Transient Exposure of Endothelial Cells to Doxorubicin Leads to Long-Lasting Vascular Endothelial Growth Factor Receptor 2 Downregulation

Figure 1. Effects of a transient exposure of endothelial cells to Doxorubicin. HUVECs were incubated with 250 nM Doxorubicin for 24 h. After extensive washing, the medium was replaced with free-drug complete medium and cells were maintained in culture for additional days. Samples were collected and analyzed at 24 h of treatment (24 h), red histogram, and after 8 days (8 d), blue histogram. NT = not treated cells, white histogram. (A) Percentage of live, apoptotic, and necrotic cells determined by cytofluorimetry. Data are shown as mean ± SEM of n = 6–9 independent experiments per condition. ns p >0.05, ** p < 0.01 *** p < 0.001, **** p < 0.0001 by unpaired Student’s t test. (B) Percentage of positive cells for Senescence-Associated β-galactosidase activity. Data are shown as mean ± SEM of n = 3 experiments per condition; *** p < 0.001, **** p < 0.0001 by unpaired Student’s t test. (C) Cell lysates analyzed by immunoblotting with the indicated antibodies. (D) Quantification of VEGFR2 protein level by densitometric analysis. Data are shown as mean ± SEM of protein level normalized to GAPDH. n = 3 independent experiments; ** p < 0.01, *** p < 0.001 by unpaired Student’s t test. (E) mRNA level…

Figure 2. VEGFR2 protein levels decrease early following Doxorubicin treatment. HUVECs were incubated with 250 nM Doxorubicin and samples were collected at the indicated time during the treatment, red histogram. White histogram represents the not treated cells. (A) Representative immunoblot of HUVEC lysates analyzed using an antibody against VEGFR2. (B) Quantification of VEGFR2 protein level by densitometric analysis. Data are shown as mean ± SEM of protein level normalized to β-actin. n = 3 independent experiments per condition. ns p > 0.05, ** p < 0.01, by unpaired Student’s t test. (C) mRNA level of VEGFR2 determined by qRT-PCR. Each sample was normalized to β-tubulin and data are expressed as fold increase compared to the untreated sample. Data are shown as mean ± SEM of n = 3–4 independent experiments per condition. ns p > 0.05, ** p < 0.01 by unpaired Student’s t test.

Figure 3. Doxorubicin-induced VEGFR2 reduction is not due to an increased degradation. (A–E) HUVECs were incubated with 250 nM Doxorubicin in absence or presence of 50 µM Chloroquine (CQ). After 16 h, cells were lysed and analyzed by immunoblotting with indicated antibodies. (A) Representative immunoblot. Arrows indicate the LC3-II and LC3-I forms. (B) Quantification of LC3 protein level by densitometric analysis. Data are shown as mean ± SEM of LC3-II level normalized to LC3-I. n = 4 independent experiments per condition. ** p < 0.01, *** p < 0.001 by unpaired Student’s t test. (C) Representative immunoblot. Arrow indicates the cleaved form of VEGFR2 in the same long-exposed blot. (D,E) Quantification of the mature (VEGFR2 230–200 kDa) and cleaved VEGFR2 (VEGFR2 160 kDa) protein levels by densitometric analysis. Data are shown as mean ± SEM of protein level normalized to vinculin. n = 4 independent experiments per condition. ns p > 0,05, * p < 0.05, *** p < 0.001 by unpaired Student’s t test. (F–H) HUVECs were incubated with 250 nM Doxorubicin in absence or presence of 5 µM Lactacystin (LC). After 16 h, cells were lysed and analyzed by immunoblotting with indicated antibodies. (F)…

Figure 4. Global protein synthesis and mTOR activity during Doxorubicin treatment. (A,B) HUVECs were incubated (+), red histogram, or not (−), white histogram, with 250 nM Doxorubicin (Dox) for 16 h and then labelled for 10 min with of 90 µM Puromycin. Cells were lysed and analyzed by immunoblotting using an antibody against Puromycin. (A) Representatives immunoblot. (B) Quantification of puromycin levels by densitometric analysis. Data are shown as mean ± SEM of puromycin levels normalized to vinculin. n = 4 independent experiments per condition. *** p < 0.001 by unpaired Student’s t test. (C–E) HUVECs were incubated with 250 nM Doxorubicin and samples were collected at the indicated time during the treatment, red histogram. White histogram represents not-treated cells. (C) Representative immunoblot of HUVEC lysates analyzed using indicates antibodies. (D,E) Quantification of p53 and p21 protein levels, respectively, by densitometric analysis. Data are shown as mean ± SEM of protein levels normalized to β-actin, n = 3 independent experiments per condition. ns p > 0.05, * p < 0.05, ** p < 0.01 by unpaired Student’s t test. (F–H) HUVECs were treated (Dox, red histogram) or not (NT, white…