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First published online 29 November 2005
doi: 10.1242/jcs.02686

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Consequences of loss of PINCH2 expression in mice

¹ Max Planck Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany
² Department of Pharmacy, Center for Drug Research, University of Munich, 81377 Munich, Germany

View larger version (39K): [in a new window]   Fig. 1. (A) Targeting strategy of PINCH2. Partial map of the PINCH2 wild-type and floxed alleles, and of the knockout allele after Cre recombination. Exons and loxP sites are indicated as rectangles and triangles, respectively. The DNA fragment sizes obtained after EcoRI restriction digest, as well as the probe used for Southern blotting are indicated. (B) Southern blot on DNA samples from offspring of a PINCH2+/– intercross. (C) Northern blot on poly(A)+ RNA extracts from bladder. (D) Nonquantitative RT-PCR performed with primers hybridizing to exons 2 and 5 of the PINCH2 gene, respectively, and template RNA from bladder of PINCH2+/+ and PINCH2–/– littermates (negative image of the agarose gel). To test the efficiency of reverse transcription, the GAPDH cDNA was amplified and is shown below. (E) Western blot on protein extracts from bladder, kidney and liver of PINCH2+/+ and PINCH2–/– littermates mice.

View larger version (78K): [in a new window]   Fig. 2. Histology of tissue section of PINCH2+/+ (left column) and PINCH2–/– (right column) (A-C) Haematoxylin and Eosin staining of bladder (A), liver (B) and kidney (C) sections; sm, smooth muscle; mu, mucosa; ep, epithelium; bd, bile duct; pa, portal area; cv, central vein; gl, glomerulus. Bars, 100 µm. (D,E) Immunofluorescent staining of bladder sections using PINCH1 (D) and PINCH2 (E) antibodies (in red), counterstained with DAPI (blue); sm, smooth muscle; mu, mucosa. Bar, 200 µm.

View larger version (134K): [in a new window]   Fig. 3. PINCH1fl/fl and PINCH1–/– mouse embryonic fibroblasts (MEFs). (A) Western blot showing the absence of PINCH1 protein in the PINCH1–/– MEFs and of the PINCH2 protein in both PINCH1fl/fl and PINCH1–/– MEFs. To control the PINCH2 assay, a lysate from PINCH2+/+ liver was also blotted. (B-E) Spreading of PINCH1fl/fl (B) and PINCH1–/– (C) cells and of PINCH1–/– cells transduced with EGFP-PINCH1 (D) and EGFP-PINCH2 (E), respectively. Cells were plated on tissue culture dishes for 16 hours in the presence of serum. Bar, 20 µm.

View larger version (68K): [in a new window]   Fig. 4. Immunofluorescent staining of PINCH1fl/fl (A) and PINCH1–/– (B) cells and of PINCH1–/– cells expressing EGFP-PINCH1 (C) or EGFP-PINCH2 (D). Cells were seeded on fibronectin-coated glass coverslips in DMEM without FCS for 4 hours. PINCH1 was either detected by PINCH1 antibody (A,B) or via the EGFP fusion protein (C). PINCH2 was visualized as EGFP-PINCH2 (D). Paxillin was stained with a commercial antibody and filamentous actin by TRITC-conjugated phalloidin. Bar, 20 µm.

View larger version (99K): [in a new window]   Fig. 5. Spreading of PINCH1fl/fl and PINCH1–/– MEFs. The numbers in each frame indicate the time in minutes from the moment of plating. (A,B) Phase contrast images. PINCH1fl/fl (A) and PINCH1–/– (B) MEFs were plated in DMEM on fibronectin-coated tissue culture dishes, incubated at 37°C and 5% CO2, filmed at intervals with a CCD camera. Bar, 40 µm. (C,D) Immunofluorescence staining of PINCH1f/fl (C) and PINCH1–/– cells (D). Cells were plated on fibronectin-coated glass, incubated for the time periods indicated and then fixed and stained. Bar, 20 µm.

View larger version (14K): [in a new window]   Fig. 6. Adhesion assay with PINCH1fl/fl and PINCH1–/– cells, and with PINCH1–/– cells transduced with EGFP-PINCH1 and EGFP-PINCH2, respectively. Adhesion was tested on fibronectin (FN), vitronectin (VN) and laminin (LN). Bars indicate percentage of total number of cells that adhered at saturating concentrations. Error bars represent ±s.d. (n=3).

View larger version (77K): [in a new window]   Fig. 7. Deletion of the PINCH1 gene in primary MEFs. (A,B) Phase contrast pictures of untreated (A) and of Cre transduced PINCH1fl/fl (B) primary fibroblasts. Bar, 40 µm. (C) Genotyping PCR of the untreated (fl/fl) and of Cre transduced PINCH1fl/fl primary fibroblasts (–/–). A water sample (c-) served as a negative PCR control. (D) Western blot on protein extracts of untreated and of Cre transduced PINCH1fl/fl primary fibroblasts cultured in medium containing 10% FCS. (E,F) Immunofluorescent staining of untreated (E) and Cre-treated (F) PINCH1fl/fl primary fibroblasts. Cells were seeded on fibronectin-coated glass coverslips in the absence of FCS for 4 hours and stained with an affinity purified anti-PINCH1 antibody. Focal adhesions and F-actin were visualized with anti-vinculin antibody and with TRITC-conjugated phalloidin, respectively. Bar, 20 µm. (G) FACS analysis of untreated (fl/fl), Cre-treated PINCH1fl/fl primary fibroblasts (–/–) stained with Alexa Fluor 488-conjugated annexin V and propidium iodide (PI); a control population of PINCH1fl/fl primary MEFs was cultured in suspension without serum to induce apoptosis (c +). In each diagram, the lower-right sector includes early apoptotic cells that are positives for annexin V but not for PI; the upper-right sector shows the percentage of dead cells that are positive for both annexin V and PI. (H) Phosphorylation of PKB/Akt in the absence of PINCH1. Cells were starved in 0.2% FCS for 4 hours, and then stimulated for 10 minutes with 10% FCS. Phosphorylation of PKB/Akt was determined in lysates from untreated (fl/fl) and Cre-treated (–/–) primary MEFs either before (0) or after (S) serum stimulation.

View larger version (46K): [in a new window]   Fig. 8. Expression of deletion mutants of PINCH1 and PINCH2 in PINCH1-null cells. (A) Western blots of cell protein extracts. Lane 1: PINCH1fl/fl cells; lane 2: PINCH1–/– cells; lanes 3-9: PINCH1–/– cells expressing, EGFP (3), EGFP-PINCH1 (4), EGFP-LIM1/P1 (5), EGFP-LIM1/P1 (6), EGFP-PINCH2 (7), EGFP-LIM1/P2 (8), EGFP-LIM1/P2 (9). (B) Northern blot of parental fl/fl cells (lane 1) and PINCH1–/– cells (lane 2). (C) Detection of ILK co-immunoprecipitated with the EGFP-tagged PINCH constructs using an anti-GFP antibody. Lane 1: lysate of PINCH1fl/fl cells; lane 2: negative control co-immunoprecipitation of PINCH1fl/fl cells expressing EGFP; lanes 3-8: PINCH1–/– cells expressing, EGFP-PINCH1 (3), EGFP-LIM1/P1 (4), EGFP-LIM1/P1 (5), EGFP-PINCH2 (6), EGFP-LIM1/P2 (7), EGFP-LIM1/P2 (8). (D-G) Localization of the EGFP-tagged constructs LIM1/P1 (D), LIM1/P1 (E), LIM1/P2 (F), LIM1/P2 (G); FAs were visualized by vinculin staining and arrowheads indicate points of partial co-localization of the constructs with vinculin. Bar, 20 µm.

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