1. Ligand Screen for cAMP Assays in Primary B Cells and RAW264.7 Cells Keng-Mean Lin, Robert Hsueh, Madhusudan Natarajan, Paul Sternweis Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX Ligand Screen for cAMP Assays in Primary B Cells and RAW264.7 Cells Keng-Mean Lin, Robert Hsueh, Madhusudan Natarajan, Paul Sternweis Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX BACKGROUND METHODS RESULTS INTRODUCTION Primary B cells Experimental Designs Ligands. Stimulating ligands are separated based on the cell types. –Primary B cells: 2MA, BLC, CGS, ELC, LPA, PGE, S1P, SDF, TER –RAW264.7 cells: PGE, ISO Non-stimulating ligands are selected from the ligands that are not responsive in the cAMP assay but are responsive in at least one of the other assays with the exception of TNF, DIM and LPS for primary B cells. Non-stimulating ligands are also separated based on the cell types. –Primary B cells: AIG, M3A, SLC, 40L, IFB, I10, I04, TNF, LPS –RAW264.7 cells: 2MA, 848, C5A, GMF, I1B, I04, I06, I10, IFA, IFB, IFG, LPA, LPS, MC1, MCF, M1A, P2C, P3C, PAF, S1P, TGF, UDP 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 plated at 1.5 x 10 6 cells/sample, and B cells are treated with ligands for 20 sec, 40 sec, 1.5 min, 5 min, and 20 min. The amounts of cAMP in the samples are determined by an enzyme based immunoassay kit, cAMP Biotrak EIA (Amersham Biosciences). Results are calculated as cAMP in fmol/ 1 x 10 6 cells. The detailed protocols are described in AFCS protocol # PP00000140 and PP00000015. Results shown in the graphs are expressed as stimulation fold as defined by the following formula: Fold = cAMP of samples treated with ligand / cAMP of samples treated with medium from the same experiment. RAW264.7 cells– RAW264.7 cells are plated at 60,000 cells/well in a 96-well tissue culture plate overnight. After treatment with ligands for indicated time, samples are processed as described in the AFCS protocol# PP00000175. Aliquots of random samples are assayed for protein amounts. Results for the cAMP assay are expressed as pmol/mg of protein. Results shown in the graphs are expressed as fold changes. Fold = cAMP of ligand- treated samples/cAMP of samples treated with medium alone. Figure1. Experimental designs for dual ligand screen in primary B cells and RAW264.7 cells. Addition1 is added to the cells, and after incubation for 5 minutes, addition2 is applied to the cells. After addition2 is added, cells are harvested at different time points and processed for the assay. Measurements from treated cells are calculated and normalized with the results from resting cells. Buffer or 1 st ligand 2 nd ligand or the combination of two ligands Incubation (5 min) Sample harvesting at different time points Addition1Addition2 A. Simultaneous additions B. Delayed additions Table 1. Summary of the cAMP dual ligand screen in primary B cells for simultaneous additions (A) and delayed additions (B). Percent: indicates the percent difference from the additive prediction (e.g.; 75% means the combination was 75% higher or lower than additive). Filled boxes indicate a high probability call (red, >additive; green, additive; green, <additive; yellow, additive. Pairs with numbers 1,2 or 3 require that number of additional experiments. Primary B cell Summary Table Stimulating Ligand Pairs (a)(b)(c) (d)(e)(f) (a)(b)(c) (d)(e)(f) (a)(b)(c) (d)(e)(f) (C) Additive interactions (A) More than additive interactions (B) More than additive interactions Figure 2. Examples of cAMP double ligand screen for stimulating ligand pairs in primary B cells. (A) LPA+TER, more than additive interactions; (B) S1P+TER, more than additive interactions; (C) 2MA+BLC, additive interactions. Upper panels of each group (a, b, and c) show the cAMP fold (stimulated over baseline samples) for simultaneous addition (panel a) or stimulating ligands alone (panels b and c). Bottom panels of each group (d, e, and f) represent normalized cAMP fold (actual cAMP fold / calculated additive fold) for simultaneous addition (panel d) or delayed addition (panels e and f). Each point represents mean ± standard deviation from at least three experiments. Cell Preparation and Analysis Laboratory, Dallas, TX. Julie Collins, Richard Davis, Audra Wendt Katherine Hawes, Jason Polasek, Amy Pope, Meghdad Rahdar, Melissa Stalder, Acknowledgements 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. Non-stimulating Paired with Stimulating Ligands Figure 3. Examples of cAMP dual ligand screen for non-stimulating ligands paired with stimulating ligand in primary B cells. (A) TER + M3A, more than additive interactions; (B) PGE+AIG, less than additive interactions. Panels a of each group show the cAMP fold (cAMP of the stimulated samples / cAMP of baseline samples). Green lines are the basal samples; black lines are samples treated with the stimulating ligand only; red lines are samples treated with non-stimulating and stimulating ligands added simultaneously; blue lines are samples treated with non- stimulating ligands for 5 min followed by the stimulating ligands. Central panels (b) and right panels (c) of each group show the relative cAMP (cAMP fold of the ligand pairs / cAMP fold of the stimulating ligand) for the simultaneous additions (panels b) and delayed addition (panels c), respectively. Each point represents mean ± standard deviation from at least three experiments. Relative cAMP(TER+LIG/ TER alone) Relative cAMP(TER+LIG/ TER alone) (A) More than additive interactions TER and M3A Simultaneous TER and M3A M3A, then delayed TER Relative cAMP(PGE+LIG/ PGE alone) Relative cAMP(PGE+LIG/ PGE alone) (B) Less than additive interactions PGE and AIG Simultaneous PGE and AIG AIG, then delayed PGE

2. FUTURE GOALS RESULTS RAW264.7 Summary Table 1. 1.Complete the data curation in primary B cells and RAW264.7 cells. 2. 2.Plan and execute experiments with targeted RNA i knock-down cells for cAMP assays. 3. 3.Initiate the triple ligand screen in RAW264.7 cells for selected ligand combinations. SUMMARY RAW264.7 Cells Ligand Screen for cAMP Assays in Primary B Cells and RAW264.7 Cells Keng-Mean Lin, Robert Hsueh, Madhusudan Natarajan, Paul Sternweis Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX Ligand Screen for cAMP Assays in Primary B Cells and RAW264.7 Cells Keng-Mean Lin, Robert Hsueh, Madhusudan Natarajan, Paul Sternweis Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX 1. 1.Dual ligand screen for cAMP assays in both primary B cells and RAW264.7 cells is in the data curation stage. 2. 2.2MA and S1P induce cAMP accumulation in RAW264.7, but are not categorized as stimulating ligands for RAW264.7 because of their low and inconsistent responses. Instead they are used as non-stimulating ligands in RAW264.7 cells. 3. 3.Non-additive interactions between S1P and TER (ISO) or PGE have been observed in both primary B cells and RAW264.7 cells, suggesting that these two cell types may have similar pathways. 4. 4.A minimum of 50 non-additive interactions for B cells and 15 non-additive interactions for RAW264.7 cells exist in the cAMP dual ligand screen. 5. 5.With the development of the RNA i technology in RAW264.7 cells, the cAMP assay will be a valuable tool to detect relevant early signaling pathways in targeted gene knock-down cells. Figure 4. Examples of cAMP double ligand screen for non-stimulating ligands paired with stimulating ligand in RAW264.7 cells. (A) ISO + S1P, more than additive interactions; (B) ISO+2MA, more than additive interactions. Left panels (a) of each group show the cAMP stimulation fold (cAMP of the stimulated samples / cAMP of baseline samples). Green lines are the basal samples; black lines are samples treated with the stimulating ligand only; red lines are samples treated with non-stimulating and stimulating ligands added simultaneously; blue lines are samples treated with non-stimulating ligands for 5 min followed by the stimulating ligands. Central panels (b) and right panels (c) of each group show the relative cAMP (cAMP fold of the ligand pairs / cAMP fold of the stimulating ligand) for the simultaneous additions (panels b) and delayed additions (panels c), respectively. Each point represents mean ± standard deviation from at least three experiments. Figure 5. Examples of cAMP dual ligand screen for non-stimulating ligands paired with stimulating ligand in RAW264.7 cells. (A) ISO + UDP, more than additive interactions; (B) PGE+UDP, less than additive interactions. Left panels (a) of each group show the cAMP stimulation fold (cAMP of the stimulated samples / cAMP of baseline samples). Green lines are the basal samples; black lines are samples treated with the stimulating ligand only; red lines are samples treated with non-stimulating and stimulating ligands added simultaneously; blue lines are samples treated with non-stimulating ligands for 5 min followed by the stimulating ligands. Central panels (b) and right panels (c) of each group show the relative cAMP (cAMP fold of the ligand pairs / cAMP fold of the stimulating ligand) for the simultaneous additions (panels b) and delayed additions (panels c), respectively. Each point represents mean ± standard deviation from at least three experiments. Table 2. Summary of the cAMP double ligand screen in RAW264.7 cells for two stimulating ligands, ISO (A) and PGE (B). Simultaneous and delayed additions are included for each ligand. Percent: indicates the percent difference from the additive prediction (e.g.; 75% means the combination was 75% higher or lower than additive). Filled boxes indicate a high probability call (red, >additive; green, additive; green, <additive; yellow, additive. AB 6. Proposed interaction networks for cAMP pathways. Small rectangle boxes indicate ligands used in the assay. Ligands on the left with solid arrows pointing toward the “cAMP” box are the stimulating ligands. Dotted arrows indicate possible interactions: +, enhancement; -, inhibition; ?, not certain. Dotted boxes (circle or square) are proposed converging points from different ligands. Figure 6. Proposed interaction networks for cAMP pathways. Small rectangle boxes indicate ligands used in the assay. Ligands on the left with solid arrows pointing toward the “cAMP” box are the stimulating ligands. Dotted arrows indicate possible interactions: +, enhancement; -, inhibition; ?, not certain. Dotted boxes (circle or square) are proposed converging points from different ligands. ELC SDF BLC 2MA S1P LPA TER PGE + + - W 40L AIG + M3a DIM X I04I10 cAMP Potential integrative pathways to cAMP CGS + LPSTNF SLC IFB Independent? + + + + + + + + + Z - - - + - Figure 7. Combined summary of the calcium and cAMP dual ligand screen in primary B cells. Ligands are listed in the yellow boxes, and ligands in green 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). (a) (b) (c) (A) More than additive interactions (a) (b) (c) (B) More than additive interactions (a) (b) (c) (A) More than additive interactions (a)(b)(c) (B) Less than additive interactions