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: (A) NFKB1 gene expression; (B) NFkBp50 Western blot analysis; (C) phosphorylated (p)-NF?Bp50 Western blot analysis; (D) NFKB2 gene expression; (E) p-NF?Bp52 Western blot analysis; (F) NFKB3 gene expression; (G) NF?Bp65 Western blot analysis; (H) p-NFkBp65 Western blot analysis; (I) NFKBIA gene expression; (J) I?Ba Western blot analysis; (K) p- I?Bß Western blot analysis; (L) NFKBIB gene expression; (M) NFKBIE gene expression; (N) IKBKG gene expression. The position (arrow) and molecular size (in kD) of the investigated proteins are indicated on each of the Western blot membrane images. Cofilin 1 was used as loading control. The vertical lines indicate group separation giving n = 5 per group.