Fig. 5

Ovarian cancer ascites stimulate MUC16 expression and release through Akt. a Meso 7 cells were incubated with FBS 10% (control), OV401 10% or 3 different ovarian cancer ascites at 10%. The expression of Akt and phosphorylated Akt were determined by immunoblot using anti-Akt and anti-phospho-Akt antibodies. Representative images from two independent experiments. Densitometric quantification of phosphorylated Akt normalized to total Akt is represented between the two blots. b OVC346 ascites was heat-inactivated by heating at 100 °C for 10 min followed by centrifugation at 13,000 rpm for 15 min. Meso7 cells were incubated with OVC346 (10%) or heat-inactivated OVC346 ascites (10%). Akt and phosphorylated Akt expression were assessed by immunoblot after 24 h. Expression of phosphorylated Akt was normalized to total Akt and densitometric quantification is shown between the blots. Representative images from two independent experiments. c Meso 7 cells were incubated with increasing concentrations of OVC346 ascites (0 to 10% v/v) and Akt and phosphorylated Akt expression were determined by immunoblot. d Meso7 cells were incubated with OV401 (10%) or ovarian cancer ascites (10%) in the presence or absence of Akt1/2 inhibitor (5 μM). MUC16, Akt and phosphorylated Akt expression were determined by immunoblot. Representative images from two independent experiments. Expression of phosphorylated Akt was normalized to total Akt and MUC16 expression was normalized to housekeeping gene GAPDH. e MUC16 release was measured in supernatants of Meso7 cells incubated with three different ovarian cancer ascites in the presence or absence of Akt1/2 inhibitor (5 μM). Bars represent standard deviation from two independent experiments conducted in duplicates. f Meso7 cells were incubated with OVC346 or OVC690 (10%) in the presence or absence of Akt1/2 inhibitor. MUC16 mRNA levels were quantified by ddPCR. MUC16 concentration data from ddPCR experiments are expressed as ratio against reference genes for each sample. Results are from two independent experiments conducted in duplicates. g Meso7 cells were incubated with benign fluid OV401 or ovarian cancer ascites OVC346 or OVC690 (10%). Expression of phosphorylated FAK, phosphorylated and total c-Src was determined by immunoblot. Representative images from two independent experiments. Densitometric quantification of phosphorylated FAK normalized to GAPDH is shown between the blots. h Cells were incubated with β1 or β5 integrin-blocking antibodies (5 μg/ml) for 30 min before adding 10% OVC508 ascites overnight. Cells were then washed 3 times and fresh FBS and hormone-free medium was added. MUC16 release was then measured in supernatants after 24 h. Data are from a single experiment