rK5-induced autophagy in HUVECs is independent of nutrient deprivation-induced stress response. (A) LAMP1-GFP–transfected HUVECs were treated with different concentrations of rK5 cultured in M199 containing 5% FBS and supplemented with 40 ng/mL VEGF (□). After 24 hours of treatment, random fields of confocal microscopy images were recorded at 600× magnification and processed as described in “Materials and methods” to determine the number of autolysosomes per cell. Transfected HUVECs in serum-reduced medium (■) was used as a positive control. Values are shown as means ± SE. (B) LAMP1-GFP–transfected HUVECs cultured either in 5% FBS supplemented with VEGF or 10% FBS medium were treated with rK5 in the presence or absence of 3-MA. The number of LAMP1-GFP⁺ vesicles per cell was quantified. Values are presented as fold increase when compared with the control group (**P < .001; n = 3). The number of vesicles per cell in the control group was considered the basal level (1.0). Values are shown as means ± SE. (C) Representative confocal images of LAMP1-GFP–transfected HUVECs cultured in 5% FBS with (Cii-v) or without (Ci) VEGF. Transfected cells were treated with rK5 (1.5 μg/mL) in the presence (Ciii) or absence (Civ) of 5 mM 3-MA. LAMP1-GFP⁺ vesicles (left column) were colocalized with mitochondria (Mitotracker Red staining). Confocal images were recorded at 600× magnification. (D) Representative confocal images of HUVECs cultured in 10% FBS in the absence (Di) or presence (Diii) of rK5. As a control, heat-inactivated rK5 (Dii) was used. Control (Dv) and rK5-treated cultures (Div) were exposed to 5 mM 3-MA. LAMP1-GFP–labeled cells from different treatment groups (left column) were merged with the DAPI and Mitotracker Red images.