Fig 5. Time course of GFP expression in HEK-293S cells at 30°C, 33°C and 37°C.
(A) Point ‘a’ represents the 5th hour after transfection at which the medium was replaced (all the data were collected from cells transfected with 2 μg GFP plasmid/35 mm dish using calcium phosphate). Three sets of experiments began as reflected by three color-coded lines as in panels A-D, and in each set, triplicate dishes were used in our experiments. Specifically, blue line represents maintaining a culture at a 33°C incubator. Red line refers to the culture that first stayed at a 37°C incubator overnight (or 19 hours precisely) and then transferred to the 33°C incubator; this was labeled as point ‘b’ (note that point b correlates to day 1 in panels B-D). Black line represents the control (37°C culture). Point ‘c’ was the time (day 6) that the experiment ended. (B) The number of cells was counted from 34.8x bright-field images taken at days 1, 2, 3, and 6. Each data point was the average cell number counts of three 34.8x images taken at randomly selected locations of a dish. (C) The percentage of green cells in each experiment. Note that all data points were collected from the 34.8x images as in B. Both bright-field and fluorescence images were used for the determination of the transfection efficiency for each experiment. (D) The ratio of the green fluorescence intensity (F/Fday 1). Fday 1 is the fluorescence intensity for each culture at the 24th hour. F stands for the fluorescence intensity at any particular time (day). All of the F/Fday 1 values were collected from the fluorescence images as in (C). (E) The plot of the F/F37°C values for days 1–3. All of the cells used for these experiments were transfected with GFP and maintained at 37°C for one day. Then, one set of the dishes were transferred to 33°C (hollow square), whereas another set dishes were transferred to 30°C (solid square). Therefore, this plot displaces the differential GFP expression on two different temperature scales, i.e., 33°C and 30°C, relative to 37°C.