Fig 2: Validation of proteomics findings. Normalized expression levels of CFL1, PPIA, and KLK7 were determined in shNT and shFLG LSEs generated from independent biological samples. A and B, Representative immunoblots (n = 3; Fig 5, A) and associated densitometric quantification (n = 3; Fig 5, B). MW, Molecular weight. C, TaqMan mRNA expression levels (n = 8). Scatter plots of log10-transformed data (means ± 95% CIs).

Fig 3: KLK5 silencing inhibits invasion of PC9 lung adenocarcinoma cells. (a) KLK5 expression is effectively suppressed by two targeted lentiviral shRNA constructs, KLK5-KD1 and KLK5-KD2, relative to cells transduced with the non-target control virus (NT), as assessed by qRT/PCR normalized to GAPDH expression. (b) KLK7 expression is effectively suppressed by two targeted lentiviral shRNA constructs, KLK7-KD1 and KLK7-KD2, relative to cells transduced with the non-target control virus (NT), as assessed by qRT/PCR normalized to GAPDH expression. (c) Knockdown of KLK5 significantly inhibits PC9 cellular invasion in Matrigel transwell assays. (d) By contrast, knockdown of KLK7 has no significant impact on PC9 cellular invasion in Matrigel transwell assays. Representative images of the invasion assays are shown above each condition. Error bars show SEM. **P = 0.01; ***P = 0.001.

Fig 4: Klk7 gene deficiency increases the brain Aß levels and amyloid pathology in AppNL -G-F/ NL -G-F mice A–CBiochemical analyses of human Aß in Tris buffer-soluble (A), SDS-soluble (B), and formic acid-soluble (C) fractions of the brains of 3-month-old male App NL-G-F/NL-G-F ; Klk7 -/- mice. Arrowheads indicate total Aß. Quantification of relative levels of Aß is shown in Appendix Fig S6.DImmunohistochemical analysis of the brains of 3-month-old male App NL-G-F/NL-G-F ; Klk7 -/- mice using the 82E1 antibody (red). Magnified images were shown below. Scale bar, 100 µm.EQuantification results of cortical 82E1-positive total Aß plaques (left) and the cortical thioflavin S-positive Aß plaques (right) are shown (n = 3 or 4, mean ± s.e.m., ***P < 0.001 by Student's t-test). Source data are available online for this figure.

Fig 5: NFAT5 affects the expression of Mmp3, Klk5 and Asprv1 protease genes in KCs. (A) Left panels, results of qRT-PCR assays showing the effect of NFAT5 on the expression of Mmp3, Klk7 and Asprv1 genes upon transduction of KCs obtained from the tails of adult ice (T) or from the skin of newborn (NB) mice. Right panels, effect of NFAT5 ablation on Mmp3 and Klk7 expression. Results of 3 independent NGS assays are shown. (B) Expression of protease genes in KCs cultured for 1 or 3 weeks (ws). NGS data of three independent batches of KCs cultured for 1 or 3 ws are shown. (C–E) Detection of NFAT5 expression upon transduction of KCs. Cultured KCs were transduced with retroviruses expressing NFAT5, or with an ‘empty’ EGZ virus. (C) Detection of human NFAT5 RNA by RT-PCR assays in tail (T) and NB KCs upon transduction. (D) Western blots showing the expression of NFAT5 upon transduction of KCs from newborn and tails of adult mice. (E) Fluorescence microscopy of NFAT5-GFP expression in transduced adult KCs. (*p<0.05).