(A) Western blot analysis of PINCH1, EGFP-PINCH1, and EGFP expression in MEFs. β-Actin served as loading control. (B) Mean ± SD results from 2D or 3D clonogenic survival assays of nonirradiated or irradiated (0–6 Gy X-rays) lrECM cell cultures (n = 3; *P < 0.05, **P < 0.01, t test). (C) Representative images of 2D and 3D lrECM colony formation of nonirradiated or irradiated PINCH1^fl/fl and PINCH1^–/– MEF cultures at 11 days after plating. Scale bars: 50 μm. (D) Experimental in vivo setup. Subcutaneous allograft PINCH1^fl/fl and PINCH1^–/– tumors were grown in immunocompromised mice. After tumor formation (diameter approximately 6 mm), tumors were locally irradiated with increasing single doses of 26–62 Gy under homogeneous hypoxia (200 kV X-rays, 0.5 mm copper filter, dose rate approximately 1.3 Gy/min). Tumor volume was measured over a time period of 210 days after irradiation. (E and F) Volume of PINCH1^fl/fl and PINCH1^–/– allograft tumors in immunocompromised mice plotted against time after irradiation (mean ± SEM; n = 10–18 per group), and tumor recurrence–free survival of PINCH1^fl/fl and PINCH1^–/– tumor-bearing mice (Kaplan-Meier statistics and log-rank test) after irradiation (see also Supplemental Figure 3).

(A) Western blotting for total and phosphorylated amounts of Akt1, FoxO1, and FoxO4. β-Actin served as loading control (see also Supplemental Figure 5). (B–G) Immunohistochemistry for total (B and C) or S473 (D and E) or T308 (F and G) phosphorylated Akt1 in PINCH1^fl/fl and PINCH1^–/– allografts. Representative images are shown. Scale bars: 50 μm. (H) Western blot analysis of total and phosphorylated amounts of Akt1 (S473, T308) in protein lysates of PINCH1^fl/fl and PINCH1^–/– allografts tumors (#, animal no.). Fold changes were calculated from densitometric analysis of protein bands of S473 or T308 phosphorylated Akt1 (mean ± SD; n = 7; **P < 0.01, t test). (I) Western blot analysis of Akt1 kinase assay using Akt1 immunoprecipitates from 2D and 3D lrECM cell cultures. Fold changes were calculated from densitometric analysis of protein bands of phosphorylated GSK fusion protein (mean ± SD; n = 3; **P < 0.01, t test). (J) siRNA-mediated PINCH1 (P1) knockdown in PINCH1^fl/fl and EGFP-PINCH1 MEFs and Western blot analysis of total and phosphorylated amounts of Akt1 (S473, T308). co, nonspecific siRNA control. (K) siRNAs targeting Akt1 in unirradiated or irradiated (0–6 Gy) PINCH1^fl/fl and EGFP-PINCH1 MEF for clonogenic survival (mean ± SD; n = 3; *P < 0.05, **P < 0.01, t test). siRNA-mediated Akt1 depletion was confirmed by Western blotting. β-Actin served as loading control.

(A) Analysis of PINCH1 mRNA expression in normal and tumor tissues from Oncomine data sets. List of tumor entities is displayed in Supplemental Table 3. Colorectal carcinomas have been analyzed selectively. (B) PINCH1 protein expression in biopsies of human colorectal carcinomas. Columns show mean PINCH1 staining intensities and statistical analysis including tumor grading 2 to 4 (mean ± SD; n = 24; c² test). Scale bar: 100 μm. (C and D) Clonogenic survival was measured in irradiated (0–6 Gy X-rays) (C) or 1 hour cisplatin- or 24 hours gemcitabine-treated (D) human colorectal (DLD1, HCT15), lung (A549, H1299), cervix (Hela), skin (A431), and pancreatic (PaTu, MiaPaCa2) carcinoma cell lines under PINCH1 knockdown. Data are mean ± SD (n = 3; **P < 0.01, t test). (E) PINCH1 knockdown cultures of human cancer cell lines were examined for total and phosphorylated amounts of the indicated proteins. P1, PINCH1.

(A) Western blot analysis of Akt1 and PINCH1 upon single or combined siRNA-mediated Akt1 and PINCH1 knockdown in DLD1 cells. Radiation survival of DLD1 PINCH1 knockdown cells was determined upon Akt1 siRNA knockdown (mean ± SD; n = 3; *P < 0.05, **P < 0.01, t test). (B) Total and phosphorylated amounts of exogenous RFP-Akt1 (WT, S473D/T308D, S473A, S473A/T308A or ΔPH) and associated signaling molecules were evaluated in DLD1 PINCH1 knockdown cultures by Western blotting. (C) Clonogenic radiation survival (0–6 Gy X-rays) was measured in DLD1 PINCH1 knockdown cultures transiently transfected with RFP-Akt1 plasmids expressing WT, S473D/T308D, S473A, S473A/T308A, or ΔPH (mean ± SD; n = 3; *P < 0.05, **P < 0.01, t test). Representative images demonstrate colony formation under tested experimental conditions.

(A) Protein phosphatase activity from MEFs left untreated or treated with okadaic acid (OA) or tautomycetin (Tau). Data are mean ± SD (n = 4; **P < 0.01, t test). (B) Protein phosphatase activity of PINCH1 knockdown DLD1 and control cultures. Data are mean ± SD (n = 4; **P < 0.01, t test). (C) Sequence homology search for the putative binding sequence KFVEF. (D) Schematic of mouse PINCH1 WT, PINCH1 V304A/F306A, PINCH1 K302A/V304A/F306A, and PINCH1ΔLIM5 inserted into pEGFP-C1 vector. (E and F) EGFP immunoprecipitates from PINCH1^–/– MEF transfected with EGFP, EGFP-PINCH1 WT, V304A/F306A or K302A/V304A/F306A mutated KFVEF motifs, or EGFP-PINCH1ΔLIM5 were examined for total amounts of indicated proteins and protein phosphatase activity (mean ± SD; n = 3; **P < 0.01, t test).

(A) Western blot of EGFP, Akt1 (S473, T308) and ILK from PINCH1^–/– MEF transfected with EGFP, EGFP-PINCH1 WT, V304A/F306A or K302A/V304A/F306A mutated KFVEF motifs, or EGFP-PINCH1ΔLIM5. β-Actin served as loading control. (B) Confocal fluorescence images on the subcellular localization of the different PINCH1 mutants described in A and of the putative Akt1 S473 phosphorylator ILK. Scale bar: 20 μm. (C) Clonogenic radiation survival of 2 Gy–irradiated PINCH1^–/– MEF transfected with EGFP, EGFP-PINCH1 WT, V304A/F306A or K302A/V304A/F306A mutated KFVEF motifs, or EGFP-PINCH1ΔLIM5. Data are mean ± SD (n = 3; **P < 0.01, t test). (D) Proposed mechanisms how PINCH1 interacts with PP1α via the LIM5 domain of PINCH1. This interaction inhibits Akt1 dephosphorylation and contributes to cellular radioresistance.