Supplementary figures from A Raf-Competitive K-Ras Binder Can Fail to Functionally Antagonize Signaling
Authors: Kauke Monique J. , Tisdale Alison W. , Kelly Ryan L. , Braun Christian J. , Hemann Michael T. , Wittrup K. Dane 
Published: April 2023

<p>Supplementary Figure S1. Cell lines constitutively express R11.1.6 or control proteins YW1 and EGFP. a, Flow cytometry histograms of human cancer cell lines A431, HT-29, LS180, and HPAF-II, transduced to constitutively express EGFP, R11.1.6 (and an EGFP reporter), YW1 (and an EGFP reporter), or fusions of EGFP to R11.1.6/YW1. b, Expression of R11.1.6/YW1, as measured by quantitative RT-PCR, in cancer cell lines transduced with R11.1.6/YW1 and the EGFP reporter, confirming that not only are these cell lines expressing the EGFP reporter, as seen in a, but also the R11.1.6/YW1 proteins. Expression levels are normalized to beta actin (ACTB). Supplementary Figure S2. Cell lines constitutively expressing R11.1.6 form colonies in a clonogenic assay, do not preferentially respond to PI3K inhibitors, and proliferate. a, Images of colony formation of cancer cell lines A431, HT-29, LS180, and HPAF-II constitutively expressing R11.1.6, YW1, or EGFP. b, Proliferation after 24 hours of cancer cell lines HT-29, LS180, and HPAF-II constitutively expressing EGFP fusions of R11.1.6/YW1 in the presence of PI3K inhibitors wortmannin and ZSTK474. Error bars represent SEM of n = 3 biological replicates. c, For human cancer cell lines HT-29 and LS180, the percentage of cells positive for EGFP following infection with lentivirus for constitutive expression of R11.1.6/YW1 (and an EGFP reporter). The fraction of cells expressing R11.1.6 does not decrease over time, suggesting R11.1.6-mediated K-Ras antagonism to be inconsequential for proliferation. Supplementary Figure S3. Cell lines express EGFP-R11.1.6/YW1 upon induction with doxycycline. Flow cytometry histograms of inducible human cancer cell lines showing EGFP fusion protein expression after 48 hours upon addition of doxycycline (dox) at various concentrations. Supplementary Figure S4. Model variables, parameters, and equations. a, Table of the model variables, as depicted in Figure 3a. b, Table of the model parameters. c, Differential equations of the competitive binding of K-Ras by R11.1.6 and Raf. Supplementary Figure S5. Quantification of EGFP-R11.1.6 in inducible cancer cell lines. a, Measured by flow cytometry, the number of copies of EGFP-R11.1.6 molecules expressed by each inducible cancer cell line following induction with 2000 ng/mL doxycycline for 48 hours. Error bars represent SEM of n = 3 biological replicates. b, Measured by western blot, the number of copies of EGFP-R11.1.6 molecules expressed by each inducible cancer cell line following induction with 2000 ng/mL doxycycline for 48 hours. c, Comparison between western blot and flow cytometry measurements of the number of EGFP-R11.1.6 copies per cell for all cell lines. Supplementary Figure S6. Model construction. a, Model output at equilibrium of the fraction of K-Ras molecules occupied by R11.1.6 as a function of the number of Raf and R11.1.6 molecules per cell. A dotted line is shown to indicate the number of Raf molecules per cell as presented in the literature (26). b, Model output at equilibrium of the total number of K-Ras - Raf complexes within the cell as a function of the number of Raf and R11.1.6 molecules per cell. The dotted line is as in a. c, Model output at equilibrium of the fraction of K-Ras - Raf complexes that remain intact in the presence of R11.1.6, shown again as a function of the number of Raf and R11.1.6 molecules per cell. This was calculated by normalizing the curves in b at various levels of R11.1.6 to the curve at which R11.1.6 is 0 (i.e. baseline, where there is no competition for binding of K-Ras). The dotted line is as in a. d, Model output at equilibrium of the fraction of K-Ras - Raf complexes that are inhibited by the presence of R11.1.6, shown again as a function of the number of Raf and R11.1.6 molecules per cell. This was calculated by subtracting the curves shown in c from 1. The dotted line is as in a. Supplementary Figure S7. Effect of Raf membrane localization on R11.1.6 competition with Raf. Model-derived heatmap depicting the fraction of inhibition of K-Ras - Raf complex formation by R11.1.6 when Raf membrane localization is not included in the simulation. The number of K-Ras molecules per cell was held constant at 105. Dotted lines indicate the average number of EGFP-R11.1.6 molecules per cell as measured in Fig. 3b and the number of Raf molecules per cell as presented in the literature (26).</p>