Fig 1: Effect of DEPTOR expression on the antitumor and inhibitory effects of thalidomide on VEGF in MM cells. (A and C) Effect of thalidomide on the proliferation of RPMI 8226 and MM.1S. (B and D) Effects of thalidomide on DEPTOR and VEGF protein expression of RPMI8226 and MM.1S. (E) Effects of thalidomide on the proliferation of RPMI8226 transfected with lentivirus GFP or DEP3. (F) Effects of thalidomide on the apoptosis of RPMI8226 cells transfected with lentivirus GFP or DEP3. (G) Effects of thalidomide and 3-MA on autophagy of RPMI8226 detected by monodansylcadaverine (MDC) staining (scale bar, 10 µm). (H and I) RPMI8226 transfected with lentivirus GFP and DEP3 for 5 days were treated with thalidomide and 3-MA. (H) Cell lysates were examined by western blot analysis for the levels of the indicated proteins. (I) VEGF in culture media was collected and analyzed by ELISA. Error bars indicate the standard deviation from 3 independent experiments. *P<0.05, **P<0.01. VEGF, vascular endothelial growth factor; MM, multiple myeloma.
Fig 2: Autophagy inducer and mitochondrial-specific antioxidants reversed the effects of DEPTOR on the angiogenesis of MM cells. RPMI 8226 and MM.1S cells were transfected with lentivirus DEP3 for 5 days and exposed to SMER28 (10 µM) or Mito-TEMPO (10 µM) for 24 h. (A-D) The mitochondrial and cytoplasmic fractions of the cultured cells were separated using the Mitochondria/Cytosol Fractionation kit. Protein expression of Cyt c in the (A) mitochondria and (B) cytoplasm of the indicated groups for RPMI8226 cells was examined by western blot analysis, respectively. Primary antibodies with COXIV and ß-actin was used as the mitochondrial and cytosolic loading control, respectively. Protein expression of Cyt c in the (C) mitochondria and (D) cytoplasm of the indicated groups for MM.1S was examined by western blot analysis, respectively. Primary antibodies with COXIV and ß-actin was used as the mitochondrial and cytosolic loading control, respectively. Protein expression levels of DEPTOR and I?B-a in the cytoplasm of the indicated groups for (E) RPMI8226 and (F) MM.1S cells were examined by western blot analysis, respectively. (G-L) The nuclear and cytoplasmic protein fractions of the cultured cells were isolated using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Protein expressions of (G) p-p50 and p-p65 in the nuclear, (H) p50 and p65 in the whole cell lysates of the indicated groups for RPMI8226 cells was examined by western blot analysis, respectively. LaminB and ß-actin was used as the loading control for the nuclear extracts and the whole cell lysates, respectively. (I) Quantification of p-p50 subunit and p-p65 subunit was normalized for total p50 protein and total p65 protein (G and H), respectively. Protein expression levels of (J) p-p50 and p-p65 in the nuclear, (K) p50 and p65 in the whole cell lysates of the indicated groups for MM.1S was examined by western blot analysis, respectively. Lamin B and ß-actin was used as the loading controls for the nuclear extracts and the whole cell lysates, respectively. (L) Quantification of p-p50 subunit and p-p65 subunit was normalized for total p50 protein and total p65 protein (J and K), respectively. Error bars indicate the standard deviation from 3 independent experiments. *P<0.05 compared with GFP group, **P<0.01 compared with GFP group. MM, multiple myeloma; VEGF, vascular endothelial growth factor; Cyt C, cytochrome C; mtROS, mitochondrial reactive oxygen species; p-p50, phospho-p50; p-p65, phospho-p65.
Fig 3: Association between DEPTOR and VEGF expression levels in MM. (A-C) Expression of DEPTOR in 3 datasets (Barretina CellLine, Garnett CellLine and Wooster CellLine) from the Oncomine database. The asterisks (*) indicate statistical significance. (D) Meta-analysis of the 3 Oncomine datasets on the DEPTOR expression difference between MM and other cancers. (E) Potential pathways of DEPTOR involved in the regulation of MM cells identified by Gene Set Enrichment Analysis. (F) Western blot analysis of DEPTOR and VEGF with indicated antibodies from MM cells lines (MM.1S, RPMI8226 and U266). (G) VEGF expression in the conditioned media of myeloma cell lines. (H) Representative western blot analysis of DEPTOR and VEGF from primary MM cells from 9 of 22 patients with newly diagnosed MM. (I) Correlation analysis between DEPTOR and VEGF relative expression levels in 22 patients with newly diagnosed MM. (J) Representative image of MVD with anti-CD34 immunostaining in bone marrow tissues of from patients with newly diagnosed MM with a high or low DEPTOR expression (scale bar, 1 mm). Microvessels are stained brown. (K) Correlation analysis between the relative expression level of DEPTOR and bone marrow MVD in 22 patients with newly diagnosed MM. **P<0.01. VEGF, vascular endothelial growth factor; MM, multiple myeloma; MVD, microvessel density.
Fig 4: Autophagy inducer and mitochondrial-specific antioxidants reversed the effects of DEPTOR on the angiogenesis of MM cells. RPMI8226 and MM.1S cells were transfected with lentivirus DEP3 for 5 days and exposed to SMER28 (10 µM) or Mito-TEMPO (10 µM) for 24 h. (A) Cell lysates were examined by western blot analysis for the levels of the indicated proteins. (B) Levels of IL-6 and VEGF in medium supernatant in the indicated groups were examined by ELISA. (C) mtROS levels in the indicated groups stained with the MitoSOX probe were evaluated using flow cytometric analysis. *P<0.05, **P<0.01. VEGF, vascular endothelial growth factor; MM, multiple myeloma; mtROS, mitochondrial reactive oxygen species.
Fig 5: DEPTOR disruption increases autophagy-related angiogenesis in MM cells. The RPMI8226 and MM.1s cells were infected with lentivirus GFP or DEP3 for 5 days; the U226 cells were transfected with blank vector (GV143) or DEPTOR overexpressed vector (GV143-DEPTOR) for 2 days. (A) Their culture media were collected and analyzed for tube formation of HUVECs (scale bar, 10 µm). The numbers of tubes per view are shown as means ± SD (n=6). (B) RPMI8226 and MM.1S cells were treated with 3-MA (20 µM) for 24 h, while U226 cells were treated with rapamycin (100 nM) for 24 h. Cell lysates were examined by western blot analysis for the levels of the indicated proteins. Error bars indicate the standard deviation from 3 independent experiments. (C) RPMI8226 and MM.1S cells were transfected with lentivirus GFP and DEP3 for 5 days, while U266 cells were transfected with GV143 or GV143-DEPTOR for 2 days. Cell lysates were ere examined by western blot analysis for the levels of the indicated proteins. Error bars indicate the standard deviation from 3 independent experiments. (D) IL-6 and VEGF in culture media were collected from cells treated as in (B and C) and analyzed by ELISA. Error bars indicate the standard deviation from 3 independent experiments. *P<0.05, **P<0.01. VEGF, vascular endothelial growth factor; MM, multiple myeloma; HUVECs, human umbilical vein endothelial cells.
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