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. 2013 Oct 22;104(12):1683–1689. doi: 10.1111/cas.12277

Figure 2.

Figure 2

Ly101‐4B inhibited the proliferation, suppressed the expression of heat shock factor 1 (HSF1) and increased the apoptosis in SKOV3 cells. (a) Structure of Ly101‐4B. (b) Quantitative PCR was performed to detect transcripts of HSF1 in SKOV3 cells. The internal standard of 18s rRNA was used and the relative transcript concentration was normalized to mock cells, which were treated with DMSO. Results represent the average of three independent experiments, and error bars indicate the standard deviations (***, P < 0.001). (c) HSF1 protein was detected by western blot assay. “Mock” represents cells treated with DMSO and “Ly101‐4B” represents cells treated with 60 μM for 48 h. β‐actin was applied as a reference. (d) MTT assay was performed to determine the cell viability. An equal amount of DMSO was used as mock control. Each experiment was performed in triplicate, and error bars indicate the standard deviations (***, P < 0.001). (e) SKOV3 cells were treated with 60 μM Ly101‐4B for 48 h, followed by flow cytometry analysis. AnnexinV‐FITC(+)/PI(−) indicated the early apoptotic cells, while annexinV‐FITC(+)/PI(+) labeled the late apoptotic cells. Numbers indicate the percentages of each cell type. Three independent experiments were performed, and similar results were obtained. The images represent one of the three independent experiments. (f) The cleaved form of caspase9 (p35 segment) was detected by western blot assay. “Mock” represents cells treated with DMSO, and “Ly101‐4B” represents cells treated with 60 μM Ly101‐4B for 48 h. β‐actin was applied as loading control. (g) HSP27, HSP70 and HSP90 protein were analyzed by western blot assay, using β‐actin as a reference.