Fig. 1

Dose-responses of Mn on cell viability and autophagy flux. (A) Stable HeLa cells overexpressing mRFP-GFP-LC3, and immortalized rat dopaminergic neuronal cells N27 were exposed to MnCl₂ (62.5 µM to 2mM) for 24 h, or (B) 48 h. Cell viability was evaluated using Calcein AM dye. Data represent mean ± SEM, n = 3 independent experiments with 4 replicates per experiment. (C) Schematic diagram illustrating the autophagy flux pathway and the construct used to create the mRFP-GFP-LC3 stable HeLa reporter cells. With this cell model, autophagosomes appear yellow due to the colocalization of RFP and GFP signals. Red signal indicates the flux is functional because the green signal is quenched by the acidic environment of the lysosomes, which fuse with autophagosomes. These stable cells were treated with vehicle control or Mn (15.6–250 µM) for 24 h. (D) Representative images of cells treated with different doses of Mn were captured using confocal microscopy. (E) Green and red vesicles per cell were quantified using Fiji. Green vesicles represent autophagosomes. The number of autolysosomes was calculated by subtracting the number of green from the red puncta per cell. Scale bar = 20 μm. (F) Stable HeLa cells were treated with MnCl₂ (125 µM), chloroquine (CQ) or both. (G) The number of autophagosomes and autolysosomes were quantified. The combination of Mn with CQ did not significantly alter the numbers of autophagosome (P = 0.9941) and autolysosome(P > 0.9999), as compared to CQ (50 µM) only, a dose that did not completely inhibit autophagy. Data represent mean ± SEM. (H) N27 cells were treated with 62.5 or 125 µM of MnCl₂ for 24 h, then incubated with LysoSensor Yellow/Blue. The ratio of acidic (yellow) vs. neutral (blue) pH of lysosomes were quantified using a plate reader. (I) N27 cells were treated with Mn 125 µM, CQ50 µM or both for 24 h, followed by immunoblotting for Atg5, a marker for autophagosomes, using Jess (ProteinSimple, Inc.) (J) Total protein per capillary was used as loading control to normalize the levels of Atg5. Mn did not further increase the accumulation of autophagosomes when combined with CQ versus CQ alone (P = 0.9998). All data represent mean ± SEM (n = 3–4 independent experiments with approximately 30 cells per experiment for E and G) and groups were compared by using Kruskal-Wallis ANOVA test with Dunn’s post-hoc test; ns: non-significant