Fig. 4

Effect of multifunctional nanoparticles on autophagosome formation. a Signals of LC3B puncta detected in 4T1 cells cultured in RPMI-1640 medium with different formulations for 4 h (a 5% glucose solution was used as control), as determined by immunofluorescence assay with an anti-LC3B antibody, followed by confocal microscopy. LC3B-positive puncta were detected in the cytoplasm by fluorescein-conjugated ImmunoPure goat anti-rabbit IgG (green). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Enlarged boxes highlight LC3B signals. Scale bar: 5 μm. b Number of LC3B-positive puncta per cell quantified from ~20 cells treated with different formulations. The means ± SD are from 3 independent experiments. One-way ANOVA was performed. *P < 0.05; **P < 0.01. (n = 3 independent experiments.) c Transmission electron micrographs of autophagosomes in the cytoplasm of HeLa cells treated with CpG+OVA+PTX-N/A. Scale bar, 500 nm. d Confocal microscopy images of HeLa cells transfected with the autophagy dual fluorescent reporter mCherry-GFP-LC3B and cultured with different formulations in Dulbecco’s modified Eagle medium for 4 h. Scale bar: 5 μm. e Number of autophagosomes and autolysosomes in HeLa cells treated with different formulations (>15 cells/experiment). Co-localized dots were counted. Data are presented as means ± SD. * P < 0.05, ** P<0.01 (n = 3 independent experiments). f Representative Western blots for LC3B-II and SQSTM1. g Relative protein levels of LC3B-II and SQSTM1, normalized to levels of GAPDH. Cells without any treatment were set as the control group. Data are shown as mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01. CpG, immunopotentiator; CQ, chloroquine; N, nanoparticles; N/A, nanoparticles coated with atezolizumab; OVA, ovalbumin; PTX, paclitaxel; S, solution