Fig 2: Characterization of ID8EV and ID8hPON2 cells.a) Expression of hPON2 protein was analyzed by western blotting in ID8EV and ID8hPON2 stable cell lines. b) ID8EV and ID8hPON2 cell fractions were subjected to western blotting for PON2 and COX IV proteins. c) Colocalization of mitochondrial marker and PON2. ID8EV and ID8hPON2 cell were fixed and stained with Mito Tracker® Red (a mitochondrial marker, red) and PON2 antibody (green). The merged image of hPON2 and mitochondria (yellow) is shown in the bottom panel. d) Colocalization of ER marker and PON2. ID8EV and ID8hPON2 cell were fixed and stained for Calnexin (green), an ER marker, and hPON2 (red). The merged image of hPON2 and Calnexin is shown in the bottom panel. e) Mitochondria were isolated from ID8EV and ID8hPON2 cells and respiratory complex II + III assay was carried out as described in Materials and methods section. The results are expressed as Cytochrome c reduced/min/mg protein. f) mitochondrial superoxide levels were measured as described in the methods and values were expressed as fold change over the ID8EV. *p < 0.05 compared to IDEV cells. g) ID8EV and ID8hPON2 cells were cultured for 48 h, serum starved for 12 h, and cell viability was measured as described in the method section. h) 5 × 103 ID8EV and ID8hPON2 cells were cultured for 48 h, serum starved for 12 h and cell proliferation was assessed using BrdU by spectrophotometry as described in the Materials and methods section

Fig 3: PON2 overexpression reduces IGF-1 expression and inhibits cell proliferation in ID8 cells.a) 3 × 105 ID8EV cells or ID8hPON2 cells were cultured on six-well plates in DMEM medium containing high glucose and l-glutamine (2 mM) and supplemented with 4% fetal bovine serum (FBS), penicillin (100 U ml-1), streptomycin (100 µg ml-1), and 1× ITS liquid media supplement (10 µg ml-1 insulin, 5 µg ml-1 transferin, and 5 ng ml-1 sodium selenite). Cells were incubated at 37 °C for 24 h in 5% CO2 and then starved for 12 h in serum free media. Total RNA was isolated and cDNA was prepared as described under materials and methods. IGF-1 mRNA was quantified by qPCR and normalized to cyclophilin. b) 3 × 105 ID8EV and ID8hPON2cells were grown 1–3 days and each day serum starved for 12 h, then cell culture supernatants were collected and IGF-1 protein levels were quantified by ELISA as described under Materials and methods. hPON2 siRNA or scrambled siRNA was transfected into SKOV3, HeLa, and A549 cells at 70% confluence. PON2 protein expression was quantified after 2 days as described under the Materials and methods (c). Total RNA was isolated and IGF-1 was quantified by qPCR (d). e) 5 × 103 ID8EV and ID8hPON2 cells were grown as indicated in b and cell proliferation was assessed using Brdu by spectrophotometry as described in the Materials and methods. f) ID8EV and ID8hPON2 cells were grown and serum starved as in b and each day conditioned media were collected. Subsequently, the media were incubated with ID8 cells for 48 h, and cell proliferation was assessed as described in the Materials and methods section. g) ID8EV and ID8hPON2 cells were grown for 2 days and serum starved for 12 h with 500 nM IGF-1 antibody (# ab9572, Abcam) or without IGF-1 antibody and cell proliferation was assessed as described in the materials and methods section. h) Following hPON2 siRNA transfection with SKOV3, HeLa, and A549 cells, cell proliferation was assessed using FITC Brdu. Brdu-positive cells were quantified using flow cytometer. *p < 0.05, compared to ID8EV. Values were expressed as fold (n = 3)

Fig 4: PON2 reduces the IGF-1 level via C-Jun transcription factor in ID8 cells.ID8EV and ID8hPON2 cell culture lysates were prepared and utilized for chromatin immunoprecipitation assays with C-Jun (a) and p53 (b) antibodies. c) Equal number of ID8hPON2 cells were plated in six-well plates. At 60 % confluence level, ID8hPON2 cells were transfected with either hPON2 siRNA or scrambled siRNA, and PON2 expression analyzed by western blotting. d) Data in c are quantified using ImageJ software. e) Four six-well plates of cells were pooled from PON2-siRNA-treated ID8hPON2 or scrambled siRNA-treated ID8hPON2 cells. Chromatin immunoprecipitation assay was performed and C-Jun promoter was quantified (e) as described in the materials and methods section. Cell culture supernatants obtained from cells described under (e) and IGF-1 level was analyzed by ELISA (f), g) cell proliferation, and h) mitochondrial superoxide levels were measured as described in the method section. *p < 0.05, compared to ID8EV. Values were expressed as fold changes (n = 3)

Fig 5: PON2 protein is induced in early stage ovarian cancer.Immunohistochemistry was performed on ovarian carcinoma tissue array slides (US Biomax Rockville, MD, USA) using PON2 antibody as described under the Materials and methods section. PON2 protein expression was quantified using Definiens Tissue Studio Software. Representative photographs of the staining are shown in a, and the quantification of the data is presented in b. Values are expressed as percentage of PON2-positive staining cells. Total protein lysates from matched normal and ovarian tumor tissues [stage I (n = 7), stage II (n = 10), stage III (n = 12), and stage IV (n = 15)] were subjected western blotting analyses using hPON2 antibody. Representative blots (two sets from each stage) are shown in c. The data were quantified using Image J software and presented in d