JCS02686 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Splicing of PINCH2 mRNA after the targeted deletion of exons 3 and 4. The bands amplified by RT-PCR using RNA from bladder of PINCH2 wild-type and knockout mice (shown in Fig. 1D) were cloned and sequenced. The figure shows the sequences of the two bands in the splicing site at the 3¢ of exon 2., The translation of the two mRNAs is reported below each sequence using the single letter amino acid code (black characters). Deletion of exons 3 and 4 in the PINCH2-null mice leads exon 2 to splice with exon 5 in place of exon 3, causing a frame shift that introduces a premature stop codon. The reading frame that is normally translated in the wild-type mRNA is given  (in blue characters) below the translation of exon 5 for the knockout mRNA.

• Supplemental Figure 2 -

  Fig. S2. The role of PINCH1 for migration and polarization of fibroblasts. (A) PINCH1fl/fl and PINCH1^–/– fibroblasts were grown to confluence overnight. A scratch was performed using the tip of a blunted glass capillary. Pictures from the same area of the scratch were taken at indicated time points. Bar, 50 mm. (B) Migration was calculated by measuring the remaining width of the scratch. Data shown are from a single representative experiment repeated five times. Error bars indicate mean ± s.d. from 10 areas measured per time point. (C) Number of polarized PINCH1fl/fl and PINCH1^–/– fibroblasts at the indicated time points in a scratch assay. Five areas of each scratch were randomly chosen and the percentage of cells polarized towards the scratch was calculated. The figure shows data from a single representative experiment repeated three times. Error bars indicate mean ± s.d. from the five chosen fields. (D) Migration of PINCH1fl/fl and PINCH1^–/– fibroblasts in a fibronectin-coated Transwell chamber. Cells on the bottom of the membrane were counted in images from five randomly chosen fields using 10´ magnification. Bars represent number of migrated cells in a single representative experiment repeated four times. Error bars indicate mean ± s.d.