Extended Data Fig. 8: Mechanisms by which Cyclin A/B RxL Macrocycles Promote SAC Activation.
From: Targeting G1–S-checkpoint-compromised cancers with cyclin A/B RxL inhibitors
a, Dose response assay of NCI-H1048 cells treated with increasing concentrations (0 nM, 1.5 nM, 3 nM, 6 nM, 12 nM) of the Mps1 inhibitor (BAY-1217389). n = 2 biological replicates. Note that 3 nM of BAY-1217389 effectively blocked phosphorylation of the MPS substrate KNL1 (see Fig. 2k) without blocking cellular proliferation and hence 3 nM of BAY-1217389 was used for all rescue experiments. b, The raw cell counts of the dose response with BAY-1217389 from a showing that NCI-H1048 cells treated with BAY-1217389 at 3 nM proliferated over the 3-day dose response assay similar to the DMSO untreated control. c-f, Dose response assays of NCI-H446 (c) or NCI-H69 (e) cells, and non-linear regression curves of cleaved PARP FACS analysis of NCI-H446 (d) or NCI-H69 (f) cells treated with increasing concentrations of CIRc-004 in presence or absence of the Mps1 inhibitor (BAY-1217389) at 3 nM for 3 days. Data are mean +/− SD. Arrows in a, c-f indicates DMSO-treated sample used for normalization. g, h, Immunoblot analysis of HCT116 (g) or NCI-H1048 (h) cells stably expressing empty vector, Flag-CDC20 WT, or Flag-CDC20 R445Q mutant treated with CIRc-004 with indicated concentrations for 24 h. For a-b, d-f, n = 3 biological replicates. For c, h, n = 2 biological replicates. i, Immunoblot analysis for cyclin B pS126 in indicated SCLC lines treated with CIRc-004 (300 nM), CIRc-005 (inactive enantiomer, 300 nM), or DMSO for 4 h. n = 3 biological replicates. j-t, NCI-H1048 (j,o,p), NCI-H69 (k,l,q,r), or NCI-H446 (m,n,s,t) cells treated with RP-6306 (Myt1 inhibitor) alone (j,k,m) or in combination with the cyclin A RxL inhibitor (CIRc-018) (l, n), cyclin A/B RxL inhibitor (CIRc-004) (o,q,s), or the cyclin B RxL inhibitor (CIRc-019) (p,r,t) for 5 days. For j-t, data are mean +/− SD of two technical replicates. n = 3 biological replicates. u, Immunoblot analysis of Wee1 after cyclin B1 IP in NCI-H1048 cells treated with CIRc-004, inactive enantiomer (I.E., CIRc-005), CIRc-018, CIRc-019, or DMSO. All inhibitors were used at 300 nM for 2 h. n = 2 biological independent experiments. v-x, Dose response assay of NCI-H1048 cells treated with Wee1 inhibitor (MK 1775, 10 nM) in combination with CIRc-018 (v), CIRc-004 (w), or CIRc-019 (x) for 5 days. Data are mean +/− SD of two technical replicates. n = 2 biological replicates.
