Fig. 2: MEK inhibition induces GATA6-dependent MHCI upregulation.

a Nine primary murine PDAC cell lines were established from spontaneous murine PDAC models (CKP, KPC, and KPCY). Created with BioRender.com. Flow cytometry analysis was used to determine GATA6 protein expression in each line. Representative histograms show GATA6 staining (GATA6 antibody, GATA6 AB, red) versus isotype control (blue) in GATA6 high (GATA6^hi) and low (GATA6_lo) expressing cell lines. The cell lines were dichotomized into GATA6^high (n = 5, orange) and GATA6_low (n = 4, blue) groups. b The parental cells (Ctrl) of three GATA6^high cell lines (60400, 511950, 70301) and three GATA6_low cell lines (60590, 60531, 511892) were treated with increasing doses of MEKi (trametinib) until 100x of the cells’ initial IC50 to generate the corresponding long-term MEKi-treated cells (MEKi). Transcriptomic profiling by RNA sequencing of the parental (Ctrl) and long-term MEKi-treated (MEKi) cells was performed. GSEA of KEGG and HALLMARKs revealed gene sets that were significantly (P-adjust < 0.05) different between parental (Ctrl) and long-term MEKi-treated (MEKi) cells within GATA6^high and GATA6_low groups, respectively. c Surface MHCI (H-2Db) expression on control (Ctrl) and MEKi-treated (MEKi) cells of GATA6^high (upper panel) and GATA6_low group (lower panel) was assessed by flow cytometry (n = 4 biological replicates for cell line 60400, 511950, 511892, 60590, and 5 biological replicates for cell line 110299, 2838c3, 60531, 6694C2). MFI mean fluorescence intensity. Mean ± SD is shown. Statistical significance was calculated by unpaired two-tailed Mann–Whitney test. d Flow cytometric analysis of GATA6 expression in GATA6 knockout (KPCY1-CRISPR-GFP v2.1-gRNA-gCas9): gGATA6_KO-3 and gGATA6_KO-4, and negative control (gNT) cell lines (n = 4 biological replicates). Statistical significance was calculated by unpaired two-tailed Mann–Whitney test. e Surface MHCI (H-2Db) expression on GATA6 knockout cell lines and negative control upon MEKi treatment (n = 4 biological replicates). MFI: mean fluorescence intensity. Mean ± SD is shown. Statistical significance was calculated by unpaired two-tailed Mann–Whitney test. f Schematic of the knock-in strategy for N-terminal AID-tagged gata6. Shown are components of the knock-in cassette, and the positions of primers for genomic PCR are marked by arrows. g Western blot showing GATA6 protein expression in 110299^WT and 110299^AID-GATA6 cells after 24 h treatment with 1 µM 5-Ph-IAA (n = 1 biological replicates). h Surface MHCI (H-2Db) expression on 110299^WT and 110299^AID-GATA6 cells treated with or without MEKi and/or 1 µM 5-Ph-IAA (n = 5 biological replicates). MFI: mean fluorescence intensity. Mean ± SD is shown. Statistical significance was calculated by One-way ANOVA and Kruskal–Wallis test.