MK 2206 br In vivo treatment of amplitude
2.4. In vivo treatment of amplitude-modulated radiofrequency electromag-netic fields
To replicate the dosimetric conditions resulting from intrabuccal ad-ministration of AM RF EMF in vivo, we designed and developed a small animal exposure system for AM RF EMF . This exposure system al-lows for control of SAR levels in the same range as those generated by intrabuccal administration . Mice were exposed to RF EMF
amplitude-modulated at the previously published breast cancer-specific  frequencies 3 h each day till the experimental endpoint. The exposure system RF output was set for delivery of a SAR level of 255 mW/kg within the brain. This SAR was selected so that it is 1) within the range of previously demonstrated in vitro activity (30–400 mW/kg)  and 2) within the range of wbSAR1g and psSAR1g in patients re-ceiving treatment with the TheraBionic device (1–352 mW/kg) . All study protocols were approved by the Wake Forest Institutional Animal Care and Use Committee (IACUC).
2.5. RNA-sequencing and miRNA array
231BrM and SKBrM3 MK 2206 treated with Sham or BCF for 7 days (n = 3/sample) and RNAs were extracted using Direct-zol™ RNA MiniPrep Plus kit (Zymo Research, Catalog number: R2072). The alignment and quality control of RNA-Seq data followed the pipeline developed by the NCI's Genomic Data Commons (GDC, https://gdc.cancer.gov/). For exosomal miRNA isolation, exosomes were collected from SKBrM3 cells treated with Sham and EMF for 7 days and miRNAs were extracted using miRNeasy Micro Kit (Qiagen, Catalog number: 217084). Expres-sion profiling of miRNA was performed by using the human miRNA chip (GeneChip miRNA 1.0 Array). Clustering and its visualization were performed using Cluster3.0 and TreeView.
2.6. [3H]thymidine incorporation assay
Growth inhibition (GI) was assessed in breast cancer cells exposed to breast-specific modulation frequencies as previously described . Briefly, cells were treated with the frequencies for 7 days, 3 h every day. At day 7, cells were washed, 3H thymidine (Amersham) was added, and cells were incubated for 4 h. After 4 h incubation, cells were washed with ice-cold PBS, fixed for 1 h with 95% methanol, rewashed in PBS, and lysed with 0.2 N NaOH. 3H thymidine incorpora-tion was measured using the Beckman Coulter scintillation counter.
2.7. MTS Cell proliferation assay
Five hundred cells/well were seeded into 96-well plates in regular growth medium. Cell viability was measured by the MTS assay accord-ing to the manufacturer's recommendations (Promega) at indicated days. r> 2.8. Colony formation assay
SKBrM3 and SKBrM3-RR cells were treated with Sham or BCF for 7 days. At day 7, cells were seeded in 6-well plate (500 cells/well), ex-posed to 5 Gy of radiation and allowed to grow in a regular medium. At day 7 (after irradiation), the colonies were fixed with ethanol for 30 min, stained with Crystal Violet (CV) for 20 min and then the number of colonies was counted manually.
2.9. Flow cytometric analysis
For CSC analysis, cells were collected after 7 days of BCF or Sham treatment, washed with PBS, and incubated with CD44-APC and ESA-
PE for 20 min followed by examining marker positive population (CD44high,ESAhigh) using BD Accuri. For calcium assay, Fluo-4 dye was
added to the media of cells that were previously exposed to Sham or BCF for 2 h and 30 mins. After Fluo-4 addition, cells were exposed to Sham or BCF 30 mins. Cells were kept at room temperature for 30 min, washed twice with HBSS, scraped off from dish, dissociated by gentle pipetting, and ran on BD Accuri.
Cells were plated (200 cells/well) in 96-well ultra-low attachment plates (Corning) with DMEM/F12 supplemented with 2% B27 (Invitrogen), 20 ng/ml EGF (Sigma-Aldrich), and 4 μg/ml insulin (Sigma-Aldrich). The number of mammospheres was counted at Day 7, and data were represented as the means ± SEM.
2.11. Quantitative RT-PCR (qRT-PCR) analysis and PCR array
Total RNA was isolated from the cells and reverse transcribed. The cDNA was then amplified with a pair of forward and reverse primers to validate the results of microarray and PCR array. The thermal cycling conditions consisted of an initial denaturation step at 95 °C for 1 min followed by 35 cycles of PCR using the following profile: 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s.
2.12. Gene set enrichment analysis (gsea)
The Gene Matrix file (.gmx) was generated using top genes thate were significantly downregulated in BCF treated SKBrM3 cells. The Gene Cluster Text file (.gct) was generated using a cohort of 710 pa-tients as described previously , and patients were separated by their status of brain metastasis. The Categorical class file (.cls) was gen-erated based on the brain-met status of each patient. The number of permutations was set to 1000.