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. 2016 Jun 20;7(29):46448–46465. doi: 10.18632/oncotarget.10186

Figure 7. AR promotes anoikis by enhancing cellular amorphosis.

Figure 7

A. AR-expressing SKhep1 cells exhibited higher stress fiber–dissolving capacity than par cells. The cells were treated with EtOH or DHT, maintained in adherent or detached conditions for 24 h, and then fixed and co-stained with rhodamine phalloidin and DAPI. Red fluorescence represents actin stress fibers and blue represents cell nuclei stained by DAPI. B. Latrunculin B1 (LtB) abolished AR-mediated anoikis. The degree of anoikis was compared among SKhep1, par, and AR (p=0.02) cells with or without pre-treatment for 3 h with 0.5 μM LtB. The percentage of cells exhibiting anoikis was compared between par and Veh-treated cells. C, D. PI3K/AKT inhibitor LY294002 (LY; C) and ROCK inhibitor (ROCK inh.; D) reduced the degree of AR-mediated anoikis. The degree of anoikis was compared among SKhep1, par, and AR cells with or without co-treatment with LY/ROCK. The percentage of cells exhibiting anoikis was compared between par and Veh-treated cells. All experiments were performed at least three times, and the data represent the mean values from three independent experiments. E. The amorphosis index is a measure of solubility of actin stress fibers in detached conditions divided by that in attached conditions. The solubility of α-actin stress fibers was determined by dividing the amount of soluble actin by the total amount of actin. F. Upper image: The par and AR cells were treated with EtOH or 10 nM DHT; soluble α-actin (cell lysate with PBS) and total actin (cell lysate with 1 μM NP-40 RIPA buffer) were then measured using an immunoblot assay with α-actin antibody. The amorphosis index was higher in DHT-treated AR cells. Lower bar graph: The optical density of the immunoblots was compared between par and AR SKhep1 cells using the equation presented in (E). The readings were compared between DHT- and EtOH-treated cells. G–H. LY (G) and ROCK (H) inhibitor abolished AR-promoted (p=0.031) cell amorphosis in DHT-treated cells. I. The degree of phosphorylation of Rho A (pRhoA) and AKT (pAKT) was significantly lower in SKAR cells in detached conditions than in SKAR cells in attached conditions. The plotted data were from the mean value of at least three independent experiments, and SD was used to show the variation in the experiments. A single * label represents significance at p < 0.05, and a double * label represents significance at p < 0.01.