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. 2012 Mar 1;8(3):297–309. doi: 10.4161/auto.18658

Figure 5.

Figure 5.

BNIP3-induced autophagy is associated with reduced lysosome abundance, which is restored by co-expression of TFEB. (A) Representative epiflourescence images (630X) demonstrating lysosome distribution (by LysoTracker red staining) in cells adenovirally transduced with Bnip3, TFEB (green, at 100 MOI), BNIP3+TFEB (at 100 MOI each) for 48 h. Nuclei are blue (Hoechst dye). Adenovirus coding for LacZ expression was added as necessary to result in equivalent MOIs (at total 200 MOI per treatment). (B) Flow cytometric analysis of LysoTracker red staining in cells treated as in (A). Control is depicted in black, BNIP3 in green, TFEB in red and BNIP3+TFEB in blue. (C) Assessment of LysotTracker red expression by flow cytometry in NRCMs expressing BNIP3, BNIP3 ΔTM, TFEB, BNIP+TFEB for 48 h; and in BNIP3 expressing cells treated for 24 h with 3MA (7 mmol/L). (D) Representative immunoblots demonstrating LC3, p62, BNIP3 (FLAG), BNIP3ΔTM (HA), TFEB (both rat TFEB and HA tagged human TFEB) and LAMP1 expression, with α-sarcomeric actin (αSA) in NRCMs adenovirally transduced with LacZ (control), BNIP3, BNIP3ΔTM, TFEB and BNIP3+TFEB as in (A) (for 48 h). (E–H) Quantitative assessment of LC3-II/α-sarcomeric actin ratio (E), total LC3 (F), p62 (G) and LAMP1 (H) abundance in NRCMs treated as in D (n = 3–7/group). p values are by post-hoc test.