br Fig Av treatment induced the activation of NF B
Fig. 2. Av treatment induced the activation of NF-κB pathway. (A) Immunofluorescence staining of Rel A in MDA-MB-231 Elafibranor (GFT505) treated with Av for 24 h. (B) Densitometry quantification of nuclear:cytoplasmic ratios of NF-κB p65 in a time course study of NF-κB nuclear translocation in MDA-MB-231 cells. (C) Western blot and (D) Densitometric analysis for NF-κB p65, p-IκBα and IκBα and GAPDH from Av-treated MDA-MB-231 cells for 24 h. Values represent the average fold change of NF-κB p65, p-IκB-α and IκB-α expression, normalized to GAPDH, and relative to control, for a total of three western blots. Results are representative of three diﬀerent independent experiments. *, **, *** indicate P < .05, P < .001, P < .0001, respectively.
vector was co-transfected with packaging plasmids, using calcium phosphate method, into 293 T cells, for the production of viral particles. The produced viral particles were used to infect MDA-MB-231 cells. Following viral transduction, MDA-MB-231 cells were sorted to at-tain > 90% Dendra-fluorescence positive cells by fluorescence-assisted cell sorting (FACS Aria-SORP, Becton Dickenson, San Jose, USA). Cells were maintained in RPMI-1640 medium (Lonza, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Sigma, USA), and incubated at 37 °C in a humidified incubator (95% air, 5% CO2). Cells were treated with bevacizumab (Avastin®, Genentech, USA) at 50 μg/ml for 24 h .
2.3. Flow cytometric analysis of CD44 expression
The expression of the cell surface glycoprotein CD44 was evaluated by flow cytometry and data were analyzed using the FlowJo software (BD Biosciences, USA). Briefly, cells were treated by Av for 24 h, har-vested and labeled with APC-tagged CD44 antibody (BioLegend, USA). Finally, cells were analyzed by BD FACS Aria II SORP (BD Biosciences, USA).
2.4. Xenograft mouse model of solid cancer metastasis
This study was approved by the Institutional Animal Care and Utilization Committee of the American University of Beirut (IACUCC# 10–07-154). Mice were xenografted with MDA-MB-231 cells using our previously established protocol . Briefly, immune-deficient NSG
mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratory, USA) were injected subdermally (s.d.) with 2 × 106 MDA-MB-231 cells. At the day of injection, mice were randomized into two experimental groups: control or Av-treated. Av (10 mg/kg) was administered in-traperitoneally (i.p.), for 4 weeks, twice per week. Cell culture media was used as a vehicle control.
2.5. Animal tissue harvesting
At weeks 5 or 9, mice were anesthetized with isoflurane and eu-thanized by cervical dislocation. Lung samples were collected and snap frozen in liquid nitrogen and then stored at −80 °C for later RNA and protein extractions.
2.6. Patients and specimens
Samples of breast carcinoma patients were retrieved from the Pathology Department at the American University of Beirut Medical Center and Hammoud Hospital University Medical Center. This study was performed according to the regulations of the IRB committee at the American University of Beirut Medical Center (reference number: PALM. FB. 01). Patients were females with no treatment history, clas-sified into 3 groups: Group 1 (11 TNBC cases), group 2 (11 ER−/PR−/ HER2+ cases) and group 3 (15 ER+/PR+/HER2− cases).
Fig. 3. Av treatment decreases transcriptional expression of VEGF. (A and B) qPCR showing mRNA levels of Cx43 and VEGF in 24 h Av-treated MDA-MB-231 cells. Data of each target mRNA were normalized to GAPDH. (C) Western blot analysis of Cx43 and GAPDH in Av-treated MDA-MB-231. Densitometry quan-tification of Cx43 and GAPDH bands were done using Image Lab software. Values represent the average fold change in Cx43 expression, normalized to GAPDH, and relative to control non-transduced cells, for a total of three western blots. Results are representative of three diﬀerent independent experiments. *, **, *** indicate P < .05, P < .001, P < .0001, respectively.
2.7. RNA extraction and quantitative PCR (qPCR)
Total RNA was isolated from cells or mouse tissues using Nucleospin RNA II Kit (Machery-Nagel, USA) or Trizol reagent (Life Technologies, USA), respectively. Total RNA from formalin-fixed paraﬃn-embedded (FFPE) breast specimens was extracted using the RecoverALL Total Nucleic Acid Isolation Kit (Ambion, USA). 1 μg of total RNA was first reverse-transcribed to cDNA using RevertAid 1st strand cDNA synthesis kit (Thermo, USA) and then amplified by qPCR using iQ SYBR Green Supermix in a CFX96 system (Bio-Rad Laboratories, USA). Human primers are listed in Supplementary Table 1. Cq was used to calcu-late the relative fold change in gene expression after normalization to the housekeeping gene, GAPDH.