![Figure 2. SOX11 promotes endothelial tube formation, cell proliferation and migration, and activation of angiogenic signaling cascades in vitro. (A) Top panel, Representative images showing the capillary tube formation by HUVECs growing on Matrigel matrix for 6 hours in the presence of control RPMI + 10% FBS, SOX11-positive or SOX11-negative CM on Matrigel matrix. Bottom panel, Quantification of the number of branching points representing tube formation by HUVECs cultured for 6 hours in RPMI + 10% FBS or SOX11-positive and SOX11-negative CM from Z138, JEKO1, and GRANTA519 MCL cell lines. SOX11-positive CM significantly promoted a prominent increase in tube formation compared with CM from SOX11-negative cells (126 vs 89 branching points, P = .0002, 121 vs 100 branching points, P = .009, and 102 vs 70 branching points, P < 1 × 10−4 in Z138, JEKO1, and GRANTA519) and RPMI + 10% FBS (126 vs 76 branching points, P < 1 × 10−4, 121 vs 76 branching points, P < 1 × 10−4, and 102 vs 76 branching points, P < 1 × 10−4 in Z138, JEKO1, and GRANTA519). Conversely, SOX11-negative CM did not have an effect on tube formation compared with RPMI + 10% FBS (89 vs 76 branching points, P = .2, 70 vs 76 branching points, P = .3 in Z138 and GRANTA519). (B) Kinetics of HUVEC growth in SOX11-positive CM (blue) or SOX11-negative CM (yellow) derived from SOX11-positive and SOX11-silenced Z138 (solid line) and JEKO1 (dashed line) MCL cell lines. Graph showing the optical density (620 nm) after staining the HUVECs with 0.2% crystal violet and measured at 620-nm wavelengths at the indicated time points (1, 3, and 5 days). (C) Graph displaying the percentage of migratory HUVEC cells toward SOX11-positive and SOX11-negative CM from Z138, JEKO1, and GRANTA519 MCL cell lines, relative to the percentage of migratory HUVEC cells toward RPMI + 10% FBS. HUVECs displayed significantly increased migration toward SOX11-positive CM cell lines (±90% migration) compared with SOX11-negative CM (±30%-70% migration, in Z138 and GRANTA519 [P < 1 × 10−4] and JEKO1 [P = .009]). (D) Left panel, WB experiments showing expression levels of basal and phosphorylated forms of FAK, Akt, and Erk1/2 proteins in HUVECs cultured in RPMI + 10% FBS or SOX11-positive and SOX11-negative CM from Z138 and GRANTA519 MCL cell lines for 3 hours. GAPDH was used as a loading control. Right panel, Fold change differences of phosphorylated forms of FAK, Akt, and Erk1/2 proteins in HUVECs cultured in RPMI + 10% FBS or SOX11-positive and SOX11-negative CM from Z138 and GRANTA519 MCL cell lines for 3 hours, correlated by quantification of GAPDH expression levels. Bar plot represents the mean percentage ± SD, n = 3. P-val is shown. The significance of difference was determined by independent samples Student t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/14/10.1182_blood-2014-04-569566/4/2235f2.jpeg?Expires=1735809564&Signature=AS-NZOZOJd0WHNPOEWga3MHTJuzL7X9D~2SAtKMzOlXfqJi3eApc7bCucmlZGHk~t60a89Tm1s1jH5f2gC5gXP-hNNsdILka58Yh3gPgsJ2cPXfx1Umjf-TVp3dfY18K4BPa-ugro~7A4gvFkmkTGu--7WfXBeQrFGg6Z0n03BFKgkQMm0mA7zrKDjPajLVrxXeYksKdFvPIK5rZtMmEoAetLXN7iegmMvGJvQPb2S3sfWwaIDCtU~4hyY3AKzzB1VMR1twxvh2C-eh3bONcEUIAeJzmDxOzOXZtRK-LrExZJpdrFjAby7S4Rw57si-YhVbqOMZ0S3y~09uMNL349Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
SOX11 promotes endothelial tube formation, cell proliferation and migration, and activation of angiogenic signaling cascades in vitro. (A) Top panel, Representative images showing the capillary tube formation by HUVECs growing on Matrigel matrix for 6 hours in the presence of control RPMI + 10% FBS, SOX11-positive or SOX11-negative CM on Matrigel matrix. Bottom panel, Quantification of the number of branching points representing tube formation by HUVECs cultured for 6 hours in RPMI + 10% FBS or SOX11-positive and SOX11-negative CM from Z138, JEKO1, and GRANTA519 MCL cell lines. SOX11-positive CM significantly promoted a prominent increase in tube formation compared with CM from SOX11-negative cells (126 vs 89 branching points, P = .0002, 121 vs 100 branching points, P = .009, and 102 vs 70 branching points, P < 1 × 10−4 in Z138, JEKO1, and GRANTA519) and RPMI + 10% FBS (126 vs 76 branching points, P < 1 × 10−4, 121 vs 76 branching points, P < 1 × 10−4, and 102 vs 76 branching points, P < 1 × 10−4 in Z138, JEKO1, and GRANTA519). Conversely, SOX11-negative CM did not have an effect on tube formation compared with RPMI + 10% FBS (89 vs 76 branching points, P = .2, 70 vs 76 branching points, P = .3 in Z138 and GRANTA519). (B) Kinetics of HUVEC growth in SOX11-positive CM (blue) or SOX11-negative CM (yellow) derived from SOX11-positive and SOX11-silenced Z138 (solid line) and JEKO1 (dashed line) MCL cell lines. Graph showing the optical density (620 nm) after staining the HUVECs with 0.2% crystal violet and measured at 620-nm wavelengths at the indicated time points (1, 3, and 5 days). (C) Graph displaying the percentage of migratory HUVEC cells toward SOX11-positive and SOX11-negative CM from Z138, JEKO1, and GRANTA519 MCL cell lines, relative to the percentage of migratory HUVEC cells toward RPMI + 10% FBS. HUVECs displayed significantly increased migration toward SOX11-positive CM cell lines (±90% migration) compared with SOX11-negative CM (±30%-70% migration, in Z138 and GRANTA519 [P < 1 × 10−4] and JEKO1 [P = .009]). (D) Left panel, WB experiments showing expression levels of basal and phosphorylated forms of FAK, Akt, and Erk1/2 proteins in HUVECs cultured in RPMI + 10% FBS or SOX11-positive and SOX11-negative CM from Z138 and GRANTA519 MCL cell lines for 3 hours. GAPDH was used as a loading control. Right panel, Fold change differences of phosphorylated forms of FAK, Akt, and Erk1/2 proteins in HUVECs cultured in RPMI + 10% FBS or SOX11-positive and SOX11-negative CM from Z138 and GRANTA519 MCL cell lines for 3 hours, correlated by quantification of GAPDH expression levels. Bar plot represents the mean percentage ± SD, n = 3. P-val is shown. The significance of difference was determined by independent samples Student t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
