Figure 4
SETD7 negatively regulates NFE2L2/ARE pathway.
(a–c) Total RNA was isolated from SETD7 knockdown Beas-2B cells to determine the transcription levels of NFE2L2, heme oxygenase 1 (HMOX1) and thioredoxin (TXN). CSE or H2O2 was added to induce oxidative stress. (d,e) NFE2L2 protein in SETD7 knockdown Beas-2B cells was quantified by western blot. H2O2 was used to induce ROS. Relative expression level of NEF2L2 was measured by comparing with the expression of Histone H3 control in each individual experiment. (f–i) Transcription of SETD7, NFE2L2, HMOX1 and TXN was quantified by qRT-PCR using total RNAs isolated from Beas-2B cells with overexpression of SETD7 and H2O2 stimulation. (j) Expression of FLAG-tagged SETD7 plasmid was confirmed by western blot using HEK293 cell lysate after 48 hs transfection. (k) Protein pull down assay was performed using lysates from HEK293 cells cotransfected with FLAG-tagged SETD7 and untagged NFE2L2 wild type vectors. Transfection of FLAG-tagged SETD7 vectors or empty FLAG-tagged vectors (control) was included in the experiments. Detection of NFE2L2 protein was performed with western blot using anti-NFE2L2 primary antibody. (l) in vitro protein pull-down assay was performed using FLAG-tagged human recombinant NFE2L2 protein and GST-tagged human recombinant SETD7 protein. Detection of both proteins was visualized by western blot using anti-SETD7 and anti-NFE2L2 antibodies. Data were expressed as mean ± standard error of the mean (SEM). n = 3. * indicates P < 0.05; ** indicates P < 0.01 (two-way ANOVA).
