The overall goal of the article is to develop a phosphospecific flow cytometry-based assay to measure the effects of the compound library on signaling pathways

The overall goal of the article is to develop a phosphospecific flow cytometry-based assay to measure the effects of the compound library on signaling pathways. Identified hits were then confirmed in their pathway selectivity assays and finally lead compound selectivity was confirmed in vivo in mice. With this background, answer the following questions pertaining to each of the figures in the journal article (20 points):

1. Fig 1: What cells were used in the primary screening, what simulants were used to induce phosphorylation of p38 and pSTAT1 and what was the outcome. How did they validate the assay – 2 point
2. Fig 2: Describe briefly (step-wise description) how the authors performed the screening of the NCI natural product library (what stimulants were used and what signaling pathways were they targeting/measuring (2 points). To identify the hits what threshold did they use and how did multiparameter kinase pathway screening help identify pathway selective inhibitors (2 point)
3. Fig 3: Describe how validation of the hits identified from initial screen was performed and what was the outcome (2 points)
4. Fig 4: Screening was performed in heterogeneous primary cell populations with a goal to identify: i) Pathway specific inhibitors; ii) cell-type specific inhibitors and iii) patterns of pathway and cell type druggability. As related to the figure 4, answer the following questions
   1. What was the origin (organ/tissue) of the primary cells and what markers were used to identify the different cell population;
   2. How did the authors demonstrate quantitative nature of the phosphoflow platform;
   3. What is Fluorescence cell barcoding and what is the advantage of its use;
   4. What is selectivity factor (SI) and how did they apply it to interpret the data in Fig. 4c,d. (4 points).
5. Fig. 5: Briefly describe the experiment and outcome of the results that lead to the identification of pathway selective compound (2 points)
6. Fig. 6 & 8: What experiment and results lead the authors to believe that the inhibitor Streptonigrin is a cell-type selective compound. What was the outcome of evaluating this compound in vivo in mice (4 points)
7. Fig. 7: What is selectivity factor clustering and how did application of this statistical tool help them identify pathways and cell types that are druggable (2 points)