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. Author manuscript; available in PMC: 2019 Feb 1.
Published in final edited form as: Cancer Res. 2017 Nov 27;78(3):742–757. doi: 10.1158/0008-5472.CAN-17-1494

Figure 4.

Figure 4

CDK inhibitor Dinaciclib induces PARP inhibitor sensitivity in TNBC (a–b) 72hr HTSA was performed on a panel of TNBC cell lines were treated with Dinaciclib in combination with Niraparib and synergism was determined using Calcusyn software (<0.9 = synergism, 0.9–1.1 = Additive, >1.1 + Antagonistic) (a) Synergistic analysis of TNBC cells treated with Niraparib (0.1–20μM) in combination with a panel of drugs. (b) Calcusyn combination index averages of Dinaciclib (5, 10, 25nM) treatment in with Niraparib (0.1–20μM) (c) Immunoblot analysis for E2F1, c-myc, p-c-myc (S62), p-c-myc (T58), RAD51 and Actin of TNBC cells treated 72hr with DMSO, Dinaciclib (5, 10, 25nM) (d) Annexin V and propidium iodide staining of MCF10A, MB231 & SUM149 treated 72hr with Dinaciclib (10nM) Niraparib (1μM). Error bars represent mean ± s.d. (n ≥ 3 independent experiments). (e) Clonogenic assay, TNBC cells treated 72hr with DMSO (ctrl) Niraparib (1uM), Dinaciclib (10nM) or combo followed by with 9-day release. (f) FACS cell cycle analysis of TNBC cells treated 72hr with Dinaciclib 10nM, Niraparib 1μM followed by 72 drug release (g) DR-GFP homologous recombination repair assay, MB231 & HCC1937 cells were transfected 24hr with DR-GFP reporter assay, then treated 72hr with Dinaciclib (10, 25nM) followed by FACS analysis for GFP+ cells. Values are normalized by control group. Error bars represent mean ± s.d. (n ≥ 3 independent experiments) (h–j) Immunofluorescence analysis of MB231 cells treated 72hr with DMSO (ctrl), Niraparib (5μM), Dinaciclib (10nM) or Combo (h) Immunofluorescence images of γH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 2.5 μm. Representative images of three independent experiments. (i) Quantification of γH2AX, RAD51 staining. Representative images of three different experiments. Data are mean ± s.d. of biological replicates. unpaired two-sided t-tests (j) Quantification of average γH2AX foci per cell. A minimum of 250 cells were counted. (k) RT-PCR analysis of DNA repair genes in MB231 cells treated with DMSO, Niraparib (1μM), Dinaciclib (10nM), Combo. ***P < 0.001, n.s. (non significant) *P < 0.05, **P < 0.01, ***P < 0.001 two-way ANOVA with Sidak post-test correcting for multiple comparisons