(A) Illustration of the knockin of a T2A-EGFP cassette into the GATA6 locus of HPAF-II cells. CDS, coding sequence. (B) EGFP transcript faithfully reflected the response of GATA6 transcription to MEK/ERK inhibition. HPAF-II GATA6-T2A-EGFP cells were treated with DMSO or 100 nM trametinib for 24 h, followed by RT-qPCR analysis. n = 2 biological replicates × 2 technical replicates/condition. (C) MEK/ERK inhibition upregulated EGFP abundance. HPAF-II GATA6-T2A-EGFP cells were treated with DMSO or the indicated inhibitors for 4 days and analyzed by flow cytometry. The MFI values of EGFP are shown. (D) Illustration of the genome-wide CRISPR screening performed in HPAF-II GATA6-T2A-EGFP cells. The top 20% GFP^hi and bottom 20% GFP^lo cell populations were sorted out for analysis. (E) Positive controls validated the CRISPR screen. sgRNAs targeting GATA6 or EP300 were depleted in the GFP^hi population. For each gene, all 4 individual sgRNAs are shown. (F) Genome-wide CRISPR screening identified positive and negative regulators of GATA6 transcription. 19,114 genes were ranked based on the gene-level, log₂-transformed fold change of sgRNAs comparing GFP^hi versus GFP^lo groups. The gene-level values were calculated for each gene using the MAGeCK algorithm combining all sgRNAs targeting that gene. No.[good sgRNAs] is the number of sgRNAs that showed consistent depletion or enrichment of that gene. (G) Cross-referencing CRISPR screen results with additional datasets identified JUNB as an ERK-regulated transcriptional repressor of GATA6. Genes annotated as transcription factors or ERK substrates were extracted from references 36 and 37. Genes whose protein abundance decreased upon ERK inhibition were extracted from reference 29. Numbers in parentheses refer to the number of genes in each dataset.