Fig 1: SRGN/CD44 axis induces CLDN1 expression via NF-?B activation. (a) NF-?B reporter assay was performed in H1299/Mock and H1299/SRGN cells. (b) Cells were cultured in serum-free medium for 48 h, and subjected to western blot analysis using anti-I?Ba (#4812, Cell Signaling Technology) and anti-p65 (sc-372, Santa Cruz Biotech). (c) H1299/SRGN cells were transiently transfected with vectors encoding dominant-negative I?B kinase a (DN-IKKa) or DN-IKKß and cultured in serum-free medium for 48 h, followed by western blot analysis of I?B and CLDN1. HA-fused DN-IKKs were detected by anti-hemagglutinin (sc-805, Santa Cruz Biotech). (d) Unsorted H1299 and CD44(-) cells stably harboring the Mock-control or SRGN-expressing vectors were cultured in serum-free medium for 48 h. Nuclear and cytosolic fractions were prepared for western blot analysis using anti-p65, anti-PARP (sc-7150, Santa Cruz Biotech) and anti-tubulin (GTX112141, GeneTex). (e) Cells described in d were cultured in serum-free medium for 48 h, and subjected to western blotting of CD44 and CLDN1. (f) H1299 CD44(-) cells stably harboring Mock-control or CD44s-expressing vectors were incubated in medium supplemented with CM collected from H1299/Mock or H1299/SRGN cells for 24 h, and subjected to western blot analysis of CLDN1. Data are presented as the mean±s.d. of three independent experiments. **P<0.01 by Student's t-test.
Fig 2: CD44 and SRGN gene expression changes in SZ were confirmed in hippocampus samples from a microarray-independent cohort using qPCR. Box plot of normalized gene expression values. (ANCOVAs using age, sex, PMI and brain pH as covariates, p < 0.05)
Fig 3: SRGN protein is expressed on pyramidal (a) and non-pyramidal (b) neurons in the human amygdala. Scale bar = 100 µm
Fig 4: SRGN elicits NSCLC aggressiveness mediated through Claudin-1 expression. (a) Cells were cultured in serum-free medium for 48 h, and western blotting was performed using anti-Vimentin (IF01, Calbiochem, Billerica, MA, USA) and anti-CLDN1 (71-7800, Invitrogen, Carlsbad, CA, USA). (b) H1299 cells were transiently transfected without or with vectors encoding GFP or CLDN1. Western blot analysis of designated proteins is shown. (c) H1299/SRGN cells stably harboring scramble-shRNA or CLDN1-shRNAs were subjected to western blot analysis of designated proteins. (d) Migration assay of H1299/SRGN cells stably harboring scramble-shRNA or CLDN1-shRNA is shown. Data are presented as the mean±s.d. of three independent experiments. *P<0.05 and **P<0.01 by Student's t-test.
Fig 5: CS-GAG modification is critical for SRGN-mediated cell migration. a Western blot analysis of SRGN in the total cell lysate and CM of H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells cultured in serum-free medium for 48 h. b Wound healing assay was performed in H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells. After 72 h, wound area (lower panel) was assessed by ImageJ and normalized to 0 h. c Boyden chamber migration assay was performed in H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells. Migrated cells were counted in 3 h. d Boyden chamber migration assay was performed in HuTu80/CD44 cells that were suspended in CM derived from H1299/Mock, H1299/SRGN or H1299/SRGN(S/A) cells, respectively. Migrated cells were counted in 5 h. e CM harvested from H1299/Mock and H1299/SRGN cells were treated with or without ChaseABC for 24 h, and subjected to western blot analysis of SRGN. HuTu80/CD44 cells were suspended in non-treated or ChaseABC-treated CM and subjected to Boyden chamber migration assay. Migrated cells were counted in 5 h. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test
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