Extended Data Fig. 4: Effects of GLUL silencing on cytoskeleton and barrier function.
From: Role of glutamine synthetase in angiogenesis beyond glutamine synthesis
a–h, Images of control (a, c, e, g) and GLULKD (b, d, f, h) ECs after staining for α-tubulin (a, b), F-actin (c, d) and nuclear staining (e, f); g and h show merged images. The images shown are representative of 3 independent experiments. i–k, Representative images of phalloidin (F-actin) + Hoechst-stained liver ECs 6 h after isolation from control (i) and MSO-treated (j) mice, and corresponding quantification of F-actin levels (k) (mean ± s.e.m.; n = 5 mice per group; *P < 0.05 versus control, Student’s t-test). l–n, Representative images of phalloidin-stained confluent monolayer control (l) and GLULKD (m) ECs aligning a scratch wound, and quantification of F-actin levels (n) (mean ± s.e.m.; n = 5 independent experiments; *P < 0.05 versus control, Student’s t-test). o, Quantification of the length of discontinuous and continuous VE-cadherin-stained junctions in control and GLULKD ECs (mean ± s.e.m.; n = 4 independent experiments; *P < 0.05 versus control, Student’s t-test). p, Quantification of VE-cadherin gap size index in control and GLULKD EC monolayers (mean ± s.e.m.; n = 4 independent experiments; *P < 0.05 versus control, Student’s t-test). q–v, Corresponding representative images of monolayer control (q, s, u) and GLULKD (r, t, v) ECs stained for VE-cadherin (q, r, u, v) and F-actin (s, t, u, v). Yellow arrows in r point to discontinuous VE-cadherin junctions and yellow asterisks indicate intracellular gaps. w, Quantification of transendothelial electrical resistance in control and GLULKD EC monolayers (mean ± s.e.m.; n = 4 independent experiments; *P < 0.05 versus control, Student’s t-test at each time point). x–z, Quantification (x) of Evans blue dye extracted from the ears of control and MSO-treated mice, induced by topical application of mustard oil (n = 4 mice for each condition, *P < 0.05; Student’s t-test), and representative pictures of the leakage of Evans blue dye into the ear tissue in control (y) and MSO-treated (z) mice. Exact P values: 0.0030 (k); 0.0036 (n); continuous control versus GLULKD: 0.0005; discontinuous control versus GLULKD: 0.0005 (o); 0.0356 (p); 0.0181 (w); 0.0002 (x). Scale bars: 20 μm (a–h, l, m), 10 μm (i, j, q–v). AU, arbitrary units.
