Fig. 3: The light dose-dependence and reversibility of POT induced endogenous protein degradation.

a COS-7 cells expressing POT-PI3K were illuminated with 488 nm laser (5 mW, 100 ms every 1 min) by FRAP. The FRAP area was within the marked Cell 1 with a circle at the diameter of 4 pixels (0.6 μm). The Cell 2 was in close proximity to the illuminated area, whereas Cell 3 was far distant. Scale bar: 10 μm. b The mCherry fluorescence in Cell 1-3 was quantified by live cell imaging (n = 3 cells). c The reversibility of POT was demonstrated through live-cell imaging in COS-7 cells expressing POT-PI3K. After 488 nm light stimulation, POT-PI3K formed clusters. The images of the recovery process were captured every minute without blue light exposure and representative time points were shown. Scale bar, 5 μm. d The formed protein clusters were measured and plotted with the conditions indicated (n = 17 cells). e The reversibility of POT-PI3K was demonstrated through immunoblotting in COS-7 cells overexpressing POT-PI3K. Cells were treated as follows: 16 h of dark (lane Dark), 12 h of dark and 4 h of pulsed blue light (1 s every 6 s, lane Light1), 4 h of pulsed blue light and 12 h of dark state (lane Recovery), and 16 h of pulsed blue light (lane Light2). f The normalized densitometric PI3K 110α and POT-PI3K over GAPDH ratio in three independent experiments. All data were presented as mean ± SEM. Statistical significance is based on two-tailed Student’s t-tests (b) or One-way ANOVA tests (d, f) and is represented with labels, ns (P > 0.05), *(P ≤ 0.05), **(P ≤ 0.01), ***(P ≤ 0.001), **** (P ≤ 0.0001).