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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 2.

Figure 2

MYC regulates DNA repair in TNBC (a) Oncoprint analysis of MYC copy number gain, BRCA 1/2, PALB2 mutations in TCGA, TNBC breast cancer cohort (b–c) MYC mRNA expression in breast cancer subtypes as determined by (b) ER/PR/HER-2 expression via IHC, or (c) PAM50 signature (d–e) MYC mRNA correlation with (d) RAD51 and (e) EIF4E mRNA expression in TCGA breast cancer (f) IHC of TNBC patient cohort stained for c-myc/RAD51 from either pre or post neoadjuvant chemotherapy treatment samples (g, h) Kaplan-Meyer curve of c-myc+/RAD51+ vs. c-myc-/RAD51- of Pre (g) and Post (h) neoadjuvant chemotherapy treated TNBC breast cancer patients (i–j) MB231 cells treated with siMYC, siNT (ctrl) for 48hrs (i) Immunoblot for c-myc, RAD51, Actin (ctrl) (j) RT-PCR analysis of c-myc, RAD51 (n = 3) (k–l) HEK293T & MCF10A cells transfected with MYC ORF cDNA (k) Immunoblot analysis for c-myc, RAD51, Actin (ctrl) (l) RT-PCR analysis of c-myc, RAD51. (m) Annexin V and propidium iodide staining of MB231 and SUM149 cells treated with Non Targeting, MYC & RAD51 siRNA 48hrs followed by Niraparib/DMSO treatment 72hrs followed by 72hr release. Error bars represent mean ± s.d. (n ≥ 3 independent experiments) (n) Clonogenic assay MB231 & SUM149 cells treated with siNT (ctrl), siMYC & siRAD51 for 48hrs followed by 48hr treatment with DMSO (ctrl), Niraparib (1, 5μM) treatment 48hrs followed by release for 6 days (o) Cell viability assay, MB231 & SUM149 cells transfected with siNT, siMYC 48hr, followed by 72hr Niraparib (2.5μM) then drug release for 7 days. Error bars represent mean ± s.d. (n ≥ 3 independent experiments) (p–q) Immunofluorescence of MB231 cells treated with siNT (ctrl), siMYC 48hr, followed by 72hr Niraparib (5μM), DMSO (ctrl) treatment (p) Immunofluorescence of γH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 10μm. Representative images of three independent experiments. (q) Quantification of γH2AX, RAD51 staining. Data are mean ± s.d. of two biological replicates (r) DR-GFP homologous recombination repair assay. MB231 & HCC1937 were treated with siNT, siMYC 48hr, followed by transfection of DR-GFP reporter assay then analyzed by FACS for GFP+ cells. Values are normalized by control group. Error bars represent mean ± s.d. (n ≥ 3 independent experiments). b, c, two-tailed unpaired t-test was performed; in b, c, the multiple t-test was performed. n.s, (non significant), *P < 0.05, **P < 0.01, ***P < 0.001