Fig. 4: Assessing the effect of AGX51 on quiescent cells and the whole proteome.

a 4T1 cells were treated with AGX51 (10 or 50 µM), when they were cultured under standard conditions (standard density, standard serum (10%)) or under quiescence-inducing conditions (high density, low serum (1%)). Following 24 h of AGX51 treatment, cell numbers were determined by cell counting and trypan blue exclusion. Means are plotted with error bars showing SD of four technical replicates. b Western blot for ID1 and ID3 on whole-cell lysates from 4T1 cells presented in (a). Actin serves as the protein loading control. Arrow indicates non-specific band. c Venn diagram of differentially expressed proteins, as identified by mass spectrometry, from three replicates of SILAC-4T1 cells treated with 40 µM AGX51 for 4 h. d Western blot for Beta-catenin and Cyclin D1 on whole-cell lysates from 4T1 cells treated with 40 µM AGX51 for 0–72 h. e Quantification of western blot shown in (d). f Structures of AGX8 and AGX51 (red asterisk indicating chiral center). g Western blot for ID3 and ID1 on whole-cell lysates from 4T1 cells treated with AGX51 and AGX8 at the indicated concentrations. h Western blot for the indicated proteins on whole-cell lysates from 4T1 cells treated with AGX51 and AGX8 at the indicated concentrations. Quantification of western blot bands shown in (g) and (h) are in Supplementary Fig. 7. See also Supplementary Figs. 4–7.