TE11 cells were grown as tumor xenografts in NOD/SCID mice. After tumor establishment, mice were dosed twice weekly with 0 (normal saline) or 1 mg/kg bortezomib i.p. (8 mice per group) for 16 days. A: photographs of representative tumors for each group at day 16. B: growth curves normalized to the start volumes. bortezomib treatment led to significant (* P<0.05) levels of tumor regression. C: Immunofluorescent staining of tumors from each group. TUNEL and Ki67 staining was done in paraffin-embedded sections (TUNEL-red, Ki67-green, DAPI-blue). Magnification 10X, scale bar: 50 μM. D: Quantification of immunofluorescence for treated and untreated tumors. Graph shows the percentage of positive cells in each group. * P<0.04.

A: Immunofluorescence showing γ-H2AX foci formation after bortezomib treatment. TE12 cells were grown on glass slides and treated with normal saline or 10nM Bortezomib for 12hrs. Slides were harvested, fixed, and permeabilized, and before being stained for phospho-H2AX (γ-H2AX-red, DAPI-blue), magnification 20 X, scale bar: 50 μM. Inset: Magnification 60 X, scale bar: 50 μM.
B: Western blot showing the downregulation of H2AX after day 4 of siRNA transfection. Proteins were extracted and resolved followed by probing for H2AX, ATM and β-actin.
C: H2AX knock-down TE12 cells and control cells were treated with 10 nM bortezomib for 24 and 48hrs. Cells were harvested, lysed and probed for caspase-3 cleavage. No difference in caspase-3 cleavage was observed in H2AX knockdown cells compared to control cells.
D: TE12 cells were treated with z-VAD-FMK (1 μM), bortezomib or a combination of both for 48hrs. Cells lysates were then harvested, resolved by Western blotting and probed for cleaved-PARP (c-PARP), cleaved caspase-3 (c-Caspase-3), total caspase-3 (Caspase-3), total PARP (PARP) and phospho-H2AX (p-H2AX). Equal protein loading was confirmed by stripping of the blot and reprobing for actin expression.

A: 3D organotypic cultures were formed in a transwell system, using a collagen and fibroblast matrix on the bottom and ESCC (TE12 cells) seeded on top. After 14 days, the cultures were treated with bortezomib (0, 0.5 μM and 1 μM). After 72hrs the reconstructs were harvested and paraffin-embedded. TUNEL staining was carried out to quantify apoptotic cells after treatment. TUNEL-green, DAPI-blue. Magnification 10X, scale bar: 100 μM.
B: Graph showing quantification of apoptotic TE12 cells in treated reconstructs (as shown in A). Total area of positive TUNEL staining (μM × 10³) was assessed using ProImage 6.0 software. Data shown is representative of three independent experiments. *P<0.05
C: Vascular networks were created using human endothelial cells (HMVEC) that were cultured and overlaid with collagen I, followed by a second overlay of collagen containing TE12 cells and esophageal fibroblasts (FEF3). After solidification of the collagen matrix, cells were treated with 0 (normal saline) or 0.5 μM of bortezomib for 72hrs. 3D-angiogenesis models were harvested and immunofluorescence staining was done, showing GFP-tagged FEF3 esophageal fibroblasts-green, HMVECs (CD31-red) and total nuclei (DAPI-blue). All representative images are shown as a 3-color merge, with the monochrome images of CD31 staining. Bortezomib treated 3D-angiogenesis model showed decreased vascular network formation in comparison to the control. Magnification 20X, scale bar: 100 μM.

A: Bortezomib treatment led to the rapid upregulation of phospho-p38 MAP kinase (p-p38) activity in TE12 cells. Irradiation of the human osteosarcoma line U2OS also led to the rapid upregulation of p38 activity. Equal protein expression was confirmed by stripping and probing the blot for actin expression.
B: Inhibition of p38 MAP kinase reverses bortezomib-induced apoptosis. TE12 cells were pre-treated with 10 μM SB 230580 for 24hrs before being treated with bortezomib (10 nM) for 48hrs. Cells were harvested, lysed and probed for caspase-3 cleavage, total caspase-3, phospho-H2AX, total H2AX and Noxa expression. Equal protein loading was confirmed by stripping the blot and probing for actin expression.
C: Knockdown of p38 MAP kinase inhibits Bortezomib induced caspase-3 cleavage. TE12 cells were transiently transfected using SMARTpool siRNA p38 MAP kinase (siRNAp38). Control cells were transfected with a scrambled siRNA sequence (siRNAcontrol) After 4 days, cells were treated with bortezomib (10 nM, 48 hrs). Protein was then extracted from the cells, resolved and probed for expression of caspase-3, cleaved caspase-3 (c-caspase-3) and total p38 MAP kinase (p38).
D: Western blot showing the positive interaction of bortezomib and radiation in the activation of p38 MAP kinase signaling. TE 12 cells plated in 10cm dishes were treated with control, 2 Gy of radiation alone, bortezomib alone, and bortezomib + radiation (2Gy). Plates were pre-treated for 4 hours with saline or bortezomib (10 nM) before irradiation. Cells were left to grow overnight before the extraction of protein, extracts were then resolved and probed for expression of phospho-p38 MAP kinase, total p38 MAP kinase, and β-actin. Radiation survival. Cells were plated in 96 well plates at the indicated number per well. Cells were then treated with either saline (control) or bortezomib (10 nM) for 4 hrs prior to radiation. Cells were left to grow for two weeks, after this time the total number of colonies were counted. The bars shown are the means obtained from six experiments (*P<0.0001).

A: In two-dimensional adherent cell culture, bortezomib reduces growth of the ESCC cell lines in a concentration-dependent fashion. Adherent TE1, TE3, TE8, TE10, TE11 and TE12 cells were treated with increasing concentrations of Bortezomib (1 nM – 10 μM) for 72 hrs before being treated with MTT. The resulting changes in absorbance were read in a plate reader at 490nm and expressed as a percentage of control absorbance. Data show the mean of three independent experiments ± SE mean.
B: Inhibition of cell growth by bortezomib is associated with G2/M cell cycle arrest. Adherent TE12 cells were treated with saline or 10 nM bortezomib for 8, 24 or 48 hrs. Cells treated with bortezomib were found to enter G2/M phase cell cycle arrest with increasing periods of time. The cell cycle profile was obtained using 15,000 cells. Data show represents mean of three independent experiments ± SE mean.
C: Bortezomib blocks growth of TE cells in three-dimensional culture. TE12 cells were grown under non-adherent conditions for 72 hrs until spheroids had formed. Spheroids were then harvested and embedded into a collagen matrix before being treated with normal saline or bortezomib (10 nM–10μM). After 72hrs, spheroids were treated with calcein-AM (viable cells, green), and ethidium bromide (dead, cells, red). Data shown is representative of three independent experiments. Magnification 4X, scale bar: 100 μM.
D: Western blot showing induction of apoptosis in cells treated with bortezomib. TE12 (ESCC) and 1205Lu (melanoma) cells were treated with 10 nM bortezomib for increasing periods of time (0–48 hrs) followed by protein extraction and probing for expression of cleaved caspase-3/PARP, p53, p21 and Noxa. β-actin is shown as a loading control.