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. 2013 Dec 13;59(2):505–517. doi: 10.1002/hep.26659

Figure 5.

Figure 5

Preferential accumulation of miR-224 was demonstrated in the autophagosome. Hep 3B cells were treated with amiodarone and chloroquine (CQ) as in Fig. 4A. Protein and RNA were extracted from the whole cell lysate and followed by autophagosome purification. (A) Protein was analyzed by western blotting for LC3, Hsp 60 (mitochondria marker), and calreticulin (endoplasmic reticulum marker) expression. (B) The quantification of miR-224 and let-7a conducted by real-time PCR is shown as mean ± SEM (n = 5). (C) Accumulation of immune-gold labeled miR-224 (18 nm gold beads) in the purified double membrane autophagosomes was detected by miRNA in situ hybridization under TEM. (D) Accumulation of immune-gold labeled let-7a (18 nm gold beads) in the autophagosomes. The arrows highlight gold labeled miR-224 in (C) and let-7a in (D). Scale bar = 100 nm. (E) Quantification was performed by counting the gold particle labeled miR-224 and let-7a in 50 autophagosomes. WC, whole cell lysate; AP, autophagosome fraction.