Polo-like kinase 1 induces epithelial-to-mesenchymal transition and promotes epithelial cell motility by activating CRAF/ERK signaling
- Virginia Commonwealth University School of Medicine, United States
Figures
Figure 1 with 3 supplements
Ectopic expression of PLK1 in RWPE-1 cells promotes cell motility.
(A) Cell lysates were prepared from the indicated PCa cell lines and subjected to Western blots in order to detect the level and activity of PLK1 protein using anti−PLK1 and anti−PLK1(pT210) …
Figure 1—figure supplement 1
Androgen receptor (AR) mRNA is expressed in RWPE-1 cells.
Total RNA from control RWPE-1 (EV) and RWPE-1–PLK1 (PLK1) cells was subjected to real-time RT-PCR. AR mRNA levels were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) and are expressed …
Figure 1—figure supplement 2
Ectopic expression of PLK1 in RWPE-1 cells induces cellular transformation and tumorigenicity.
(A) The effect of PLK1 upregulation in RWPE-1 cells on anchorage-independent growth in soft agar. RWPE-1–PLK1 (PLK1) cells and vector control (EV) cells were plated in soft agar and grown for 21 …
Figure 1—figure supplement 3
Ectopic expression of PLK1 in PrEC cells promotes cell migration and invasion.
(A) Modulated PLK1 levels in PrEC cells. PrEC cells were infected with lentivirus encoding Flag-PLK1 (PLK1) or control vector (EV). PLK1 protein levels were determined by Western blot. (B) A …
Figure 2 with 2 supplements
Overexpressing PLK1 in RWPE-1 cells induces EMT.
(A) Representative phase-contrast images of control RWPE-1 and RWPE-1–PLK1 cells. Scale bar = 50 μm. (B, C) mRNA and protein expression of different EMT markers in RWPE-1–PLK1 cells (PLK1) and …
Figure 2—figure supplement 1
The effect of PLK1 overexpression on EMT is independent of its cell cycle function.
RWPE-1–PLK1 (PLK1) cells and vector control (EV) cells were left either unsynchronized (Asyn) or synchronized at G1, S, and M phases. The expression of EMT markers in these cells was examined by …
Figure 2—figure supplement 2
Overexpression of PLK1 in PrEC cells induces EMT.
(A) Representative phase-contrast images of control PrEC (EV) and PrEC-PLK1 (PLK1) cells. Scale bar = 50 μm. (B, C) EMT marker mRNA and protein expression in PrEC cells (PLK1) and vector control …
Figure 3 with 4 supplements
Downregulation of PLK1 reverses EMT and inhibits the motility of metastatic PCa cells.
(A) Two metastatic PCa cell lines (DU145 and C4-2B) were infected with lentiviral shRNA constructs that target either the 3’-UTR of endogenous PLK1 (shPLK1#1) or serve as a control (shCTL). …
Figure 3—figure supplement 1
Partial knockdown of PLK1 in PCa cells has minimal effect on cell cycle progression and cell death.
(A) The effect of PLK1 downregulation on cell cycle distributions was analyzed by flow cytometric analysis. (B) The effect of PLK1 downregulation on apoptosis was determined by staining with annexin …
Figure 3—figure supplement 2
Downregulation of PLK1 results in EMT reversion and reduction of migration of metastatic PCa cells.
(A) Three metastatic PCa cell lines (PC3, C4-2B, and DU145) were infected with lentiviral shRNA constructs that target either the 3’-UTR of endogenous PLK1 (shPLK1#2) or serve as a control (shCTL). …
Figure 3—figure supplement 3
Validation of the role of PLK1 in induction of EMT in the ARCaP cell culture model.
(A) The level and activity of PLK1 protein in ARCaPE and ARCaPM cells were examined by Western blotting analysis using anti-PLK1 and anti-PLK1(pT210) antibodies, respectively. β-actin was used as a …
Figure 3—figure supplement 4
PLK1 contributes to physiological EMT events.
RWPE-1 and ARCaPE cells were treated with a combination of TGF-β1 (4 ng/mL) and EGF (50 ng/mL) in the presence and absence of BI2536 (1 nM) for 5 days. The EMT marker expression and morphological …
Figure 4 with 1 supplement
Both ZEB1 and ZEB2 play a causal role in PLK1-induced EMT and increased motility of prostate epithelial cells.
(A) Expression of different EMT-inducing transcription factors was examined in control and PLK1-overexpressing RWPE-1 cells by quantitative real-time RT-PCR. mRNA expression of genes of interest was …
Figure 4—figure supplement 1
Elevated PLK1 levels result in activation of ERK, and upregulation of ZEB1, ZEB2, and Fra1 in PrEC cells.
Protein levels of ZEB1 and ZEB2, Fra1, and activated and total ERK1/2 in PrEC (EV) and PrEC-PLK1 (PLK1) cells were determined by Western blotting.
Figure 5
ERK1/2 activation is essential for PLK1-induced EMT and increased motility of prostate epithelial cells.
(A) The effect of PLK1 overexpression on activation (phosphorylation) of ERK1/2 and MEK1/2 was determined by immunoblotting. (B−E) Control (EV) and PLK1-overexpressing (PLK1) RWPE-1 cells were …
Figure 6
Fra1 is a critical mediator of PLK1-induced EMT and accelerated motility of prostate epithelial cells.
(A, B) mRNA and protein expression of Fra1 were examined in control (EV) and PLK1-overexpressing (PLK1) RWPE-1 cells by real-time RT-PCR (A) and immunoblotting (B), respectively. The data are …
Figure 7
CRAF plays an essential role in PLK1-induced EMT and accelerated motility of prostate epithelial cells.
(A) The effects of PLK1 overexpression on the levels of total and phosphorylated CRAF were determined by immunoblotting. p/t: indicates densitometric intensity ratio of phosphorylated to total CRAF. …
Figure 8 with 1 supplement
PLK1-mediated phosphorylation of CRAF leads to CRAF activation and stabilization.
(A) Co-immunoprecipitation of endogenous PLK1 and CRAF from HeLa cell lysates obtained under control conditions or following nocodazole treatment to arrest cells at M phase. (B) GST pull-down assay …
Figure 8—figure supplement 1
Downregulation of PLK1 led to dramatic reduction of CRAF phosphorylation at S338, S339, and S621 that was rescued by WT PLK1, but not KM PLK1.
DU145 cells were infected with lentiviral shRNA constructs that target either the 3’-UTR of endogenous PLK1 (shPLK1#2) or serve as a control. WT or KM PLK1 was then re-expressed in PLK1 knockdown …
Videos
Video 1
Video showing the random migration of control RWPE-1 cells.
https://doi.org/10.7554/eLife.10734.007
Video 2
