Figure 1.

EC Liquid or Aerosol Exposure Impairs Autophagy Clearance

(A–C) Autophagy mRFP-EGFP-LC3 reporter images of control, menthol 1% e-liquid, and tobacco 1% liquid NSCs after 4 h. Scale bar, 20 μm.

(D–F) Autophagy reporter images of control, menthol 1% e-liquid, and menthol 6TPE aerosol-treated NSCs after 24 h. Scale bar, 20 μm.

(G and H) Quantification of total autophagosome area after 4 and 24 h of treatment.

(I) Comparison of the number of autophagosomes between 4 and 24 h of treatment. Significance of autophagosome number was determined by comparing the two time points.

(J and K) Size distribution plot revealing an increase in medium and large autophagosomes in 24-h e-liquid- and aerosol-treated NSCs.

(L) Western blot analysis of p62, an autophagy marker revealing increased expression in e-liquid and aerosol treatments.

(M–O) Autolysosome (mApple-pHluorin-LAMP1) reporter images of control, menthol 0.5% e-liquid, and menthol aerosol-treated NSCs after 24 h. Arrows point to individual autolysosomes. Scale bar, 20 μm.

(P) A schematic diagram showing the autophagosome to autolysosome maturation and quenching of pHluorin (green) fluorescence intensity upon lysosome acidification.

(Q) Red-to-green fluorescence ratio of the autolysosome reporter showing a loss of the acidity of the autolysosome.

Asterisks on top of each bar indicate the statistical significance. (*p < 0.05, **p < 0.01, ***p < 0.001). Data are represented as mean ± SEM.