Figure 6. Chemical induction of R15 expression inhibits signaling and growth of human tumor lines driven by select RAS mutations.

(A–E) Doxycycline (DOX)-inducible, R15-expressing stable lines were generated from RAS mutant tumor cells. ERK-MAPK activation was then measured ± DOX treatment by western blot analysis for pERK levels. The mutant RAS protein expressed in each tumor line is indicated above the panels. Vinculin expression was used as a control for loading.

(A, F, and K) H1915^R15; (B, G, and L) LS1034^R15; (C, H, and M) HuPT3^R15; (D, I, and N) PANC-1^R15; and (E, J, and O) CFPAC-1^R15. Data from additional tumor lines are shown in Figure S4. The experiments were repeated three times for each cell line except H1944^R15 and A375^R15, which were repeated two times.

(F–J) R15 expression reduced the proliferation of a subset of RAS mutant human tumor cells. Results are the average of triplicate wells ± SEM shown by bars.

(K–O) R15 expression reduced anchorage-independent growth of a subset of RAS mutant human tumor cells. Engineered R15 cells were plated on soft agar in the absence (−) or presence (+) of DOX and allowed to grow for 3 to 4 weeks. Graphs represent the average colony number from three wells ± SD. Colonies were counted using NIH ImageJ software. Images are representative wells from each assay. The experiments were done in three technical replicates for each cell line. p values were determined by comparison of colony numbers between −DOX and +DOX conditions using a Student’s t test. Data on additional lines are shown in Figure S4. See Figure S5 for data on the effects of R15 on AKT activation.