Figure 5
Glutamine deprivation increases macropinocytosis (A) Macropinocytosis was assessed in BxPC3 and MIAPaCa2 cells by monitoring the uptake of FITC-Dextran under either glutamine-replete or glutamine-free conditions. Scale bars, 20 μm. (B) The relative macropinocytic uptake of BxPC3 and MIAPaCa2 were quantified by image-based determination of the total macropinocytic vesicle area compared with the DAPI-stained area of cells. Data are expressed relative to the values of BxPC3 observed in the glutamine-free conditions. Data are shown as the mean of five images in each experiment ± SEM. (Error bars represent the SEM of values from five representative images per experiment) (C) Macropinocytic uptake of MIAPaCa2 cells induced by glutamine deprivation was inhibited by the treatment with EIPA (50 μM). (D) The levels of macropinocytic uptake of MIAPaCa2. MIAPaCa2 were quantified by image-based determination as shown in (B). Data are expressed relative to the values of MIAPaCa2 in the glutamine-free conditions. (E) MIAPaCa2 cells were cultured in glutamine deprivation medium for 6 days, either with or without 2% BSA and further with EIPA treatment. Growth levels were measured by cell counting. Data are expressed relative to the values observed in the glutamine-free conditions. (F) MIAPaCa2 cells expressing ATG7 shRNA and control shRNA were cultured in glutamine-free medium for 16 h after treatment with FITC-Dextran and macropinocytic degradation was determined by monitoring intracellular FITC-Dextran. Scale bars, 20 μm. (G) Levels of macropinocytic uptake of MIAPaCa2 cells were quantified by image-based determination as shown in (B). Data are relative to the values of MIAPaCa2 cells cultured in glutamine-free conditions. Error bars represent the SEM of triplicates from a representative experiment. *p < 0.05.
