P276-00 targeting CCND1/pRB mediated E2F1 pathway and causing G1 arrest in FaDu cells. (a) Western blot analysis of CCND1, pRb, CCNE1 and E2F1 in FaDU cells treated with 1 and 3 μM P276-00 for 3, 6, 12 and 24 hours.(b-c) RTQPCR based transcript profiling of various downstream targets of E2F1 pathway. Data is represented on log2 scale as mean ± S.E.M. (n=3-5). (b) Dose response - FaDu cells were exposed to 5 different concentrations of P276-00 (range: 0.1-10 μM) for 12 hours in FaDu cells. (c) Time response- cells were exposed to 4 different time points (3, 6, 12 and 24 hours) with 1 μΜ of P276-00 (d-e) Automated fluorescence imaging based dose dependent protein profiling of CCND1/pRB mediated E2F1 pathway and its downstream targets. FaDu cells (1–2 × 104 cells/ml) were treated with 8 different concentrations of P276-00 (range 0.03 to 10 μM) for 24 hours and observed using ArrayScan HCS reader after staining with respective antibody (d) Quantification of the automated fluorescence imaging data has been represented in sigmoidal dose response curve where compound concentration. is represented in log2 scale. One thousand cells were counted for each replicate well and the results were presented as an average ± S.E.M. (n = 3). EC50 values have been generated for all the 5 proteins. (e) Panel of representative images.