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. 2020 Jan 10;16(11):1949–1973. doi: 10.1080/15548627.2020.1712108

Figure 1.

Figure 1.

SQSTM1 activates autophagy in response to lipotoxicity. (A) Sqstm1 WT or sqstm1 KO MEFs were incubated with PA (500 μM) for indicated times and subjected to immunoblot analysis with antibodies against SQSTM1, KEAP1, ACTB (loading control), nuclear (N) NFE2L2, and LMNB1 (nuclear marker). (B) The densitometric analysis of KEAP1 immunoblots. (B–C) Total mRNA isolation from cells were treated as described in (A) and subjected to qRT-PCR analysis for relative mRNA expression of Keap1 (B), Gsta1, Hmox1, and Nqo1 (C). (D) Sqstm1 WT or sqstm1 KO MEFs were incubated with PA (500 μM) and BafA1 (10 nM) and subjected to immunoblot analysis with antibodies against SQSTM1, KEAP1, LC3B, and ACTB (loading control). (E) Densitometric analysis of LC3B-II immunoblots. (F) GFP-LC3B fluorescence analysis of puncta by confocal microscopy using GFP-LC3B HeLa cells transfected with control siRNA (siCON) or SQSTM1 siRNA and treated with PA for 18 h. The representative single optical sections and merge images are shown. Scale bar: 10 μm. (G) Quantitative analysis of GFP-LC3B puncta. (H) GFP-LC3B fluorescence analysis of puncta by confocal microscopy using GFP-LC3B HeLa cells infected with Ad-SQSTM1 for 18 h. The representative single optical sections and merge images are shown. Scale bar: 20 μm. (I) Quantitative analysis of GFP-LC3B puncta. (J) Immunoblot analysis with antibodies against GFP, SQSTM1, KEAP1 and ACTB. (K) Densitometric analysis of GFP-LC3B-II immunoblots. (L) The colocalization of Lysotracker and LC3B by confocal microscopy using Ad-SQSTM1-infected cells. The representative single optical sections and merge images are shown. Scale bar: 10 μm. (M) Quantitative analysis of colocalization. (N) Hepa1c1c7 cells were transfected with mRFP-GFP-LC3B plasmids and infected with Ad-SQSTM1. The representative single optical sections and merge images are shown. Scale bar: 10 μm. (O) Quantitative analysis of RFP-LC3B and YFP-LC3B. Data are presented as the mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and N.S., not significant