Supporting Information

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1 Supporting Information Nan et al /pnas SI Materials and Methods Plasmids. PAmCherry1-KRas wild type and G12D mutants were generated by subcloning the respective KRas cdna fragments into a Gateway entry vector, pentr-pamcherry1-mcs. Expression clones were then generated by LR reactions between the entry clones with Gateway destination vectors including pcdna3-dest, plentipuro-teton-cmv-dest (Addgene no ), and plenti-neo- PGK-DEST (Addgene no ). The pcdna3-dest vector was constructed by fusing the DEST cassette from plenti-puro-teton- CMV-DEST to the pcdna3 backbone. The Gateway entry vector for DD-PAmCherry1-KRas G12D was constructed by subcloning the DD (i.e., Fv1E) domain from the Ariad homodimerization kit (now available through Clontech as product no ) into pentr- PAmCherry1-KRas G12D. The Clontech InFusion kit (Clontech no ) was used for all fusion reactions. Cell Culture and Induced Expression. BHK21 cells were obtained from American Type Culture Collection (catalog no. CCL-10), and maintained in high-glucose DMEM (no ; Gibco) supplemented with 10% (vol/vol) FBS (no ; Gibco) at 37 C and 5% (vol/vol) CO 2. TRex-293 cells were obtained from Life Technologies (no. R710-7) and maintained in DMEM with 10% (vol/vol) FBS. Stable cell lines were cultured under conditions identical to the parent cell lines. For induced expression, Doxycycline was added when cells were plated; the cells were continuously exposed to Doxycycline for h before final assays with imaging or Western blotting. Western blotting revealed that the level of protein expression stabilizes at 48 h and onward. Stable Cell Lines. Stable cell lines were generated by infecting cells with Lentiviral particles generated using the ViraPower Lentiviral Packaging System (Life Technologies no. K4975) using manufacturer recommended protocols. Viral particles were collected as supernatants at 48, 60, and 72 h post transfection of 293T cells and concentrated using the Lenti-X concentrator (Clontech no ). We typically use a multiplicity of insertion(moi)of less than 1 for infection and remove noninfected cells through drug selection. Drug concentrations for selection were 2 μg/ml and 600 μg/ml for puromycin and G418 (neomycin), respectively. After selection, <1,000 cells were plated on a 10-cm dish and allowed to grow into colonal populations each containing 100 cells. Isolated clones were manually picked with a 50-μL pipette tip; the clones were individually grown in 96-well plates to confluency. The clones were subsequently expanded in 12-well plates then 10-cm dishes. In parallel, each clone was individually examined for protein expression by Western blotting, and for homogeneity in expression patterns across the population by imaging and measuring the PAmCherry1 signals. Western Blotting. For Western blotting, cells were plated in six-well plates at densities to reach confluency in h. Cells were treated as indicated and harvested using a RIPA cell lysis buffer (Thermo Scientific, no ) supplemented with a mixture of phosphatase and protease inhibitors (ThermoFisher no ) and a cell scraper. Collected cell lysates were incubated on ice for 15 min and vortexed 3 5 times in the period. The lysates were then centrifuged at 18,400 g and 4 C for 15 min on an Eppendorf 5424 centrifuge equipped with an FA aerosoltight rotor. Supernatants were collected and assayed with a BCA kit (ThermoFisher no ) to measure the total protein concentrations. Equal amounts (10 20 μg) of total proteins for each sample were mixed with a 4 NuPAGE LDS loading buffer (LifeTechnologies, no. NP0007), supplemented with β-mercaptoethanol (βme). The mixture is brought to the correct volume with water, mixed, and heated at 95 C for 5 min. After cooling down to room temperature, samples were loaded into a Bris-Tris gradient gel (4 12%, LifeTechnologies no. NP0323) and run at 200 V for 50 min at room temperature or for 100 min at 4 C. Protein transfer was performed on a wet-transfer system at 30 V for min with water cooling, using a low fluorescence PVDF membrane (EMD Milipore no. IPFL10100). Blots were incubated in blocking buffer (LiCOR no ), primary antibodies diluted in blocking buffer,washed,followed by incubation with dyesecondary antibodies for imaging on a LiCOR Odyssey imager. Antibodies used for Western blotting in this study are: pan-ras (Rabbit monoclonal, Cell Signaling Technology no. 3965), KRas (Mouse monoclonal, Abcam no. ab55391), pperk (Mouse monoclonal, Sigma-Aldrich no. M9692), tubulin (Mouse monoclonal, Life Technologies no ), and total Erk (Mouse monoclonal, Cell Signaling Technology no. 4695). Microscopy. Confocal microscopy was performed on a Zeiss LSM 710 microscope equipped with 405 nm and 561 nm lasers, for photoactivation and excitation of PAmCherry1, respectively. Cell expressing PAmCherry1 fusion proteins were first exposed to 405 nm illuminations at 4% of maximum laser power, and subsequently imaged with the 561 nm laser. PALM microscopy was performed on a custom setup as previously described. Briefly, light from a 405-nm laser (CUBE-405; Coherent) and a 561-nm laser (MGL-H-561; OptoEngine) was combined and introduced to the back of a Nikon TE-300 inverted microscope (Nikon Instruments) in a total internal reflection (TIR) scheme. The typical power density used for photoactivation (405 nm) is W/cm 2, and that for imaging (561 nm) is 1 kw/cm 2. The 405-nm laser remained on during image acquisition, with the power gradually increased to ensure complete photoactivation of all molecules within the field of view. Data Analysis. PALM image analysis was carried out using a homewritten program package in Matlab (Mathworks). Images of individual molecules were recognized using a nonmaximal suppression algorithm. Each image was then fitted to an asymmetric 2D Gaussian function using an analytical form of the Jacobian matrix to find its centroid coordinates. Fiducial markers were manually picked based on the criterion that displacement trajectories from multiple (>2) markers exhibit less than 5-nm deviation RMS. The extracted coordinates of all of the recognized molecules were corrected with the fiducial markers. Spatial pattern analysis including Ripley s K test (18) and simulation aided DBSCAN (SAD) were performed with mixed Matlab and C programs as described (14). 1of6

2 Fig. S1. Biological activity of PAmCherry1-KRas expression constructs. NIH 3T3 cells stably expressing PAmCherry1-KRas WT were not transformed (B) compared with the original NIH 3T3 cells (A). By contrast, stable expression of PAmCherry1-KRas G12D mutant caused marked transformation of the cells as indicated by the formation of numerous colonies (C). Stable cell lines were established by lentiviral infection as described in Materials and Methods. Cells were grown past confluence to allow colony formation. Fig. S2. Western blotting of BHK21 cells stably expressing PAmCherry1-KRas wild type under Dox induction. Cells were incubated with various concentrations of Dox (0, 0.5, 1, 2, 5, and 0 ng/ml, lanes 1 6, respectively) and serum starved overnight. Cells for lane 6 was treated with 10 ng/ml EGF for 15 min, and those for the other lanes were untreated; cells were then harvested for Western blotting. 2of6

3 Fig. S3. Formation of KRas clusters at high expression levels. PALM images of BHK21 cells (originated from the same clone as used in Fig.1) stably expressing PAmCherry1-KRas G12D under 5 ng/ml Dox (Top), and those (parent BHK21 cells) transiently overexpressing PAmCherry1-KRas G12D (Middle) or PAmCherry1- KRas wild type (Bottom) were taken under the same conditions as described in SI Materials and Methods. 3of6

4 Fig. S4. Dox-induced expression of PAmCherry1-KRas G12D and Raf-MAPK activation in 293TRex cells. Stable cell 293TRex cell line expressing PAmCherry1- KRas G12D was established using the same method as described for BHK21 cells. Single cell clones were isolated and assayed with both Western blotting (A) and PALM microscopy (B). Ripley s K test (C) and SAD analysis (D) both suggested formation of Ras dimers and a small population of trimers at 2 ng/ml Dox, when Raf-MAPK activation was observed in the Western blot (A). Fig. S5. Artificial dimerization of KRas G12D in TRex-293 cells activates Raf-MAPK. We established a stable TRex-293 cell line expressing DD-PAmCherry1-KRas G12D (i.e., a dimerizable KRas mutant) and isolated single cell clones for imaging and Western blotting. 4of6

5 Fig. S6. Artificial dimerization of KRas G12D rescues TRex-293 cells from serum starvation (A) and MAPK inhibition (B).TRex-293 cells expressing DD-PAmCherry1- KRas G12D under 1 ng/ml Dox were grown to confluency and serum starved (A) or treated with a MEK inhibitor Trametinib (GSK ; 50 nm) (B) inthe absence (Left) or presence (Center) of 100 nm AP To quantify the number of viable cells, we trypsinzed the cells and used mild (1,000 g) centrifugation to pellet the cells. The pelleted cells were then resuspended in PBS, stained with trypan blue and counted (unstained cells) using a hemocytometer (Right). Fig. S7. Western blotting of TRex-293 cells expressing DD-PAmCherry1-KRas G12D/Y64A double mutant. We generated TRex-293 cells stably expressing DD-PAmCherry1-KRas G12D/Y64A double mutant under tetracycline regulation by lentiviral infection, similarly to those expressing the G12D single mutant. A pool of infected cells were exposed to 0, 2, and 4 ng/ml Dox for 48 h and serum starved for 4 h before treatment with mock or AP20187 (100 nm; 15 min). DD-PAmCherry1-KRas G12D activated MAPK in the absence of AP20187 at high concentrations (e.g., 4 ng/ml) of Dox and in response to AP20187 at low Dox concentrations (e.g., 2 ng/ml). 5of6

6 Fig. S8. Comparing biological activities of DD-KRas G12D and DD-PAmCherry1-KRas G12D in TRex-293 cells. TRex-293 cells stably expressing DD-KRas G12D or DD-PAmCherry1-KRas G12D under tetracycline regulation were generated in parallel by lentiviral infection and single clones were isolated, similarly to that described in Materials and Methods. Cells from single clones were exposed to 0, 2, and 4 ng/ml Dox for 48 h and serum starved for 4 h before treatment with mock or AP20187 (100 nm; 15 min). Both DD-KRas G12D and DD-PAmCherry1-KRas G12D activated MAPK in the absence of AP20187 at high concentrations (e.g., 4 ng/ml) of Dox. Activation of MAPK was minimal at low Dox concentrations (e.g., 0 2 ng/ml) but was significantly enhanced by incubating cells with AP20187 in both cell lines. 6of6