Figure 1.
EZH2 is expressed in chondrosarcoma.
A. EZH2 expression was analyzed by immunostaining in enchondromas (n = 8) (A1), or grade 2 and 3 chondrosarcomas (n = 7; 6 samples were positive for EZH2 staining) (A2 and A3 respectively). B. EZH2 expression was analyzed in two batches of both SW1353 and CH2879 chondrosarcoma cell lines and normal chondrocytes by Western blot. Actin was used to compare protein loading.
Figure 2.
DZNep reduces EZH2 protein expression and H3K27me3 and but not mRNA.
SW1353 and CH2879 cells were treated with DZNep (1 µM) for 72 h. A) EZH2 protein expression was analyzed by Western blot. Histograms represent the quantification of three independent experiments after normalization with actin. B) H3K27me3 was also analyzed by Western blot. H3 was used to compare protein loading. Histograms represent the quantification of three independent experiments after normalization with H3. C) EZH2 mRNA expression was analyzed by RT-PCR from CH2879 treated with DZNep for 72 h. Data were expressed as means ± SEM.
Figure 3.
DZNep induces death in chondrosarcoma but not chondrocytes.
CH2878 cells (A), SW1353 (B), or chondrocytes (C) were treated with DZNep (1 µM) for 14 days. Treatment was renewed at each medium changes (at days 4, 7 and 10), and adherent cells regularly counted. The results of three independent experiments are shown. Data are expressed as means ± SEM.
Figure 4.
DZNep induces apoptosis in SW1353 and CH2879 chondrosarcoma cell lines.
Cells were treated with DZNep (1 µM) for 7 days (A and C) or 5 days (B). A) At day 7, cells were fixed and cell cycle determined by flow cytometry. Histograms represent the sub-G1 phase percentage from three independent experiments. B) At day 5, proteins were extracted and PARP protein expression was analyzed by Western blot. C) At day 7, cells were stained with Apo 2.7 antibody coupled to phycoerythrin and analyzed by flow cytometry.
Figure 5.
DZNep reduces SW1353 and CH2879 chondrosarcoma migration.
Cells were pretreated 4 days with DZNep (1 µM) and a straight scratch was made in individual 6-wells dishes with a 200 µL pipette tip. A) Microscopic observations were recorded 24 hours after scratching the cell surface. Dotted lines showed the initial mark of the scratch. B) Graph represents the percentage of filling 24 hours after the wound. Data were expressed as means ± SEM.