P62, but not NQO1, replicated Nrf2 phenotype.

<p>All western blot images were the representative image from at least three independent analyses, and Actin was use as a loading control. A) NQO1 knockdown couldn't rescue Keap1 knockdown. Cells were transfected with siRNA control (siC), siRNA against NQO1 (siN), siRNA against Keap1 (siK), and their combinations as indicated. 24 hrs later, cells were treated without or with 10% CSE for 24 hrs. Total proteins were collected for western blot analysis. B) P62 knockdown couldn't rescue Keap1 knockdown. Cells were transfected with siRNA control (siC), siRNA against P62 (siP), siRNA against Keap1 (siK), and their combinations as indicated. 24 hrs later, cells were treated without or with 10% CSE for 24 hrs. Total proteins were collected for western blot analysis. C) Effect of P62 overexpression. Cells were transfected with P62 plasmid (P62) or empty vector (PCDNA3, or PC). 24 hrs later, cells were treated without (C) or with (CSE) at increasing doses (8%, 10%, 12%) for 24 hrs. D) overexpression of P62 represses LC3B. Cells stably transfected with GFP-LC3B were transfected with P62 plasmid (P62) or empty vector (PCDNA3, or PC). These cells were treated without (C) or with (CSE) 10% CSE for 24 hrs. Live images were recorded. “punctate dots” of GFP-LC3B were the indication of autophagosomes. E) Quantification of autophagosome (GFP-LC3B+ dots) was described in Materials and Methods. *, $: p<0.05, n = 3.