Skip to main content
. 2011 Dec 1;7(12):e1002396. doi: 10.1371/journal.pgen.1002396

Figure 11. Calpain inhibitors restore P1a expression and HD formation in Ogna keratinocytes and Ogna mice.

Figure 11

(A) Immunoblotting of cell lysates from primary PlecOgna/Ogna keratinocytes grown to ∼70% confluence in KGM/0.3, and exposed to either DMSO (solvent) alone, or calpain inhibitor ALLN (10 µM) for up to 24 hours. Note, samples analyzed contained equal amounts of calpain-1 and K5. Numbers below lanes represent protein ratios relative to an arbitrary level of 1.0 assigned to the control (solvent) sample. One of three experiments is shown. (B) Primary PlecOgna/Ogna keratinocytes grown as in (A) were exposed to DMSO (solvent) alone, or calpain inhibitor MDL-28170 (50 µM) for 48 hours. The bar diagram shows proportions (%) of solvent- and MDL-28170-treated cells having formed HPCs. Data shown represent mean values of cell counts (>500/genotype) in randomly chosen optical fields from three independent experiments ±95% CI. *** P<0.001, unpaired Student's t-test. (C) Visualization of P1a on frozen sections of mouse tail skin using P1a-specific antibodies. Note that in tail skin of PlecOgna/+ mice treated with MDL-28170 there was a clear increase in P1a levels compared to mice treated with DMSO (solvent) only; even though the restored P1a levels did not reach the levels of Plec+/+ controls. Arrowheads, basal cell membrane of basal keratinocytes. Bar, 20 µm. (D) Semiquantitative confocal microscopy analysis (n = 5 per group) of P1a levels in MDL-28170- relative to DMSO (solvent)-treated samples (100%, red broken line) using identical image settings. Note that the mean intensities of P1a fluorescence signals had increased ∼2-fold (compared with untreated skin) after treating PlecOgna/+ mice for 5 days with MDL-28170. Box and whisker plot as in Figure 2. P<0.01, unpaired Student's t test. (E) Immunoblotting of tissue extracts prepared from tail skin epidermis using antibodies to calpain-1. Note partial inhibition of autoproteolytic calpain-1 cleavage upon topical treatment of PlecOgna/+ mouse tails with MDL-28170.