Ligand Screen for Calcium Assays in Primary B Cells and RAW264.7 Cells Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh

1. A. Stimulating ligand pairs Addition1 Addition2 B. Non-stimulating –stimulating ligand pairs  [Calcium] (nM) Time (sec) Table 1. Summary of the calcium dual ligand screen in primary B cells for (A) stimulating ligand pairs and (B) non-stimulating –stimulating ligand pairs for simultaneous addition experiments.Red boxes indicate greater than additive interactions; green boxes indicate less than additive interactions; white boxes indicate the interaction indicate the call has not been made.  [Calcium] (nM) A. Stimulating ligand pairs Time (sec) B. Non-stimulating –stimulating ligand pairs  [Calcium] (nM) Figure 2. Interactions between AIG and ELC with scaling. Calcium profiles within each ligand pair were scaled on the time and amplitude axis. For every ligand pair, the calculated addition of the single ligand controls (resulting in the expected trace) for all experiments were used for scaling. We used all expected traces to generate a mean expected trace. The peak amplitude for every expected trace of each individual experiment was amplitude scaled to the peak of the mean expected trace. The time to peak for each expected trace was scaled to the time to peak for the mean expected trace. The scaling factors for the expected traces for each experiment were applied to the remaining traces for that experiment (i.e., ligand1, ligand 2, delayed additions, and simultaneous treatments). Time (sec) Table 2. Summary of the calcium dual ligand screen in RAW264.7 cells for (A) stimulating ligand pairs and (B) non-stimulating –stimulating ligand pairs.Red boxes indicate greater than additive interactions; green boxes indicate less than additive interactions; white boxes indicate the call has not been made. Cell Preparation and Analysis Laboratory, Dallas, TX. Julie Collins, Richard Davis, Audra Wendt Katherine Hawes, Jason Polasek, Amy Pope, Meghdad Rahdar, Melissa Stalder, Ligand Screen for Calcium Assays in Primary B Cells and RAW264.7 Cells Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh, Paul Sternweis Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX INTRODUCTION BACKGROUND RESULTS The single ligand screen in primary B cells assess changes in calcium, cAMP, phosphoproteins and gene expression in the presence of individual ligands. The signaling pathways that evoke the above responses to each ligand may or may not be unique, raising the possibility that a ligand response may be altered in the presence of another ligand. The dual ligand screen was implemented to identify whether such interactions exist, and if so, how prevalent they are. The results of the simultaneous addition of ligands are compared to an “expected” value that equals the additive sum of each individual ligand. Interaction between pathways would be indicated by a non-additive response and vice versa. In May of 2003, AFCS decided to switch the cell system to RAW26.7 cells. RAW 264.7 is a good candidate for study because of its responsiveness to a variety of ligands, and is also amenable to genetic manipulation (introduction of RNAi, dominant negative molecules or mutant molecules) and microscopy, approaches critical to the goals of study of interactive signal transduction set by the AfCS. From the experience with the ligand screen in primary B cells, the decision was made that dual ligand screen would be implemented for RAW264.7 cells, and the data for the single ligand screen could be extracted from those of the dual ligand screen. Data presented are examples from the dual ligand screen from both primary B cells and RAW264.7 cells. Primary B cells Primary B cell Summary Table Experimental Designs Buffer or 1st ligand or the combination of two ligands A Buffer or 2nd ligand or the combination of two ligands (A) Less than additive interaction AIG - TER Measuring Calcium in response to 1st addition (10 min) Measuring Calcium in response to 2nd addition (10 min) Measuring Resting calcium (5 min) B (B) More than additive followed by less than additive interaction Figure1. Experimental designs for dual ligand screen in primary B cells and RAW264.7 cells. Panel A, experimental paradigm for the calcium dual ligand screen. Panel B, experimental template for B cells. Four ligands (L) could be used in one experiment, and L1 and L3 are stimulating ligands, whereas L2 and L4 could be either stimulating or non-stimulating ligands. ELC - LPA METHODS Ligands. Stimulating ligands are separated based on the cell types. –Primary B cells: AIG, BLC, ELC, LPA, M3A, S1P, SDF, SLC –RAW264.7 cells: 2MA, C5A, LPA, PAF, UDP Non-stimulating ligands are selected from the ligands that are not responsive in the calcium assay but are responsive in at least one of the other assays for primary B cells or RAW264.7 cells. Non-stimulating ligands are also separated based on the cell types. –Primary B cells: TER, PGE, 2MA, CGS, DIM, 40L, IFB, I10, I04, TNF, LPS –RAW264.7 cells: 848, GMF, I1B, I04, I06, I10, IFA, IFB, IFG, ISO, LPS, MC1, MCF, M1A, P2C, P3C, PGE, S1P, TGF Cells. Primary B cells. Resting splenic B cells are isolated from the male C57/BL6 mice according to the AFCS protocol# PP00000016. RAW264.7 cells. RAW264.7 cells are cultured according to the AFCS protocol# PP00000159. Experimental designs.  As shown in Figure 1. Assay procedures. Primary B cells -- B cells are loaded with 4 mM Fluo3 AM at 5 x 106 cells/ml and then assayed at 7.5 x 106 cells/ml using the Fluoroskan Ascent (Thermo Labsystems). Data are analyzed by Ascent software and Microsoft Excel and results are expressed as calcium concentration in nM. The detailed protocol is described in AFCS protocol # PP0000014000. RAW264.7 cells– RAW264.7 cells are plated at 60,000 cells/well in a black-walled clear bottom 96-well tissue culture plate overnight. RAW264.7 cells are loaded with 3 mM Fluo3 AM at room temperature for 30 min, and then recovered at 37 oC for 30 min, and then assayed with the Fluoroskan Ascent (Thermo Labsystems). The detailed protocol is described in AFCS protocol # PP0000017600. RAW264.7 Summary Table (C) Additive interaction LPA - TNF Figure 3. Examples of the calcium dual ligand screen in primary B cells. (A) AIG+TER, less than additive interaction; (B) ELC+LPA, initial peak response is more than additive and then followed by less than additive interaction; (C) LPA+TNF, additive interaction. Results shown are mean ± standard deviation from at least three experiments of the simultaneous addition. Blue lines represent observed responses; red lines represent expected (calculated) responses. Acknowledgements

2. BLC SDF ELC SLC LPA 2MA I04 I10 TER DIM PGE TNF CGS Calcium 40L • Dual ligand screen for calcium assays in both primary B cells and RAW264.7 cells is in the data curation stage. • S1P is able to induce calcium transients in RAW264.7, but it is not categorized as a stimulating ligand for RAW264.7 cells because of its low and somewhat inconsistent responses. Instead S1P is used as a non-stimulating ligand in RAW264.7 cells. • A minimum of 50 non-additive interactions for B cells and 15 non-additive interactions for RAW264.7 cells exist in the calcium dual ligand screen. • With the development of the RNAi technology in RAW264.7 cells, the calcium assay could be a very valuable tool to detect changes in the relevant early signaling pathways in targeted gene knock-down cells. A. More than additive interaction B. Additive interaction C5A-UDP C5A-PAF Potential Integrative Pathways to Ca  [Calcium] (nM) AIG-high AIG-low S1P M3A + + + + IFB + + + + ? Time (sec) LPS + -  [Calcium] (nM) Time (sec) Figure 5. Proposed interaction networks for calcium pathways. Small rectangle boxes indicate ligands used in the assay. Ligands on the top with solid arrows pointing toward the “Calcium” box are the stimulating ligands. Dotted arrows indicate possible interactions: +, enhancement; -, inhibition; ?, not certain. Dotted boxes (circle or square) are proposed converging points with different ligands. C. Less than additive interaction D. Less than additive interaction LPA-ISO PAF-UDP  [Calcium] (nM)  [Calcium] (nM)  [Calcium] (nM) Time (sec) Time (sec) Time (sec) Figure 4. Examples of the calcium dual ligand screen for simultaneous additions in RAW264.7 cells. (A) C5A+UDP, more than additive interaction; (B) C5A+PAF, additive interaction; (C) PAF+UDP, less than additive interaction; (D) LPA+ISO, less than additive interaction. Panels A to C are for stimulating ligand pairs, and blue lines in the upper and lower graphs for each panel represent the indicated ligand alone; panel D is the non-stimulating ligand (ISO) paired with stimulating ligand (LPA). Results shown are one example of each ligand pair from at least three experiments of the simultaneous additions. Blue lines ligand alone; red lines represent observed responses; black lines represent expected (calculated) responses.  [Calcium] (nM)  [Calcium] (nM) Time (sec) Time (sec) • Complete the data curation for calcium assays in primary B cells and RAW264.7 cells. • Validate the interactions observed in RAW264.7 cells with the revised template. • Plan and execute experiments with targeted RNAi knock-down cells for calcium assays. • Initiate the triple ligand screen in RAW264.7 cells for selected ligand combinations. Figure 6. Combined summary of the calcium and cAMP dual ligand screen in RAW264.7 cells. Ligands are listed at the left and bottom sides of the matrix. Ligands in blue are calcium responders, and ligands in red are the cAMP stimulators, and ligands in black are non-stimulators for either assay. Red boxes indicate greater than additive interactions; green boxes indicate less than additive interactions; white boxes indicate the interaction is either additive or the call has not been made; gray boxes mean not tested because both ligands are non-responsive in either assays. Letters inside the boxes reflect the interactions observed with cAMP assays (A) or calcium assays (C). Ligand Screen for Calcium Assays in Primary B Cells and RAW264.7 Cells Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh, Paul Sternweis Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX RESULTS RAW264.7 cells SUMMARY SUMMARY FUTURE GOALS