Figure 1.
BTZ arrests SKM-1 cells at G2/M phase.
A. SKM-1 cells were treated with vehicle (control) or 10 nM BTZ for 24 h and then stained with PI for FACS analysis. B. The distribution of cells in each cell cycle phase is shown. **P<0.01.
Figure 2.
Effect of BTZ on the expression of cell cycle regulatory proteins Wee1 and cdc25C.
SKM-1 cells were treated with 10 nM BTZ and the expression of Wee1 and cdc25C was analyzed by immunoblotting. GAPDH was used as loading control.
Figure 3.
A. Representative flow cytometry scatter plots showing cells undergoing apoptosis in response to BTZ. B. Bar graph shows percentage of SKM-1 cells undergoing apoptosis in response to 5 and 10 nM BTZ for 24 h. C. Immunoblot analysis of cleaved caspase-3. D. Immumofluorescence staining of cleaved caspase-3. Bar 50 µm. *P<0.05.
Figure 4.
BTZ induces autophagy in SKM-1 cells.
SKM-1 cells were treated with 10 nM BTZ for 24 h and immunoblotted for microtubule-associated light chain (LC3-I and LC3-II). GAPDH was used as loading control.
Figure 5.
ERK1/2 expression in SKM-1 cells.
Immunoblotting of SKM-1 cells shows that total and p-ERK1/2 are both expressed in untreated SKM-1 cells (control). However, BTZ inhibited the expression of p-ERK1/2 but not total ERK1/2. GAPDH was used as loading control.
Figure 6.
SKM-1R cells are resistant to BTZ induced apoptosis.
A. Flow cytometry scatter plots of the apoptosis assay. B. Bar graph shows percentage of SKM-1 and SKM-1R cells undergoing apoptosis in response to a 24 h treatment with 5 nM BTZ. *P<0.05.
Figure 7.
Cell viability in response to BTZ treatment.
SKM-1 and SKM-1R cells were exposed to 5 nM BTZ for 24 h and the viable cell number was evaluated by MTT assay. The result showed a reduction in cell viability by treating wild type SKM-1 cells with 5 nM BTZ with no notable effect on SKM-1R cells. *P<0.05.
Figure 8.
Expression of LC3 in BTZ resistant SKM-1R cells.
Cell lysates were subjected to immunoblot analysis using specific antibody against LC3. Conversion of LC3-I to LC3-II was not observed in SKM-1R cells treated with BTZ.
Figure 9.
ERK1/2 expression in BTZ resistant SKM-1R cells.
Immunoblotting of SKM-1R cells shows that both total and phospho-ERK1/2 are upregulated in response to BTZ treatment. Protein expression levels were quantified by ImageJ. Total and phospho-ERK1/2 levels were normalized to GAPDH.
Figure 10.
Reversal of BTZ resistance by MEK inhibitors in SKM-1R cells.
A. Flow cytometry scatter plots showing Annexin V staining of SKM-1R cells. B. Bar graph shows percentage of SKM-1R cells undergoing apoptosis in response to a 24 h exposure to 5 nM BTZ. Both PD98059 (PD) and U0126 (U0) significantly increased the percentage of cells undergoing apoptosis. *P<0.05, ***P<0.001.