Extended Data Fig. 2: Experiments Supporting Main Fig. 5.
From: Direct targeting of amplified gene loci for proapoptotic anticancer therapy
(a) ChIP analysis of γH2AX in BT474 cells detected increased DNA damage at the targeted HER2 gene following HER2-5922 treatment. Data are presented as mean ± SEM and analyzed by two-way ANOVA with Tukey test post-hoc, ***P < 0.001, n = 3 independent experiments. (b) Quantification of phosphorylated ATM by flow cytometry following treatment with HER2-205. Data are presented as mean ± SEM and analyzed by one-way ANOVA with Tukey test post-hoc, *P < 0.05, n = 3 independent experiments. (c) Analysis of HER2 gene expression by RT-PCR 12 h post-treatment with HER2-targeted TFOs (mean ± SD; two-ANOVA with Tukey test post-hoc; ns, not significant; n = 3 independent experiments). (d) Quantification of triplex-induced DNA double strand breaks using the neutral comet assay as measured by tail moment 12 h post TFO treatment (mean ± SEM; one-ANOVA with Tukey test post-hoc; ****P < 0.0001; n = 3 independent experiments). (e) Western blot analysis of activation of apoptosis as measured by cleaved PARP and pH2AX Y142 12 h following TFO treatment (representative immunoblots, n = 2 independent experiments). Western blot analysis of the phosphorylation status of HER family receptors (f) HER3, (g) HER4, and (h) EGFR (HER1) in multiple breast cancer cell lines following HER2-205 treatment (representative immunoblots, n = 2). (i) Analysis of HER2 gene expression by RT-PCR 12 h post-treatment with HER2-targeted TFOs (mean ± SEM; one-way ANOVA with Tukey test post-hoc; ns, not significant; n = 3 independent experiments). (j) Analysis of HER2 gene expression by RT-PCR 20 h following pretreatment with the transcription inhibitor, α-amanitin (mean ± SD; one-way ANOVA with Tukey test post-hoc; ****P < 0.0001; n = 4 experiments).
