https://orcid.org/0000-0002-7317-0858
Figures & data
Figure 1. FASN is expressed in migratory cells and malignant tissue
(a) Whole cell lysates were prepared from a range of cancer cell lines as illustrated. These were then subjected to SDS-PAGE and immunoblotted for FASN and the loading control HSP90. (b) Densitometry analysis was performed and relative quantification of FASN levels calculated. (c) Human primary prostate tissue was excised by surgical biopsy from seven different patients. Histopathological analysis of stained sections was performed and the approximate % of benign and malignant material was documented. Tumour percentage unknown for F2; but malignant diagnosis was confirmed for this sample. (d) proteins were extracted from the tissue samples and probed for FASN expression levels via SDS-PAGE gel electrophoresis HSP90 was used as a loading control. (e) Densitometry analysis was performed and relative quantification of FASN levels calculated. Data represents the mean values ± SEM from three independent experiments. Statistical significance was determined by student’s one-way ANOVA with Tukey’s post-hoc test. ** p < 0.01, ***p < 0.001.
Figure 2. FASN depleted cells exhibit reduced migration speed on collagen
(a) 1542, 1542 shControl, 1542 shFASN A3 and 1542 shFASN A4 cells were seeded into collagen coated wells, serum starved, stimulated with 10 ng/ml HGF and imaged for 16 h with images being taken at 5 minute intervals. Migration plots of each cell line trajectory were created (60 cells per condition). Cell tracking data was placed into a Mathematica notebook and then the speed of cell migration was calculated as described in methods. (b) PC3, PC3 shControl, PC3 shFASN A3 and PC3 shFASN A4 cells were seeded into matrigel coated wells and treated as above (a).
Figure 3. HGF-induced cell scattering is diminished in FASN depleted cells
(a) Whole cell lysates were prepared from DU145, DU145 shControl, DU145 shFASN A3 and DU145 shFASN A4. These were then subjected to SDS-PAGE and immunoblotted for FASN and the loading control HSP90. Densitometry analysis was performed and relative quantification of FASN levels calculated. (b) DU145, DU145 shControl, DU145 shFASN A3 cells colonies were seeded on coverslips, serum starved for 24 hours, then incubated with or without HGF (10 ng/ml) for 24 hours. Cells were fixed and stained for F-actin (red) and DAPI (blue). (c) For each population indicated 90 cells were counted over 3 independent experiments and scored for single cell (scattered) or within a colony. mean ± S.E.M. Statistical significance was determined by a student’s Anova Tukey test. n.s.d = No statistical difference ** p < 0.005 (d) DU145, DU145 shControl, DU145 shFASN A3 and DU145 shFASN A4 cells were seeded on matrigel and incubated for 24 hours. Cells were fixed and stained for F-actin and DAPI. Images were quantified for cell spread area 90 cells per condition over 3 independent experiments see methods for details. mean ± S.E.M. Statistical significance was determined by a student’s Anova Tukey test. n.s.d = No statistical difference * p < 0.05
Figure 4. FASN activity supports RhoU and Cdc42 regulation of cell migration
Proposed model of the impact of loss of FASN activity (and therefore palmitate production) on prostate cancer cell migration and invasion.
