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. 2013 Nov 22;8(11):e79763. doi: 10.1371/journal.pone.0079763

Figure 4. Analysis of LPP levels and sub-cellular distribution in CD and controls fibroblasts before and after P31-43 treatment.

Figure 4

A. Increased focal adhesion localization of LPP in CD fibroblasts with respect to controls, as revealed through paxillin co-staining. a) Confocal immunofluorescence images of fibroblasts from CD patients and controls treated or not with P31-43 for 30 min and stained with antibodies against paxillin (red) and LPP (green); the merge of the red and green fields is shown in yellow. Representative fields. b) Statistical analysis of the number of LPP/paxillin-merged positive focal adhesions per cell. Focal adhesions of 30 fibroblasts from several fields from 3 independent experiments with 5 patients and 5 controls were counted. Columns represent the means, and the bars represent the standard deviations. Student's t-test. * = p<0.05;*** = p<0.001. B. LPP protein levels were not increased in the fibroblasts from CD patients with respect to the controls and did not vary after 24 h treatment with P31-43. a) Western blot analysis of fibroblasts protein lysates from controls and CD patients treated or not with P31-43. The upper line was stained for LPP, and the lower line was stained for tubulin. Representative experiment. b) Densitometric analysis of western blots stained for LPP in fibroblasts from 5 CD patients and 5 controls treated or not with P31-43. LPP levels were normalized to tubulin levels. Columns represent the means, and the bars represent the standard deviations. Student's t-test. C. Decreased localization of LPP in the nuclear fraction of CD fibroblasts. a) Western blot analysis of LPP after separating the nuclear and cytosolic protein fractions. The upper lines were stained for LPP, and the lower lines were stained for tubulin and lamin A/C, which were used as loading controls for the cytosol and nuclear fractions, respectively. Representative experiment. b) Densitometric analysis of western blots stained for LPP in fibroblasts from 5 CD patients and 5 controls. The LPP levels were normalized in each protein fractions to the loading controls. Columns represent the means, and the bars represent the standard deviation. Student's t-test. * = p<0.05; ** = p<0.01.