Fig. 3: Validation of cell cycle distribution and endogenous autophagy in WT and ATG7 KO PIP-FUCCI cells with or without nanoparticle treatment.

a Representative flow cytometry data showing thorough cell cycle distribution selected from three independent biological replicates. Cells accumulated at G1 phase, S phase and G2 phase (upper panel for each cell line), as well as M phase (isolated with mitotic shake-off; lower panel for each cell line) were respectively circled in green, red, orange and purple, with corresponding percentages indicated. b Representative transmission electron microscope images of autophagic vesicles indicated by rust-red arrows. N, nucleus. Scale bars, from left to right, 10 μm, 4 μm and 1 μm for WT PIP-FUCCI cells, and 10 μm, 3 μm and 1 μm for ATG7 KO PIP-FUCCI cells. c Statistical analyses of autophagic vesicle numbers per cell. Data were presented as median (IQR; n = 10 cells/group). Two-tailed Mann-Whitney U tests with Monte Carlo approximation were used to compare autophagic vesicle number between WT and ATG7 KO PIP-FUCCI cells within each group: Control (U = 4.500, p < 0.0001, 99% CI [0.000, 0.001], r = 0.795), DiD-LIP (U = 3.000, p < 0.0001, [0.000, 0.001], r = 0.840), DiD-LNP (U = 0.000, p < 0.0001, [0.000, 0.001], r = 0.890). The level of significance was set at p < 0.05. d Western blot analysis of autophagy-related proteins including ATG7, p62 and LC3. Cancer cells were respectively incubated with DiD-labeled liposomes (DiD-LIP, at a dose equivalent to 5 μg/mL DOX) and lipid nanoparticles (DiD-LNP, at a dose equivalent to 6 μg/mL mRNA) for 2 h. The same volume of PBS was used as a control. WT PIP-FUCCI, wild-type U2OS cells stably expressing PIP-FUCCI; ATG7 KO PIP-FUCCI, ATG7 knockout U2OS cells stably expressing PIP-FUCCI. Source data are provided as a Source Data file.