Extended Data Figure 7. MAPK-PSD4-ARF6 axis mediates KRAS*-dependent SDC1 membrane localization.

a, Cells were grown in presence (ON) or absence (OFF) of doxycycline or treated with AZD8330 (100 nM) for 16-18 hours. (Top) ARF6 activity was measured with GGA3-PBD pull down assay. GTP and GDP used as positive and negative controls, respectively. (Bottom insert) ARF6 activity was calculated as ratio of captured Arf6:input Arf6/vinculin.

b, iKras p53^L/+ tumor cells were grown in the presence (ON) or absence (OFF) of doxycycline, or treated with AZD8330 (100 nM) for 16-18 hours. Cell lysates were used for measurement of PIPK activity (n=3 biological replicates; Data are mean + s.d.). P-values were determined by unpaired two-sided Student’s t-test.

c, Representative images of morphology change in iKras p53^L/+ tumor cells with dominant negative Arf6 (Arf^T27N) or constitutively active Arf6 (Arf^Q67L). Experiments were repeated 3 times with similar results.

d, iKras p53^L/+ tumor cells stably expressing Arf6^Q67L or its empty vector (Vec) were grown in the presence (ON) or absence (OFF) of doxycycline for 48 hours and surface Sdc1 were measured with FACS using anti-Sdc1 antibody (Top). The fluorescence intensity of surface SDC1 is shown (Bottom) (n=4 biological replicates; Data are mean + s.d.). P-values were determined by paired two-sided Student’s t-test.

e, iKras p53^L/+ tumor cells stably expressing Arf6^T27N or empty vector (Vec) were grown in the presence (ON) or absence (OFF) of doxycycline for 48 hours and surface Sdc1 were measured with FACS using anti-Sdc1 antibody. Representative histograms (top) and bar figure of fluorescence intensity were shown (bottom) (n=3 biological replicates; Data are mean + s.d.). P-values were determined by paired two-sided Student’s t-test.

f, mRNA expression of GAPs and GEFs of Arf6 in iKras p53^L/+ tumor cell microarray dataset upon Kras^G12D inactivation (n=4 biological replicates). P-values were determined by unpaired two-sided Student’s t-test.

g, iKras p53^L/+ tumor cells were grown in the presence (ON) or absence (OFF) of doxycycline, or treated with Trametinib (50 nM) for 16-18 hours and Psd4 mRNA level was measured by qPCR (n=3). P-values were determined by unpaired two-sided Student’s t-test.

h, MiaPaCa2 cells harboring doxycycline-inducible shRNA targeting human KRAS were grown in the absence (OFF) or presence (ON) of doxycycline, or treated with Trametinib (50 nM), AZD8330 (50 nM), or BKM120 (100 nM) for 18 hours. Cell lysates were blotted for PSD4, phosphor-ERK and KRAS. Arrow: band for PSD4. Experiments were repeated twice with similar results.

i, iKras p53^L/+ tumor cells stably expressing Psd4 or its empty vector were grown in the presence (ON) or absence (OFF) of doxycycline for 48 hours. ARF6 activity was measured by GGA3-PBD pull down assay. Input lysates were immunoblotted to validate expression of Arf6, p-Erk, p-Mek, Psd4, and Kras. Experiments were repeated twice with similar results.

j, iKras p53^L/+ tumor cells stably expressing Psd4 or its empty vector (Vec) were grown in the presence (ON) or absence (OFF) of doxycycline for 48 hours and surface Sdc1 was measured by FACS using anti-Sdc1 antibody. Representative histograms of FACS analysis (top) and bar figure of fluorescence intensity of surface Sdc1 (bottom) were shown in the plot (n=3 biological replicates; Data are mean + s.d.). P-values were determined by paired two-sided Student’s t-test.