p62/SQSTM1 knockdown had little effect on the Nrf2-NQO1 pathway in oral cancer cells.

<p>However, p62/SQSTM1 knockdown affected the growth of the cells. (A) p62/SQSTM1 was abundantly expressed in SAS and CAL27 oral cancer cells. HeLa, endocervical carcinoma cells; TIG-108 and 121, normal human fibroblasts. (B) p62/SQSTM1 knockdown was performed by two kinds of shRNAs (sh-p62(1) and sh-p62(2)). shRNA for luciferase was used as control knockdown (sh-control). p62/SQSTM1 expression was significantly decreased by sh-p62(1) and sh-p62(2). Under no irradiation or 10 Gy X-ray irradiation, expressions of Nrf2 (pS40), Keap1, NQO1 and HO-1 were subtly affected by p62/SQSTM1 knockdown. Similar amounts of each cell protein were loaded in each lane of the SDS-PAGE. α-tubulin was used a loading control. Left and right panels indicate the data on SAS and CAL27, respectively. Three independent experiments were repeated, and the blotting photographs are representative ones. (C) p62/SQSTM1 knockdowns and X-ray irradiation were similarly performed. No radiation (upper); The effects of shRNA (sh-p62(1) and sh-p62(2)) were partial under normal culture condition. X-ray irradiations of 5 Gy (middle) and 10 Gy (lower); The cancer cell growth was significantly inhibited by two kinds of shRNA for p62/SQSTM1. Left and middle panels indicate the data on SAS and CAL27, respectively. The data on TIG-121 are displayed as a reference at the right panels. In the WST-8 assays, mean absorbance values (OD450) ± SE are shown vertically, and the number of days after exposure to radiation is indicated horizontally. The values are derived from quadruplicate experiments (*, <i>p</i><0.05, one-way factorial ANOVA and multiple comparison tests accompanied by Scheffe's significance test).