Figure 4

Mn²⁺-mediates an increase in autophagy, which is protective during Mn²⁺ stress.

(a) SH-SY5Y cells were transfected with tandem fluorescence LC3 containing GFP and RFP for 24 h. Cells treated with 0.5 mM Mn²⁺ for 6 h had a significantly increased (***p < 0.001) number of red LC3 puncta per cell (at least 50 cells per experiment). 0.5 mM Fe²⁺ treatment did not show any significant increase in LC3 puncta and co-treatment of 0.5 mM Fe²⁺ and 0.5 mM Mn²⁺ substantially (***p < 0.001) reduced LC3 puncta compared to Mn²⁺ alone. Treatment with NH₄Cl neutralized lysosome pH, allowing the appearance of yellow LC3 puncta indicating autophagic flux. EBSS was used as a positive control for autophagy induction. Bar chart shows the quantification of average LC3 red puncta per cell for various treatments (n = 3, Student’s t-test). (b) Representative western blot of ATG5^+/+ and ATG5^−/− MEF with or without NH₄Cl treatment. NH₄Cl inhibited autophagy degradation, allowing the accumulation and visualization of the LC3-II protein in normal ATG5^+/+ MEF but not ATG5^−/−. (c) MTT assay of ATG5^+/+ and ATG5^−/− MEF treated with Mn²⁺ for 24 h showing the significant reduction (*p < 0.05, n = 3, two-way ANOVA, Bonferroni post hoc test) in ATG5^−/− cell viability at 2 mM Mn²⁺ compared to ATG5^+/+ MEF. Corresponding phase contrast images of 2 mM Mn²⁺ treatment for 24 h showed similar increased vulnerability of ATG5^−/− MEF.