Fig. 5.

Effects of on multiple molecules on cellular viability and miR-223 regulation of these molecules. a Effects of multiple molecules on cellular viability. The cells were pre-treated with NF-κB inhibitor (PDTC) or mTOR inhibitor Ku-0063794 (abbreviated as Ku) for 1 h, or transfected with siRNA specific to hsp70 gene or non-specific siRNA (mock) for 24 h and subsequently washed to stop transfection. Cells were then treated with celastrol (4 μM for MCF-7 and 2 μM for PC3) or DMSO for 24 h, before the numbers of viable cells were quantified with flow cytometry. b Effects of miR-223 alteration on multiple molecules. Cells were transfected with miR-223-Down antagomir (shortened to anti-miR-223) or miR-223 mimics or corresponding mock agent (mock transfection) for 24 h, then the cells were washed to stop transfections. Some of the cells were used immediately to extract protein; the remaining ones were used for culture with celastrol (4 μM for MCF-7 and 2 μM for PC3) or DMSO for the indicated time, before proteins were extracted. The obtained proteins were used to detect phosphorylated NF-κB (p-NF-κB), p-mTOR, and p-HSF-1, as well as whole levels of HSP70 by Western blot, with respective primary antibodies and corresponding secondary antibodies being used. The mean of relative expressions of specific proteins in different treatments is displayed by heat map (detailed in the legend of Fig. 3). Tests were repeated three times. Cel represents celastrol. Data in A panel are presented as mean ± SD, statistics were determined between different treatments (without celastrol) and DMSO control, or between different treatments (plus celastrol) and celastrol alone * indicates P < 0.05, while ** indicates P < 0.01