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Fig. 2 | BMC Cancer

Fig. 2

From: Ascites from ovarian cancer patients stimulates MUC16 mucin expression and secretion in human peritoneal mesothelial cells through an Akt-dependent pathway

Fig. 2

Ovarian cancer ascites stimulates MUC16 expression and release in HPMCs. a Meso7 HPMCs were cultured for 24 h in the presence of different ascites (10% v/v), TNFα (25 ng/ml) or FBS 10% (control). Cell lysates were obtained and immunoblot were performed to assess MUC16 expression. Tubulin was used as a loading control. b Immunofluorescence detection of MUC16 in Meso7 HPMCs. Meso7 cells were primed with OVC439 and OVC690 ascites (10% v/v) for 24 h and cells were fixed with 3.7% formaldahyde, permeabilized with 0.1% triton and stained with anti-CA125 M11 antibody (left panel). OVCAR3 cells were treated similarly. Magnification of Meso7 treated with OVC690 ascites and OVCAR3 cells stained with anti-CA125 M11 antibody (right panel). c Schematic representation of HPMCs experimental approach for the measurement of MUC16 in conditioned medium. HPMCs were incubated in medium containing 10% FBS until confluence was reached. Cells were then washed, starved for 4 h, treated with different ascites or benign fluids as indicated and incubated overnight. Cells were then washed 3 times and incubated in serum-free medium for an additional 24 h or more. The conditioned medium was collected, centrifuged and the supernatants were assessed for MUC16 detection. d Levels of MUC16 in the supernatant of conditioned medium of treated Meso7. Levels were standardized according to the total protein concentration. Results are from two independent experiments performed in duplicates. * indicates P <  0.001. e Ascites were heat-inactivated by heating at 100 °C for 10 min followed by centrifugation at 13,000 rpm for 15 min. HPMCs were incubated with inactivated ascites or medium containing 10% FBS (control). Results are from two independent experiments performed in duplicates. f Meso7 cells were starved in medium without FBS nor hormones for 4 h and then incubated 4 or 8 h with medium containing either 10% FBS (control), 10% benign fluid OV401 or 10% ascites (OVC346, OVC690). MUC16 mRNA levels were quantified by ddPCR. MUC16 concentration data from ddPCR experiments are expressed as ratio against reference genes for each sample. P > 0.05. Results are from a single experiment performed in triplicates. g Immunohistochemistry of MUC16 on human omentum section, in benign disease versus ovarian cancer conditions (Picture taken at 10X magnification, left panel). The counterstaining was done with haematoxylin to allow nuclei visualization. Representative images from omental biopsies from two ovarian cancer and one patient with a benign disease were obtained and multiple section were analyzed. Enlarged representative images (40X magnification; right panel) shows the localisation of MUC16 at the mesothelium in ovarian cancer omentum

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