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. 2021 Oct 17;18(6):1318–1337. doi: 10.1080/15548627.2021.1974175

Figure 3.

Figure 3.

Decrease of SQSTM1 blocked autophagic flux in keratinocytes. (A) RT-qPCR analysis of SQSTM1 mRNA expression levels in control and SQSTM1 knockdown HaCaT cells (n = 3 independent experiments). (B, C) western blot analysis of SQSTM1, KEAP1, and NFE2L2 expression levels in control and SQSTM1 knockdown HaCaT cells. Quantification results of protein expression are shown (n = 3 independent experiments). (D, E) western blot analysis of KEAP1 and NFE2L2 expression levels in HaCaT cells treated with high glucose (30 mM) or mannitol (osmotic control) for 72 h. Quantification results of protein expression are shown (n = 3 independent experiments). (F, G, and H) the HaCaT cells were infected with adenovirus harboring tandem fluorescent mRFP-GFP-MAP1LC3B/LC3B for 24 h, followed by transfection with siRNAs in normal-glucose culture medium. Representative images of the HaCaT cells expressing mRFP-GFP-MAP1LC3B/LC3B are shown. Green: GFP puncta, red: mRFP puncta, scale bar: 10 μm. Semi-quantitative analysis of autophagosomes (AP, yellow puncta in merged images) and autolysosomes (AL, red-only puncta in merged images). n = 10 randomly selected fields/conditions from 3 independent experiments. The data were presented as mean ± s.d. and analyzed by student’s t-test (E), or one-way ANOVA with Fisher’s LSD post hoc analysis (A, C, and H), or one-way MANOVA with Fisher’s LSD post hoc analysis (G). *P < 0.05, **P < 0.01, ***P < 0.001.