Effect of GA on localization and protein level of HSPs in OS 143B cells.

<p>OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90 protein were determined by immunofluorescence. <b>A</b>. GA did not change the mitochondrial localization of Hsp60, however, it decreased the mitochondrial pool of the protein. Cell nuclei were shown in blue while Hsp60, AIF immunoreactivities in green and red, respectively. Merged image of all kinds of staining (orange) was also presented. <b>B</b>. GA upregulated Hsp70 protein level. Cell nuclei were shown in blue, while Hsp70 immunoreactivity in green. Merged image of both was also presented. <b>C</b>. GA upregulated Hsp90 protein level. Cell nuclei were shown in blue while Hsp90 immunoreactivity in red. Merged image of both was also presented. Original magnification ×40. Each experiment was performed at least three times. The representative data were shown.</p

GA affected HSPs gene expression in OS 143B cells.

<p>OS 143B cells were treated with GA (4 µM) for 6 h; the levels of Hsp60, Hsp70, Hsp90AA1, Hsp90AB1, Hsp90B1 transcript were then determined by means of Real Time PCR. Treatment with GA resulted in upregulation of Hsp60 (A), Hsp70 (B), Hsp90AA1 (C), Hsp90AB1 (D) transcript, however it did not impact Hsp90B1 gene expression (E). Values are mean ± SE of three independent experiments, relative mRNA levels of HSP/beta-actin are presented. The data were analyzed by Student's t-test using GraphPad Prism Software version 6.02. *P<0.01, **P<0.001 vs. control.</p

GA affected HSPs protein levels in OS 143B cells.

<p>OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90, Hsp90AA1, Hsp90AB1proteins were determined by Western blotting. <b>A</b>. GA decreased the level of Hsp60 and induced post-translational modification of Hsp60 partially derived from the hyperacetylated isoform. <b>B–E</b>. GA upregulated Hsp70 (B), total cellular Hsp90 (C), Hsp90AA1 (D), Hsp90AB1 (E) protein levels. Each experiment was performed at least three times. The representative data are shown. Densitometric analysis of HSP/beta-actin was performed using Quantity one 4.5.2 software.</p

The effect of GA on proliferation and induction of cell death of OS 143B cells.

<p><b>A</b>. GA inhibits OS 143B cell growth. OS 143B cells were treated for 24 h with serial GA dilutions (within the range of 0.8 µM–50 µM). The cell viability was then determined by means of MTT assay. Data from at least three independent experiments are presented as mean ± SE. The absence of error bar denotes a line thickness greater than the error. Data were analyzed by GraphPad Prism Software version 6.02 performing One-way ANOVA combined with Dunett's Multiple Comparison Test. *P<0.01 vs. control. <b>B–D</b>. GA induced cell death of OS 143B cells. 143B OS cells were treated with 4 µM GA for 24 h, the cells were then harvested and the percentage of apoptotic and necrotic cells was determined performing double PI-Annexin V staining. <b>B</b>. Annexin V, PI-live/dead dot plots showing apoptosis and necrosis before and after treatment with GA. Plots are representative of five individual experiments. <b>C–D</b>. Total apoptotic (C) and necrotic (D) cell number before and after treatment with GA. Data from at least three independent experiments are presented as mean ± SE. Data were analyzed using GraphPad Prism (GraphPad Software, Inc., version 6.02, USA). Significant differences between groups were determined by Student's t-test. *P<0.01, ***P<0.0001 vs. control.</p