Figure 4
From: Transcription factors NRF2 and HSF1 have opposing functions in autophagy
Knockdown of NRF2 inhibits autophagy. (a) Immunoblotting analysis of NRF2, p62 and LC3B in lysates from U2OS cells, which had been transfected with either control siRNA (siCTL) or NRF2 siRNA (siNRF2) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, and supplemented with 10 nM bafilomycin A1 (Baf-A1) or vehicle (0.1% DMSO) for the last 2 h. The levels of β-actin served as a loading control. For detection of LC3B, proteins from cell lysates were resolved using 13% SDS-PAGE and transferred onto 0.45 µm PVDF membranes, whereas for detection of NRF2 and actin, proteins were blotted onto 0.45 µm NC membranes. (b–d) Real-time PCR analysis of NRF2 (b), HSF1 (c), and p62 (d) from a parallel experiment described in (a) except without Baf-A1 treatment. The mRNA levels of 18 S were used for normalization. The data are represented by mean + SD from three independent transfections. Student’s t-test was used to test for statistical significance, where *represents p < 0.05 when comparing siCTL and siNRF2 for each treatment pair.
