Fig 2: Contribution of endogenous Ang II to muscle VEGF production and endothelial cell-muscle cell crosstalk.C2C12 myotubes and confluent cultures of microvascular endothelial cells were lysed, and endogenous basal levels of angiotensinogen, AT1R and AT2R were assessed by Western blot analysis (A). In all blots, two independent cultures are shown for each cell type. C2C12 myotubes (n = 3 per condition) were treated overnight with the AT1R inhibitor Losartan (0.1 µM or 1 µM) and VEGF transcript levels were assessed by qPCR (B). Representative image and the quantification of RT-PCR analysis of VEGFA isoform expression (VEGF120 ~170 bp; VEGF164 ~300 bp and VEGF180 = not detected) in C2C12 myotubes and skeletal muscle endothelial cells (C). Two independent samples are shown for both C2C12 and endothelial cells (n = 6 for C2C12 and n = 13 for endothelial cells). Lysates of matrix bound proteins were assessed for VEGF and tubulin protein expression by Western blotting (D). Cell extract (Ex) was used as a comparator with extracts of matrix (M) alone. Three independent samples of matrix-associated protein extracts are shown. Tubulin was detectable within cell extract, but not in matrix-derived extracts. In (E), endothelial cells alone or previously treated with an inhibitor of the VEGFR2, were incubated on matrix which previously contained C2C12 myoblasts with or without losartan treatment. Representative image and quantification of phosphorylated p38 levels (n = 6 for E and E+M, n = 5 for E+ML and n = 4 for EV+M). Values are presented as mean ± SEM. One way ANOVA followed by Tukey’s multiple comparison test and student’s t-test were used to assess statistical significance which was set as p<0.05. In panel B, *denotes p<0.05 vs. untreated cells. In panel C, *denotes p<0.05 vs. C2C12 cells and in panel E, *denotes p<0.05 vs. endothelial cells. E – endothelial cell, Ex - cell extract, Coll- type I collagen matrix, M - matrix lysate, E+M – endothelial cells + matrix from untreated C2C12, E+ML – endothelial cells + matrix from losartan-treated C2C12 cells and EV+M – endothelial cells pretreated with a VEGFR2 inhibitor + matrix from untreated C2C12.

Fig 3: OTUD6B-AS1 was associated with aggressive pathological parameters and promoted breast cancer progression. Expression of OTUD6B-AS1 was positively correlated with axillary lymph node metastasis (A), larger tumor size (B), advanced tumor stage (C, one way ANOVA, P = 0.0096; stage I vs. II, P = 0.0050; stage I vs. III, P = 0.0162; stage II vs. III, not significant), and high Ki-67 expression (D). The OTUD6B-AS1 overexpression plasmid significantly upregulated OTUD6B-AS1 expression in breast cancer BT474 cells (E). OTUD6B-AS1 promoted breast cancer cell proliferation (F), wound healing (G, 40× magnification), migration, and invasion (H, I, 200× magnification). OTUD6B-AS1 increased the tube formation ability of HUVEC (J, 40× magnification). OTUD6B-AS1 decreased E-cadherin expression and increased the expression of HIF-1a, MMP1, SMAD5, Snail, Twist1, and VEGFA (K, cropped gels blots are used).