Fig 2: GzmB cleaves TNC in vitro.A total of 500 ng of commercial (A) or lab-generated (B) rhTNC was incubated with (or without) 50 nM rhGzmB for several time points at 37°C and analyzed by Western blot probing for TNC (upper panel) and GzmB (lower panel). GzmB generates 3 TNC peptides of distinct molecular weights, identified as *1, *2, and *3. (C) Expected repartition of the effective cleavage sites for GzmB in TNC based on the in silico prediction and the in vitro digestion. (D) A total of 50 nM of rhGzmB was preincubated at 37°C for 1 hour with 300 nM of SA3N before the addition of rhTNC (500 ng). Digestions were performed for 24, 48, and 72 hours followed by Western blot analysis probing for TNC, SA3N, and GzmB. Images shown are representative of at least 3 independently repeated experiments.

Fig 3: In silico prediction identifies TNC as a potentially novel GzmB substrate.Schematic diagram of predicted granzyme B (GzmB) cleavage sites on human TNC (hTNC). Letters represent the single–amino acid code of the tetrapeptide preceding as well as the dipeptide following the expected cleavage site. Arrows represent the location of the expected cleavage site (after aspartic acid, D) and correspond to the amino acid location referenced between brackets.

Fig 4: TNC fragments detected in patients with RA align with in silico prediction and in vitro digestion by GzmB.GzmB (A) and TNC (B) quantification in healthy controls (HC, n = 5), rheumatoid arthritis (RA, n = 12), osteoarthritis (OA, n = 7), and inflammatory arthritis (IA, n = 6) patient SF represented as means ± SD. Statistical analyses were performed using nonparametric ANOVA (Kruskal-Wallis rank-sum test) followed by Dunn’s multiple comparisons test with Bonferroni’s P value adjustment. (C) Correlation (Spearman rank-order) between TNC and GzmB levels in HC, RA, OA, and IA patients’ SF (n = 30). (D) Western blot analysis demonstrating the presence of 2 TNC fragments (70 kDa and 30 kDa) in the SF of 12 patients with RA. *=P < 0.05, **=P < 0.01.

Fig 5: Atrophic patients with COPD upregulate tenascin C and downregulate fibronectin post-pulmonary rehabilitation (PR). Comparisons between nonatrophic and atrophic patients with COPD pre- and post-PR. Boxplot graphs depict the median (black line) and lower and upper quartiles for: a) tenascin C protein levels and b) mRNA expression (TNC); c) fibronectin protein levels and d) mRNA expression (FN). The values for each individual participant are represented as black data points. Protein levels were measured with ELISA. mRNA expression was measured with quantitative real-time PCR; data are presented as fold changes relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).