Fig. 2

Increased ATGL levels induce an aggressive phenotype in HeLa cells. HeLa cells were transfected with pATGL for 48 h. (a) Morphological change of HeLa cells transfected with pcDNA™4/HisMaxC and pcDNA™4/HisMaxC-ATGL for 48 h. (b) RT-qPCR analysis of E-cadherin, N-cadherin, Vimentin and Fibronectin mRNA. β-Actin (ACTB) was used as reference control. Data are shown as fold change vs. CTRL represented by a dashed line in the bar graph (n = 4; * p < 0.05 vs. CTRL). (c) Western blot analysis of mesenchymal marker N-cadherin levels. β-Actin and ATGL were used as loading and transfection control, respectively. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL. The Western blots reported are representative of three independent experiments that gave similar results. (d) Western blot analysis of active β-catenin levels on nuclear extracts. Lamin B1 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as nuclear and cytosolic purity control, respectively. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL for each fraction. The Western blots reported are representative of three independent experiments that gave similar results. (e) RT-qPCR analysis of VEGF mRNA. ACTB was used as reference control. Data are showed as fold change vs. CTRL (n = 4; * p < 0.05 vs. CTRL). (f) Wound Healing assay. HeLa cells were seeded onto 12-well plates and transfected with pATGL for 48 h. The monolayer was wounded with a plastic tip and monitored under bright-field microscope, taking images at 0, 8, and 16 h from “wound” formation; images, representative of three independent experiments that gave similar results. (g) Patatin Like Phospholipase Domain Containing 2 (PNPLA2) gene expression in different grade of cervical cancer was assessed by Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo, accession number GSE63514) through an Affymetrix Human Genome Array (100 samples of cervical cancer subdivided for grade vs. normal cervical epithelium) (* p < 0.05; ** p < 0.01; *** p < 0.001 as indicated)