Table 1.
Primer Sequences used for real-time RT-PCR.
Figure 1.
Aldehyde dehydrogenase 1 (ALDH1) expression in sarcoma cell lines using the Aldefluor® assay.
Fluorescence versus forward scatter was shown in a density blot from (A) DEAB control cells and (B) ALDH1-expressing cells (called ALDH1high). (C) ALDH1 expression in % of gated cells. The highest proportion of ALDH1high 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 ALDH1high population generated a significant higher account of ALDH1high cells. (E) The enhanced ALDH activity was also demonstrated by western blot.
Table 2.
Overview of all results including the corresponding significances.
Figure 2.
Proliferation analysis of ALDH1high and ALDH1low sarcoma cells.
The immunohistochemical analysis using anti-Ki-67 proliferation marker revealed a decreased proliferation level of (A) SW-1353 ALDH1low cells and compared to (B) SW-1353 ALDH1high cells. (C–E) Dynamic proliferation curves for ALDH1high and ALDH1low cells seeded at 10,000 cells per well measured with the xCELLigence system.
Figure 3.
Clonogenic activity of ALDH1high and ALDH1low cells.
(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.
Figure 4.
Relative mRNA expression of stemness markers and ABC transporters genes in ALDH1high and ALDH1low cells.
The expression level was normalized (ΔCt) to the expression of mRNA for GAPDH, ACTB, and hprt-n as an internal control and compared to the corresponding ΔCt (ΔΔCt) in controls. The normalized expression levels from (A) c-Myc, (B) β-catenin, and (C) SOX-2 were shown. (D) ABCG2 was more highly expressed in ALDH1high than in ALDH1low cells, whereas the p values for (E) ABCA2 were not significant. (F) ALDH1high SW-1353 cells also showed a significant higher expression of ABCB1.
Figure 5.
Analysis of the cytotoxic effect of chemotherapeutic agents on ALDH1high and ALDH1low populations sorted from (A–C) SW-982 and (D–F) SW-1353 cells.
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.