Fig 4. Assessment of the effect of combining autophagy inducers on chemosensitivity in CT26 murine colorectal cells.

(A) The induction of autophagy in CT26 cells, following lithium treatment (30 mM) for 24 hours, was assessed with the Cyto-ID autophagy detection kit. (B) Viable cells following treatment with lithium alone or in combination with 5-fluorouracil (5-FU) (20 μM) for 24 hours were counted and equal numbers reseeded in triplicate, in the absence of drug. (i) Cells were allowed to grow for 14 days, then were fixed and stained and colonies quantified using the Odyssey Infrared Imaging System (Li-COR). (ii) Data is presented graphically as the mean +/- SEM of three independent experiments. Asterisks indicate a significant difference in the number of colonies formed in combination treated cells compared to single agent treated cells (* p < 0.05) (unpaired t-test). (C) Morphological features of CT26 cells were examined following treatment for 24 hours. Panels to the right show the morphology induced in response to lithium alone (upper) and in combination with 5-FU (lower). Lower left panel shows morphology in cells treated with 5-FU alone. Arrows indicate the accumulation of vesicles in lithium, 5-FU and combination treated cells (Magnification 40x). (D) Cells were treated with either lithium (30 mM), 5-FU (20 μM) or a combination of both for 24 hours and autophagy levels assessed with the Cyto-ID autophagy detection kit. Representative image of FACS analysis with insert showing corresponding mean fluorescence intensity (n = 3).