Fig. 3: Inhibition by intracellularly expressed 12VC1 monobody of signaling and proliferation of RAS mutant-driven cancer cells.

a Effects of 12VC1 expression on ERK activation (24 h. induction with 4.3 µg/mL dox in quarter decrements). The numbers under the pERK panel indicate the ratio of pERK signal to the total ERK signal normalized to the no dox sample. Representative data shown, and experiment was reproduced at least one additional time with similar results. b Percent-change of monobody expressing population after 72 h of dox induction relative to 24 h of induction (mean ± SD). A negative percentage signifies a decrease in population. The p-values for the differences between 12VC1 and MB(Neg) expression, as determined with two-tailed unpaired t-test for H358, H23, PATU8902, H441, HPAF-II, A375, and HEK293T are 0.002, <0.001, <0.001, <0.001, 0.9, 0.006, and 0.55, respectively. Error bars represent s.d.; biological replicates, n = 4 for H358 and HEK293T and n = 3 for the other cell lines. c The effects of monobody expression on tumor growth in a mouse xenograft model. Tumors were developed from subcutaneously injected PATU8902 cells that express 12VC1 or MB(Neg) under a dox-inducible promoter. Mouse were given dox-containing feeds on the day indicated with the black arrow through the end of the experiment. Plots show the effect on average tumor sizes over time (n = 5, biological replicates, mean ± SEM). Extracted tumor weights at the end of the experiment are also shown (n = 5, biological replicates, mean ± SEM), average tumor sizes were compared using two-tailed unpaired t-test, p-value = 0.03, 0.71, respectively for 12VC1 and MB(Neg) tumors with and without doxycycline. d Immunoblotting for monobody expression and ERK activation in lysates from each tumor, numbered 1 through 5 at the end of the experiment. The lanes labeled P are the lysates of equivalent cells grown in plastic dishes confirming that these cells exhibited detectable pERK levels and dox-inducible expression of monobodies. The pERK levels of the tumor lysates were quantified relative to that of the cultured cells in the absence of dox (the leftmost lanes). Note that the cells grown in dishes and those in xenograft are distinct samples, and thus the pERK levels between these two types of samples may be substantially different. Immunoblotting has been technically replicated three times with similar results.