Cystatin C deficiency affects cathepsin expression and activity.

<p>A/B: K14-HPV16 transgenic <i>Cstc^+/+</i> and <i>Cstc^−/−</i> mouse chest (A) and ear (B) skin tissue real-time PCR analysis for cathepsins K, L, S, and B. Number of mice per group is indicated in each bar. C/D: K14-HPV16 transgenic <i>Cstc^+/+</i> and <i>Cstc^−/−</i> mouse chest (A) and ear (B) skin tissue extract immunoblot analysis for cathepsins K, L, and S. Actin immunoblots were used for protein loading controls. E/F: Densitometric analysis for the cathepsin immunoblot signals from the chest (E) and ear (F) shown in C and D. Data are mean ± SEM. p<0.05 was considered statistically significant, non-parametric Mann-Whitney <i>U</i> test. NS: no significant difference.</p

Immunostaining for inflammatory cells in chest and ear skin tumor sections.

<p>CD4⁺ T cells (<b>A</b>) and Mac-3⁺ macrophages (<b>B</b>) in K14-HPV16 transgenic <i>Cstc^+/+</i> and <i>Cstc^−/−</i> mouse chest and ear skin tissue sections. Number of mice per group is indicated in each bar. Representative sections are shown to the right. Both dermis and epidermis (ED) are indicated. Data are mean ± SEM. Scale bar: 100 µm.</p

Mouse chest and ear skin tissue extract elastase and collagenase assays.

<p>K14-HPV16 transgenic <i>Cstc^−/−</i> mouse chest and ear skin tissue extracts showed significantly increased elastase (A) and collagenase (B) activities compared those of the <i>Cstc^+/+</i> mice. Number of mice per group is indicated in each bar. Data are mean ± SEM.</p

Immunoblot analysis for CatS and cystatin C in mouse chest skin tissue extracts.

<p>Chest skin tissue extracts from K14-HPV transgenic <i>Cstc^+/+</i> mice at different stages (1 month, 3 months, and 6 months) were analyzed for CatS (A) and cystatin C (B) expression. Cystatin C immunoblot analysis confirmed the absence of cystatin C in K14-HPV transgenic <i>Cstc^−/−</i> mice in chest skin tissue extracts from 3-month-old K14-HPV transgenic <i>Cstc^+/+</i> and <i>Cstc^−/−</i> mice (C). Equal protein loading (30 µg/lane) was confirmed with a β-actin immunoblot.</p

Immunostaining for CatS in chest and ear skin tumor sections.

<p>Number of mice per experimental group is indicated in each bar. Representative sections are shown to the right. Both dermis and epidermis (ED) are indicated. Data are mean ± SEM. Data are presented as % of CatS-positive area. Scale bar: 100 µm.</p

Immunostaining for apoptotic and proliferating cells in chest and ear skin tumor sections.

<p>K14-HPV16 transgenic <i>Cstc^+/+</i> and <i>Cstc^−/−</i> mouse chest and ear skin tissue section immunostaining for TUNEL (A) and Ki67 (B). Representative sections are shown in the right panels. Dermis and epidermis (ED) are indicated. Arrowheads indicate hair sebaceous units. Data are mean ± SEM. Scale bar: 100 µm.</p

Serum protease levels.

<p>Serum levels of CatS (A), CatL (B), and MMP-9 (C) in K14-HPV16 transgenic <i>Cstc^+/+</i> and <i>Cstc^−/−</i> mice. Number of mice per experimental group is indicated in each bar. Data are mean ± SEM. NS: no significant difference.</p

Immunostaining for microvessels in chest and ear skin tumor sections.

<p>K14-HPV16 transgenic <i>Cstc^+/+</i> and <i>Cstc^−/−</i> mouse chest and ear skin tissue section immunostaining for laminin-5 fragment γ2 (A) and CD31 (B). Number of mice per experimental group is indicated in each bar. Representative sections are shown in the right panels. Dermis and epidermis (ED) are indicated. Data are mean ± SEM. Scale bar: 100 µm.</p

CysC enhances total lysosomal-dependent protein degradation in serum-deprived neuronal cells.

<p><b>A.</b> Effect of increasing concentrations of CysC on total rates of protein degradation. N2a cells were labeled for 2 days with [³H]-leucine. After extensive washing, cells were incubated in serum-containing or serum-free media. Removal of serum maximally activates lysosomal degradation. The cells maintained in serum-free media were supplemented or not with increasing concentrations of CysC as labeled. The rate of total protein degradation at the indicated times was calculated as the percentage of total radiolabeled protein transformed into soluble amino acids. <b>B.</b> Effect of inhibition of lysosomal proteolysis on the CysC-induced increase in protein degradation. N2a cells were labeled as in A and then maintained in serum-free media and supplemented or not with CysC. Where indicated 20 mM NH₄Cl and 100 µM leupeptin were added to inhibit lysosomal proteolysis. Protein degradation was calculated as in A. <b>C</b>. Effect of CysC on macroautophagy-dependent proteolysis. N2a cells labeled as in A and maintained in serum-free media were supplemented or not with CysC. Half of the cells were treated with 10 mM 3MA to inhibit macroautophagy. The percentage of lysosomal degradation that results from autophagic degradation (3MA sensitive), in the presence or absence of CysC was calculated. Values are mean and SED of triplicate wells in 3–4 different experiments. One way ANOVA for differences between CysC treated and untreated samples were significant for *<i>p</i> = 0.05; **<i>p</i> = 0.001 and between control and ammonium chloride treated samples were significant for <b>⁺</b><i>p</i> = 0.01.</p

In vitro neuroprotection by either extracellular or endogenouse human CysC.

<p><b>A.</b> Light microscopy pictures of N2a cells incubated for 48 hours in medium containing serum or in serum-free medium in the absence or presence of different concentrations of CysC. Scale bar represents 100 µm. <b>B.</b> Neuronal survival was measured by counting live cells, and expressed as percentage of neuronal survival in cultures incubated in serum-containing medium. Data are the mean and SEM of 4 experiments. <b>C. D.</b> Primary rat cortical neurons were cultured in neurobasal medium in the presence (<b>C</b>) or absence (<b>D</b>) of B27-supplement and different concentrations of human CysC for 24 hours. Cell survival analyzed by the MTS assay is expressed as percentage of live cells in cultures incubated in B27-supplemented medium without CysC. Data are the mean and SEM of 3 experiments. F and P values determined by one way ANOVA for (C) were 85.09 and <0.0001 and for (D) 34.00 and <0.0001. <b>E</b> Primary cortical neurons isolated from brains of CysC knockout (CysCko), transgenic mice overexpressing human CysC (CysCtg), or wild type (WT) mice were incubated in B27-supplement containing or lacking media. Survival is expressed as percentage of live cells in wild type cultures incubated in B27-supplemented medium. For groups incubated with B27 the F and P values determined by one way ANOVA were 20.60 and 0.0007 and for groups incubated without B27 were 68.93 and <0.0001.</p