The tubulin deacetylases, HDAC6 and SIRT2, negatively regulate EC lumen and tube formation in 3D matrices. (A) ECs were treated with siRNAs directed to HDAC6 and SIRT2 and their combination, which leads to increased EC lumen formation and increased tubulin. Data are shown as mean EC lumenal area ± standard deviation (SD; n = 6). Statistical significance *P < .05, **P < .005. (B,C) ECs were induced to express HDAC6 vs SIRT2 (vs control GFP) using recombinant adenoviruses, leading to markedly reduced lumen formation after 24 hours of culture. This was assessed by photography (C) and marked loss of acetylated tubulin (D-E) in culture lysates, which correlates with a lack of lumen formation (B-C). Western blots were performed to assess protein knockdown and the levels of acetylated tubulin in the indicated siRNA-treated EC cultures undergoing lumen formation (F-H). Lysates were made after 24 hours of culture. Data are shown as mean EC lumenal area ± SD (n = 6). Statistical significance *P < .05, **P < .005.