Fig 1.
Expression of PTRF in PC3 cells reduces cell motility and FAK stabilization in FAs.
(A) Transwell migration assay shows that PC3-GFP-PTRF cells migrate slower than wild-type PC3 and PC3-GFP cells. (B) Representative confocal images of PC3, PC3-GFP and PC3-GFP-PTRF cells and quantification of FAs per cell in each of the cell lines show that the number of FAs per cell is not significantly affected by PTRF expression in the cell. (C) Fluorescence Recovery After Photobleaching (FRAP) assay shows that FAK-GFP intensity recovery level is increased with PTRF-mCherry co-transfection compared with FAK-GFP alone or FAK-GFP and mCherry co-transfection. The FAK-GFP intensity recovery curve graph of one representative experiment and a bar graph of the FAK-GFP mobile fraction (calculated based on the fluorescence recovery plateau) summarized from all experiments are shown. (n≥3; ***: p<0.001; **: p<0.01; *: p<0.05.)
Fig 2.
Expression of PTRF does not affect pCav1 in PC3 cells.
Western blot shows expression levels of GFP, GFP-PTRF, Cav1 and pCav1 in PC3 wild-type cells (PC3), PC3 cells stably transfected with GFP (PC3-GFP) and PC3 cells stably transfected with GFP-PTRF (PC3-GFP-PTRF). Western blot band intensity of Cav1 and pCav1 is quantified and normalized to that of β-actin and shows no significant difference of Cav1 expression or phosphorylation between PC3, PC3-GFP and PC3-GFP-PTRF cells. (n≥3; ***: p<0.001.)
Fig 3.
Exogenous Gal3 restores FAK stabilization in FAs and cell migration of PTRF-expressing PC3 cells in a dose-dependent manner.
(A) FRAP assay of FAK-GFP shows the FA-associated FAK stability in PC3 cells transfected with FAK-GFP alone or FAK-GFP plus PTRF-mCherry and subjected to Gal3-His treatment at the indicated concentrations (1, 1.5, 2, 3, or 4 μg/ml). Reduced FAK stabilization in FAs of PTRF-expressing PC3 cells is restored by Gal3-His in a dose-dependent manner. A bar graph of the FAK-GFP mobile fraction summarized from all experiments and a representative FAK-GFP recovery curve graph from one experiment (showing NT and 2 μg/ml Gal3-His treatment only) are shown. (B) Quantification of PC3, PC3-GFP and PC3-GFP-PTRF cell migration using the transwell assay with no treatment or treated with Gal3-His (1, 1.5, 2, 3 or 4 μg/ml) shows that 2 μg/ml Gal3-His treatment increases the cell migration of all cell lines to a similar extent. Other concentrations of Gal3 increase migration of PC3-GFP-PTRF cells to a lesser extent, but do not affect migration of PC3 or PC3-GFP cells. (NT: non-treated; n≥3; *: p<0.05; **: p<0.01; ***: p<0.001; n.s.: not significant.)
Fig 4.
Exogenous Gal3 restores FAK stabilization in FAs of Gal3 knockdown cells.
(A) Western blot shows the efficiency of Gal3 siRNA knockdown. (B) FRAP assay shows FA-associated FAK-GFP stability in PC3 cells transfected with no siRNA, scramble control siRNA (siCTL) or the siRNA against human Gal3 (siGal3), and subjected to 2 μg/ml Gal3-His treatment. The FAK-GFP intensity recovery curve graphs of one representative experiment and a bar graph of the FAK-GFP mobile fraction summarized from all experiments are shown. (n = 3; ***: p<0.001.)
Fig 5.
Exogenous Gal3 does not restore FAK stabilization in FAs of Cav1 knockdown cells.
(A) Western blot shows the efficiency of Cav1 siRNA knockdown. (B) FRAP assay of FAK-GFP shows FA-associated FAK stability of PC3 cells transfected with no siRNA, scramble control siRNA (siCTL) or the siRNA against human Cav1 (siCav1), and subjected to 2 μg/ml Gal3-His treatment. The FAK-GFP intensity recovery curve graphs of one representative experiment and a bar graph of the FAK-GFP mobile fraction summarized from all experiments are shown. (n = 3; ***: p<0.001).
Fig 6.
Summarized working models of affecting Gal3-pCav1 function.
Extracellular Gal3 and non-caveolar pCav1 each stabilizes FAK within FAs dependent on each other. Expression of PTRF disrupts this function through three possible ways: 1) by recruiting pCav1 away from non-caveolar Cav1 scaffolds and into caveolae; 2) by direct interaction with non-caveolar pCav1; 3) by affecting Gal3 secretion and thus extracellular Gal3 concentration. Through which pathway PTRF affects Gal3-pCav1 function on FA dynamics remains to be studied.