Extended Data Fig. 2: Genetic depletion of Mek1/2 in established PDAC.
a, Genetic strategy to delete Mek1 and Mek2 by 4-hydroxytamoxifen (4OHT)-mediated CreERT2 activation. Pdx1-Flp;FSF-KrasG12D/+;FSF-R26CAG-CreERT2/+ mice were crossed with mice harboring loxP-flanked Mek1 and Mek2 alleles. This allowed MEK1/2 deletion in established PDAC by tamoxifen administration in vitro and in vivo after orthotopic transplantation. b, Genotyping PCR of PDAC cells with indicated genotypes to analyze recombination of the floxed Mek1 allele. Non-recombined mutant, recombined mutant and wild-type PCR products are indicated on the right side. Representative gel of three independent experiments. c, Western blot analysis of MEK1 and MEK2 expression in primary PDAC cell cultures with indicated genotypes after 4 days of tamoxifen (4-OHT) and vehicle (ethanol, EtOH) treatment. HSP90 served as loading control. Representative gel of three independent experiments. d, Clonogenic assays of mPDAC cells with indicated genotypes. Control cells treated with vehicle (ethanol; EtOH) are shown in the upper row, 4-OHT treated cells in the lower row. e, Schematic representation of the experimental set-up to test the effect of Mek1/2 knockout in vivo by tamoxifen administration using syngeneic immunocompetent PDAC models. mPDAC cells with conditional Mek1lox/lox;Mek2lox/lox alleles were used for the orthotopic transplantation experiments. f, Waterfall plot showing tumor response of vehicle and tamoxifen treated animals after one week of treatment (fold-change compared to baseline before treatment based on MRI-volumetric measurements, y-axis). Each bar represents one mouse. P values calculated with two-tailed unpaired t test. g, Kaplan-Meier survival curve of vehicle and tamoxifen treated PDAC models. Number of mice is indicated in the corresponding panels. P value was calculated with log-rank (Mantel-Cox) test. h, Representative images of HE and IHC for MEK1, MEK2 and KI67 of tissue sections of tumors from orthotopically transplanted Mek1lox/lox;Mek2lox/lox models treated with vehicle or tamoxifen. Representative pictures of three independent experiments. Scale bars, 100 µm.
