P<0.01. (F) Quantification of microvessel density in faction zone of ad-ILK and ad-null animals. n=6 per group. *P<0.01.

Fig 5: ILK knockout in combination with bosutinib treatment causes cell adhesion defects. A, Western blot analysis of cell lines grown in 2D. Data are representative of three independent experimental replicates. B, Adhesion challenge: IncuCyte cell confluence was normalized to DMSO PX459v2. DMSO, solid fill; bosutinib, clear fill. C, 2D 8-point dilution for half-inhibitory concentrations (EC50) for PND-1186. Endpoint quantification using normalized cell counts form Hoechst-stained images. D, Cell-by-cell mask and a “rounded cells” classifier whereby area <600 μm2 and eccentricity <0.65, one hour post drug addition. E, IncuCyte S3 cell-by-cell quantification for MDA-MB-231 cells plated at 8,000 cells per well. Arrow, drug addition. A bosutinib EC20 (0.9 μmol/L) concentration was used in all experiments. ***, P < 0.001.