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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: Nat Cell Biol. 2021 Apr 1;23(4):377–390. doi: 10.1038/s41556-021-00654-5

Extended Data Fig. 7. EGFR plays a central role in generating pulsatile ERK dynamics in PDO-KRASG12V .

Extended Data Fig. 7

a, Fluctuating ERK dynamics in PDO-KRASG12V, abolished by afatinib at 50nM concentration. Shown traces are representative for 14 single-cell analyses from 3 PDOs. Experiment performed twice.

b, As a, loss of ERK activity oscillations, observed in PDO-KRASG12V upon administration of pan-HER inhibitors lapatinib (1μM, 21 cells), dacometinib (500nM, 12 cells), or EGFR-specific inhibitors erlotinib (500nM, 17 cells) and gefinitib (500nM, 18 cells). Two independent experiments performed. ~5 organoids per condition.

c, As a, loss of ERK oscillations, observed in PDO-KRASG12V upon anti-EGFR antibody cetuximab (500 ng/ml). Residual ERK activity was sensitive to trametinib (MEKi). Experiment performed once; 30 cells analyzed.

d, Oscillating ERK dynamics persist in PDO-KRASG12V despite HER2-inhibitor CP-724714 (5μM). Three traces representative for 24 single-cell analyses, from one experiment with 4 organoids.

e, FRET-trace demonstrating that afatinib (200nM) instantly interrupts rising phase of pulsatile ERK (arrow). Representative for >20 observations in various PDO-lines.

f, Shp2-inhibitor SHP099 (5μM) abrogates autonomous ERK activity in PDO-KRASG12C, but not BRAFV600E(#4). Shown are representative multi-cellular z-plane analyses. Experiment performed once; 3 organoids. f’ BRAFV600E(#4) is similarly unresponsive to drugs targeting Src (KX2, 500nM), FAK (PND-1186, 500nM) and cKit/PDGFR/Bcr-Abl (dovitinib, 100nM; imatinib, 1.0 μM; masitinib, 100nM; pazopanib, 250nM). Shown are 2x-normalized ratios (mean±s.d.). n numbers represent PDOs and are indicated in the graph per group. Experiment performed once.

g, Adapted pInducer20 for doxycyclin-inducible expression of HRASN17 and P2A-coupled reporter mKate2-NLS (TRE2, Tet-Responsive Element). Western blot demonstrating doxycyclin-mediated induction of HRASN17 expression (general anti-RAS antibody) in BRAFV600E(#3)(EKAREN5+pInducer). S.E., short exposure; L.E., long exposure. Vinculin as loading control. Experiment performed once.

h, Western blot analyses on indicated PDOs illustrating pan-HER inhibition on components of the linear EGFR-MAPK-pathway. Pharmacological treatments as in Fig. 4e. Experiment performed once.