Fig. 2: DCAP blocks the autophagic flux without impairing mitochondrial potential. | Cell Death & Disease

Fig. 2: DCAP blocks the autophagic flux without impairing mitochondrial potential.

From: A broad-spectrum antibiotic, DCAP, reduces uropathogenic Escherichia coli infection and enhances vorinostat anticancer activity by modulating autophagy

Fig. 2

a–e Immunoblot analysis of LC3, SQSTM1, AMPK phosphorylated in T172 (pAMPK) and ULK1 phosphorylated in S555 (pULK1) in U2OS cells treated 24 h with three increasing doses of DCAP (1, 5 and 10 μM) or CCCP (5, 10 and 20 μM). GAPDH was used as a loading control. Densitometry analysis of LC3-II (b), SQSTM1 (c), pAMPK (d) and pULK1 (e) is reported as relative protein levels normalized by GAPDH. Vehicle (DMSO) sample value was set to 1 (dotted lines in the graphs). Shown as mean ± s.e.m., n = 3. *P < 0.05; **P < 0.01; ***P < 0.001 DCAP versus CCCP (two-way ANOVA with Bonferroni post test). f Loss of mitochondrial membrane potential (ΔΨm) was quantified by measuring the amount of monomeric JC-10 dye after 24 h of treatment with vehicle (DMSO), 10 µM DCAP or 20 µM CCCP. These doses were adopted as they produced a comparable LC3-II induction (b). Shown as mean ± s.e.m., n = 6. ***P < 0.001, CCCP versus vehicle (one-way ANOVA). g Immunoblot analysis of cytosolic and organelles-enriched (vacuolar) preparations from cells treated 24 h with vehicle (DMSO), 10 µM DCAP or 25 µM chloroquine (CQ). Autophagic LC3-II and lysosomal LAMP1 proteins were used to confirm the enrichment in autophagosomes and autophagolysosomes in vacuolar fractions. Cytoplasmic TUBULIN protein was adopted to evaluate potential cytosolic contaminations in the organelles-enriched fraction. Immunoblot with antibodies against the autophagic receptors, SQSTM1 and NBR1, showed accumulation of these proteins in vacuolar fractions of DCAP- and CQ-treated cells. h Autophagosomal degradation of a SQSTM1 protein fused with a Red Fluorescent Protein (RFP) was observed by live fluorescent microscopy in SQSTM1-RFP expressing cells treated with DMSO, 10 µM DCAP or 25 µM CQ. Representative images before addition of compound (time 0) and at 6, 12, and 24 h post-treatment are shown. DCAP and CQ treatment produced accumulation of SQSTM1-fluorescent dots, overtime. Scale bar in the 24 h frame = 5 µm

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