Figure 1.
c-Rel Knockout Enhances TNF-α-Induced RelA-Dependent Proinflammatory Gene Expression
(A) Wild-type or c-Rel knockout MEFs (3 × 105 at time of harvest) were treated in a six-well plate with 100 ng/mL TNF-α for 30 min or 3 h.
(B) Wild-type or c-Rel knockout MEFs were cultured and treated as in (A).
(C) Wild-type MEFs were transduced with lentivirus expressing three distinct CRISPR guide sequences against c-Rel, G1, G2 and G3, selected with hygromycin and examined for the knockdown of c-Rel by western blotting.
(D) Pool of four clones from c-Rel CRISPR G1 knockout MEFs was treated as above, and qPCR was performed on indicated genes.
(E) BMDM cells generated from wild-type and c-Rel knockout mice were treated as above, and qPCR were performed on indicated genes.
(F) c-Rel knockout MEFs reconstituted with human-c-Rel by lentiviral transduction (pLM), wild-type (WT), and c-Rel knockout (KO) cell lysates were analyzed by western blotting. pLCγ1 was used as a loading control for whole-cell lysates.
(G) c-Rel knockout MEFs re-expressing human c-Rel (pLM), wild-type, and c-Rel knockout MEFs (4 × 105 at time of harvest) were treated in a six-well plate with 100 ng/mL TNF-α for 30 min or 3 h. Samples were then analyzed by qPCR to determine the abundance of indicated mRNAs relative to that of ribosomal protein L32 (L32).
Data are representative of at least three independent experiments performed in triplicates, presented as mean ± standard error of mean (SEM) (n = 3). p Values were obtained by unpaired Student’s t test; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. See also Figure S1.