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. 2013 May 3;7:56. doi: 10.3389/fncel.2013.00056

FIGURE 2.

FIGURE 2

Methylene blue induces macroautophagy in HT22 cells. (A) Immunocytochemical staining of LC3B showed perinuclear aggregation of LC3B after 24 h treatment of methylene blue. (B) Representative Western blots showed that methylene blue increased both levels of LC3B-I and LC3B-II in a dose- and time-dependent manner. (C) Compared with 6-h serum deprivation, methylene blue robustly increased LC3B-II level. (D) Flow cytometry analysis indicated that chloroquine partially reversed methylene blue-induced anti-apoptosis after serum deprivation. (E) Western blot showed chloroquine increased LC3B-II level and stabilized p62 level in methylene blue-treated cells, suggesting chloroquine inhibited methylene blue-induced macroautophagy. (F,G) Calcein-AM assay demonstrated that methylene blue pretreatment significantly enhanced cell viability after serum deprivation which was blocked by chloroquine. *, p < 0.05; **, p < 0.01. N, normal; U, untreated; SD, serum deprivation; MB, methylene blue; C, chloroquine. n = 3 in each group, each study has been repeated at least three time.