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. 2016 Apr 27;7(23):35144–35158. doi: 10.18632/oncotarget.9059

Figure 2. Stress hormone increases MTOC via GR.

Figure 2

A. cortisol obviously induced γ-tubulin in various cell lines. Each cells were treated with indicating dose of cortisol for 72 hr. Western blot was performed for measuring γ-tubulin expression. Actin was used for loading control. B. GHs induce γ-tubulin in regardless of FST. A498 (VHL deficient cell line) were incubated with cortisone (5 μM) and cortisol (5 μM) with/without FST (3 μM) for 24 hr. However, BRCA1 expression was increased by FST, although reduction of BRCA1 expression by GH was not affected. In addition, p53 expression was not altered by FST or GH. Actin was used for loading control. C and D. The number of MTOC is increased by cortisone treatment (CORT; 5 μM) in VHL-intact C2V cells. About 50 cells were counted in each conditions to check average MTOC numbers and representative pictures were shown. Cells were stained with anti-γ-tubulin antibody (Red) to detect MTOC and DAPI (Blue) for DNA. E. GR and cortisone can induce MTOC amplification. After GR transfection or cortisone (5 μM) treatment, HEK293 cells were stained with anti γ-tubulin antibody (Red) and DAPI (Blue). F. Addictive effect of GH and GR on MTOC amplification. About 50 numbers of cells were counted in each condition to estimate MTOC numbers. G. Overexpressed GR can provide the resistance to Taxol in VHL-positive C2V cells. GR was transfected to C2V cells and Taxol (Tax; 3 μM) was treated. 72 hr later, cell viability was measured by MTT assay. H. GR blocks Rad51-mediated Taxol-sensitization. Re-sensitization to Taxol by Rad51 overexpression in VHL-deficient A498 was erased by GR overexpression. A498 cells were transfected with GR and/or Rad51 and incubated with Taxol for 72 hr. Cell viability was determined by MTT assay. I. GR overcomes the Rad51-induced γ-tubulin reduction. In addition, Rad51 is obviously reduced by GR overexpression. GFP-tagged GR and HA-tagged Rad51 were transfected to HEK 293 cells. After 72hr, protein levels were detected with anti-GFP and HA antibodies. J. Obvious reduction of Rad51 by cortisone treatment in GR transfected HEK 293 cells. GFP-tagged GR was transfected to HEK 293 cells and cortisone (5 μM) was treated. After 72hr, western blot was performed. K. GR expression was not altered by VHL. GFP-tagged GR and HA-tagged VHL were transfected to HEK 293 cells and cortisone (5 μM) was treated. After 72hr, western blot was performed. Actin was used as loading control.