Timecourse of gene expression in HeLa cells (parental cells and clonal knockouts of MED12 and MED24) following induction by TNFa or IL-1b

Activation of nuclear factor kappa B (NF-kB) by inflammatory signals results in nuclear translocation of the transcription factor p65 and induction of gene expression. We identified MED12 and MED24 in an optical pooled CRISPR knockout screen in HeLa-Cas9 cells for genes affecting the activation and/or relaxation of p65 to the cytoplasm following induction by either TNFa or IL-1b. We generated isogenic clonal knockout HeLa-Cas9 lines using crRNA transfection and confirmed that loss of MED12 or MED24 results in delayed relaxation of p65, assayed by live-cell imaging of a p65-mNeonGreen fluorescent fusion. Overall design: Biological replicates were defined as replicate clonal knockout cell lines (for MED12 and MED24 knockouts) or replicate stimulations (for parental control cells) (n = 3 or 4 biological replicates).