Extended Data Fig. 3. Pharmacologic and genetic modulation of drug sensitivity in classical and mesenchymal PDAC cell cultures.
a, b, Clonogenic assays of a representative human (left) and mouse (right) PDAC cell culture showing antagonism to the trametinib/nintedanib (T/N) combination. Cell cultures were treated with indicated concentrations of T/N.
c, Western blot of phospho-ERK and ERK in T/N (10 nM trametinib + 2 µM nintedanib) and vehicle treated classical and mesenchymal primary mPDAC cell lines. HSP90 served as loading control. Classical cell lines are marked in yellow, mesenchymal in blue. Representative gels of three independent experiments.
d, Clonogenic assays using increased drug concentrations of the T/N combination of three of the most antagonistic cell lines, as depicted in figure 2, panel (g).
e, Doxycycline-induced overexpression of KRASG12D in mouse PDAC cells. 2259 mPDAC cells representative of the classical subtype was transduced with lentivirus carrying doxycycline-inducible KRASG12D or GFP-control expression constructs. KRASG12D or GFP expression were induced by doxycycline (100 ng/ml) for one or 14 days.
f, Western blot of phospho-ERK and total ERK in cells overexpressing KRASG12D or GFP for one day. HSP90 served as loading control.
g, Expression of the marker gene Cdh1 for epithelial cell differentiation was evaluated by qRT-PCR (normalized to Cyclophilin B). Data are shown as mean ±SD; n=3 biological replicates. P value was calculated with two-tailed unpaired t test.
h, Representative picture of three independent experiments of the morphological changes of PDAC cells upon KRASG12D induction for one or 14 days of doxycycline treatment. Scale bars, 200 µm.
i, Representative clonogenic assays of mPDAC cells treated with the indicated concentrations of trametinib and nintedanib upon KRASG12D (right panel) or GFP (left panel) overexpression.
j, Bliss synergy scores for the mPDAC cell line treated with the combination of trametinib and nintedanib upon KRASG12D or GFP overexpression.