Analysis of the mitochondria-caspase dependent pathway.

<p>A,B) Relative gene expression analysis of the pro- and anti-apoptotic markers Bax, Bak, Bcl-2, and Bcl-xl in Cal-78 and SW-1353 cells treated with the respective IC₅₀ values of bortezomib for 24 h (<i>n</i> = 10). C) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27, HSP60, and HSP70 as well as HTRA, Livin, p27, and p53 in both cell lines. D) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment.</p

Bortezomib induces autophagy in human chondrosarcoma cells.

<p>A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC₅₀ values of bortezomib for 24 h. C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II. D) Quantification of the relative LC3B protein expression.</p

Apoptotic induction of bortezomib.

<p>A,C) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3/7 activity than untreated cells. Untreated control cells served as reference value (ratio = 1). B,D) Cleavage of caspase-3 was detected after 24 h of bortezomib treatment by flow cytometry. The y-axis denotes cell counts and the x-axis represents fluorescence intensity of the APC antibody. The black filled histogram represents untreated control cells, the striated histogram represents 5 nM, and the checkered histogram shows 10 nM bortezomib treated cells.</p

Analysis of TRAIL/death receptor pathway.

<p>A,B) Relative gene expression analysis of IGF1R, Fas, and the death receptors TRAILR-1 and TRAILR-2 after bortezomib treatment with the respective IC₅₀ concentrations for 48 h. Untreated control cells served as reference value (ratio = 1). C) The human apoptosis antibody protein array supported the important role of the IGF binding proteins (IGFBP-1 to IGFBP-6), the TNF receptor family, and the TRAIL receptors in both cell lines. D) Western blot analysis showed the downregulation of Fas and TNF-R1.</p

Primer Sequences used for real-time RT-PCR.

<p>Primer Sequences used for real-time RT-PCR.</p

Aldehyde dehydrogenase 1 (ALDH1) expression in sarcoma cell lines using the Aldefluor® assay.

<p>Fluorescence versus forward scatter was shown in a density blot from (A) DEAB control cells and (B) ALDH1-expressing cells (called ALDH1^high). (C) ALDH1 expression in % of gated cells. The highest proportion of ALDH1^high cells is represented by SW-684 cells (1.77±0.9%; n = 12), SW-982 cells (2.23±1.0%; n = 11), and SW-1353 cells (2.69±1.3%; n = 8). (D) After two weeks cultured the ALDH1^high population generated a significant higher account of ALDH1^high cells. (E) The enhanced ALDH activity was also demonstrated by western blot.</p

Proliferation analysis of ALDH1^high and ALDH1^low sarcoma cells.

<p>The immunohistochemical analysis using anti-Ki-67 proliferation marker revealed a decreased proliferation level of (A) SW-1353 ALDH1^low cells and compared to (B) SW-1353 ALDH1^high cells. (C–E) Dynamic proliferation curves for ALDH1^high and ALDH1^low cells seeded at 10,000 cells per well measured with the xCELLigence system.</p

Clonogenic activity of ALDH1^high and ALDH1^low cells.

<p>(A) The quantitation of the clone formation efficiency from SW-684, SW-982, and SW-1353 cells. Data from five independent experiments represent average colony count/well after 14 days. (B) Representative colony forming units from all three cell lines.</p

Analysis of the cytotoxic effect of chemotherapeutic agents on ALDH1^high and ALDH1^low populations sorted from (A–C) SW-982 and (D–F) SW-1353 cells.

<p>Both subopulations were treated with 0–5.0 µM doxorubicin, 0–5.0 µM epirubicin, 1–100 µM cisplatin, and measured after 48 h. Mean value ±SD of all measurements was fitted according the Hill equation. Significant differences on the individual concentrations were incorporated in the curves.</p

Overview of all results including the corresponding significances.

<p>Overview of all results including the corresponding significances.</p