Effects of PINCH1 knockout on signal transduction under adhesion and suspension conditions.

<p>(A) Western blot on total cell lysates from <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEF grown on polystyrene plus FCS (first lane), in suspension plus FCS (second lane) or in suspension without FCS (third lane). (B) Densitometric analysis from protein bands shown in ‘A’ after normalization to total protein or β-Actin expression and subsequently to adhesion plus FCS conditions of <i>PINCH1</i>^fl/fl MEF ( = 1). FCS, fetal calf serum; Susp, suspension.</p

PINCH1 depletion sensitizes human tumor cells to irradiation independent from adhesion.

<p>siRNA-mediated PINCH1 depletion was confirmed by Western blotting in human HNSCC cell lines HTB43 (A) and HTB35 (B) cells. Measurement of clonogenic radiation survival (0–6 Gy X-ray) upon PINCH1 knockdown is shown in adherent and suspension (1 h) cultures (mean±SD; <i>n</i> = 3; * <i>P</i><0.05, ** <i>P</i><0.01; t-test). Poly-S, Polystyrene; Co, control.</p

Characterization of PINCH1, ILK and α-Parvin expression in different human tumor and immortalized mouse cell lines.

<p>(A) Western blot on total cell lysates of human and mouse cell lines from different origin (mouse = <i>PINCH1</i>^fl/fl MEF, <i>PINCH1</i>^−/− MEF, <i>ILK</i>^fl/fl renal fibroblasts, <i>ILK</i>^−/− renal fibroblasts; human primary fibroblasts = HSF1, HSF2, CCD32; human cancers = HTB35 (cervix), HTB43 (pharynx), CCL221 (colon), PATU8902 (pancreas), A549, SKMES1 (lung), U138MG, A172 (brain), MDAMB231 (breast), HL60, Jurkat (leukemia). (B) Correlation between the protein expressions of ILK versus PINCH1 versus Parvin in the different cell lines as analyzed in ‘A’.</p

PINCH1 determines radiation cell survival in a substratum-independent manner.

<p>(A) Western blot on <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEF lysates. (B) Measurement of clonogenic survival in MEFs irradiated (0–6 Gy X-ray) at 24h or 48h after plating (mean±SD; <i>n</i> = 3; * <i>P</i><0.05; t-test). (C and D) Clonogenic survival in non-irradiated and irradiated (4 Gy) <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− and <i>PINCH1</i>^−/− reconstituted with EGFP-PINCH1 MEFs (mean±SD; <i>n</i> = 3; t-test). (E) Total cell numbers from colony formation assays at the time point of assay termination ( = day 8). (F) Cell numbers of 15 poly-S colonies were counted. Results are mean±SD (<i>n</i> = 3; t-test). Polystyrene, Poly-S; poly-L-Lysine, Poly-L; Fibronectin, FN.</p

Colony cell numbers and cell morphology remain unaltered upon PINCH1 depletion.

<p>(A) and (B) Cell numbers of 15 colonies were counted in PINCH1 knockdown and control HTB43 and HTB35 cell cultures. Results show mean±SD (<i>n</i> = 3; t-test; n.s., not significant). (C) and (D) show cellular morphology in PINCH1 depleted and control cell colonies of HTB43 and HTB35 tumor cell lines. Photographs illustrate (<i>i</i>) representative colony growth in 35-mm wells, (<i>ii</i>) a single representative colony (magnification = 10×), and (<i>iii</i>) a representative zoom (magnification = 40×) from the edge of a colony. Co, control.</p

Plating efficiencies of HTB43 and HTB35 under siRNA-mediated PINCH1 knockdown.

<p>*Poly-S, polystyrene.</p

PINCH1 depletion does not modify tumor cell adhesion.

<p>Adhesion (cells per field) of (A) HTB43 and (B) HTB35 cells upon PINCH1 knockdown. At 48 h after siRNA transfection, cells were kept in suspension for 1 h on agarose prior to plating. Indicated time points were analyzed for the number of adherent cells as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013056#s2" target="_blank">Materials and Methods</a>”. Results show mean±SD (<i>n</i> = 3). (C) and (D) display representative microscopic images and (E) and (F) f-actin immunofluorescence stainings (Phalloidin) of HTB43 and HTB35 cells, respectively, at indicated time point after plating.</p

Adhesion and suspension preconditioning mediates similar radiation cell survival of <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEF.

<p>(A) Clonogenic survival of <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEFs irradiated under adhesion to Poly-L or after 1h in suspension with 0–6 Gy X-rays. After irradiation, suspension cell cultures were plated on Poly-L or FN for colony formation (mean±SD; <i>n</i> = 3; t-test). (B) Colony formation was determined in cells held in suspension for 1h prior to a 4-Gy X-ray irradiation. Then, cells were plated on FN at indicated time point (mean±SD; <i>n</i> = 3; t-test). (C) Cells were kept in suspension for 1h with (+FCS) or without FCS (-FCS) prior to 4-Gy irradiation. Subsequently, cell plating to FN was performed under two conditions: +FCS = plating with FCS; -FCS (for 6 h) = FCS was added 6 h after plating. Poly-L-Lysine, Poly-L; Fibronectin, FN; FCS, fetal calf serum. * <i>P</i><0.05. ns, not significant.</p

Plating efficiencies of <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEFs under indicated growth conditions.

<p>*Poly-S, polystyrene; Poly-L, poly-L-lysine; FN, Fibronectin.</p

Signal transduction modification in adherent and suspension tumor cell lines after PINCH1 knockdown.

<p>(A) Western blot on total cell lysates from PINCH1 depleted HTB43 and HTB35 cells grown under adhesion or in suspension. (B) Densitometric analysis from protein bands shown in ‘A’ after normalization to total protein or β-Actin expression and subsequently to adhesion conditions of siRNA control cells ( = 1). Co, control; P1, PINCH1.</p