Fig 2: ROCK activity in FLAG‐FKBP51‐expressing U2OS cells. (a) U2OS cells were transfected with FLAG‐FKBP51 or the FLAG‐mock expression vector for 24 h. Next, cells were immunostained with anti‐cortactin antibody (green) and phalloidin‐ATTO565 (red). Nuclei were stained with Hoechst 33258 (blue). (b) The immunoblot analysis of ROCK1 in U2OS cells treated with FLAG‐FKBP51 or the FLAG vector (left panel), and the relative ROCK activity with or without the ROCK inhibitor Y27632 (10 μM) or ATP (125 μM) (n = 3) (right panel).

Fig 3: Cell motility and invasion assay following knockdown of FKBP51. (a) U2OS cells were treated with siRNA‐FKBP51 or siRNA‐control for 48 h. Next, proliferation was measured using the WST‐1 assay. Relative fluorescence units (RFU) indicate the relative amount of proliferation. Column graphs show the mean ± SEM from five samples. (b) U2OS cells were treated with siRNA‐FKBP51 or siRNA‐control for 48 h and then subjected to the wound‐healing assay. Phase contrast images are shown for 0 and 24 h. The dotted lines indicate cells at the start of the experiment, and white lines show the tips of migrated cells after 24 h. (c) Column graphs show the mean ± SEM from three samples. (d) U2OS cells were treated with siRNA‐FKBP51 or siRNA‐control for 48 h and then subjected to the cell invasion assay. Relative fluorescence units (RFU) indicate the relative amount of proliferation. Column graphs show the mean ± SEM from three samples.

Fig 4: The AKT inhibitor inhibited the proliferation and cycle of AML-M5 cells. (A) FKBP51-knockdown U937 cells and negative control cells were treated with different concentrations of AI and a CCK-8 assay was performed. The absorbance at 450 nm of each group was used to reflect cell proliferation. (B, C) The effect of the AKT inhibitor on the cell cycle in FKBP51-knockdown U937 cells and control cells. (D) After treating FKBP51-knockdown U937 cells and negative control cells with AKT inhibitors (1.8, 5.4 and 9 μM) for 24 hours, western blot analysis of total and phosphorylated (Ser473) AKT and AKT downstream targets (p27, BAX, BCL-2, total and phosphorylated (Ser256) Foxo1, and total and phosphorylated (Ser9) GSK3β) was performed. NC-sh, negative control for sh-FKBP51; sh-FKBP51, shRNA-FKBP51; AI, AKT inhibitor. All cells were treated with LPS (1 µg/ml) for 15 min before protein extraction. Each experiment was performed in triplicate. Statistical differences from negative control cells are indicated by asterisks (* for p < 0.05, ** for p < 0.01).

Fig 5: The effect of FKBP51 on the expression level of AKT pathway-related proteins. (A) Phosphorylated (Ser473) and total AKT and AKT downstream targets (P21, P27, BAX, BCL-2, total and phosphorylated Foxo1A, and total and phosphorylated GSK3β) in AML-M5 cells with different expression levels of FKBP51 were detected by western blot. GAPDH was used an internal control. (B, C) Statistical results of AKT-related protein levels in FKBP51 overexpressed THP-1 cells and FKBP51-knockdown U937 cells as well as their corresponding control cells. All cells were treated with LPS (1 μg/ml) for 15 minutes before protein extraction. Each experiment was performed in triplicate. Statistical differences from negative control cells are indicated by asterisks (* for p < 0.05, ** for p < 0.01).