Figure 4. Pharmacological inhibition of the autophagic process with 3-methyladenine or E64/pepstatinA protects cultured cortical neurons against kainate-hypoxia treatment. (A) Representative immunoblots against LC3 and SQSTM1 and the corresponding quantifications confirm that 3-methyladenine (3-MA) prevents accumulation of LC3-II (KaHx: 164 ± 14%, 3-MA KaHx: 95 ± 14; Welch ANOVA, n = 5 independent experiments) and reduction of SQSTM1 (KaHx: 65 ± 8%, 3-MA KaHx: 110 ± 12%; Welch ANOVA, n = 5 independent experiments) following exposure to KaHx. (B and C) Quantification of PI-positive nuclei as a percentage of all nuclei (Hoechst-stained) demonstrates that inhibition of the autophagic process with (B) 3-MA (ct: 6.6 ± 1.4, 3-MA: 8.4 ± 1.9, KaHx: 54.8 ± 2, 3-MA KaHx: 24.6 ± 4.8, Steel-Dwass test, n = 3 independent experiments) or (C) E64/PepstatinA (PepA) (ct: 7.9 ± 0.1, E64/PepA: 12.5 ± 1, KaHx: 60 ± 3.8, E64/PepA KaHx: 26.7 ± 7.4, Tukey-Kramer test, n = 4 independent experiments) is neuroprotective. Values are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.