FIG 9.

The lymphangiogenic activity of a B-cell growth-promoting p17 variant (NHL-a105) is supported by autophagy. (A) LN-LECs were cultured for 16 h in complete medium (normal) or under serum-starved (stressed) conditions and then cultured for 12 h in the absence or presence of 10 ng/ml of GST, p24, p17, or NHL-a105 in complete medium. (B) LN-LECs were serum starved for 16 h in the presence or absence of 3-MA (5 mM) or after nucleofection with siScramble or siBeclin-1. After starvation, LN-LECs were stimulated or not with NHL-a105 (10 ng/ml). (C) Tube formation assay of LN-LECs serum starved for 16 h in the presence or absence of CA-074Me (50 μM) or serum-starved LN-LECs after nucleofection with siScramble, siGRASP55, or siRab8a. Cells were then treated or not for 12 h with NHL-a105 (10 ng/ml). Values reported for tube formation are the means ± SDs of three independent experiments with similar results. Statistical analysis was performed by one-way ANOVA; a Bonferroni posttest was used to compare data. (D) LN-LECs were serum starved for 16 h in the presence or absence of 3-MA (5 mM) or CA-074Me (50 μM). In some experiments, LN-LECs were nucleofected with siScramble, siGRASP55, or siRab8a for 24 h and then serum starved for 16 h. Then, cells were treated or not with 10 ng/ml of NHL-a105. After 6 h of stimulation, culture supernatants were collected and analyzed for the presence of ET-1 by quantitative ELISA. Bars represent the means ± SDs of triplicate samples. Statistical analysis was performed by one-way ANOVA. A Bonferroni posttest was used to compare data (**, P < 0.01; ***, P < 0.001). NT, not treated.