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  • br mRNAs were quantified with

    2019-09-16


    mRNAs were quantified with real-time quantitative Lovastatin using Power SybrGreen® Master Mix (Applied Biosystems) and gene-specific primers described in Table 1. Commercially available primers (Qiagen, Hilden, Germany) were used to quantify BCL2 (Hs_BCL2-1-SG Quanti-Tect Primer #249900), ESR1 (Hs_ESR1-1-SG QuantiTect Primer #044492), TNFSF11, the gene for RANKL, (Hs_TNFSF11-1-SG Quanti-Tect Primer #PPH01048F), and SNAI1 (Hs_SNAI1–1-SG QuantiTect Primer #010010) mRNA. All other primers were self-designed using the National Center for Biotechnology Information PrimerBLAST [78] and ordered from ThermoFisher Scientific (Waltham, MA). Exact sequences for all ThermoFisher primers are given in Table 1. RNA quantities were determined by the standard curve method. mRNA levels were normal-ized to levels of glyceralde-3-phosphate dehydrogenase (GAPDH) mRNA. mRNA for BAX, BCL2, CXCR4, CXCL12, OPG (TNFRSF11B), and RANKL (TNFRSF11) are presented as ratios of BAX to BCL2, CXCR4 to CXCL12, and OPG to RANKL.
    2.3.2.3. Total p53 content. Total p53 was measured 24 h post-cell treatment with vitamin D metabolites as described above using a sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Human Total p53 DuoSet® IC). Cells were plated in 24 well plates, treated with vitamin D3 metabolite for 15 min, and incubated for 24 h in complete media. Conditioned media were collected, and cell layers were lysed by sonication (40A for 5 s) in 200 μL lysis buffer according to manufacturer's protocol. Lysates were centrifuged at 10,000g for 15 min; the supernatant was collected and assayed for total protein content (ThermoFisher, Pierce 660 nm Protein Assay) and total p53 content following the manufacturer's instructions. Data are presented as a ratio of nanograms of total p53 to total protein content.
    2.3.2.4. DNA fragmentation. DNA fragmentation was assessed by colorimetric terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (Trevigen, TiterTAC™ in situ microplate TUNEL assay). Cells were incubated with vehicle, 24R,25(OH)2D3, or 24S,25(OH)2D3 for 15 min followed by incubation in fresh media for  BBA - General Subjects xxx (xxxx) xxx–xxx
    24 h. After fixing the cultures with 3.7% sucrose-buffered formaldehyde, samples were processed for TUNEL staining following manufacturer's instructions. Final absorbance was detected at 450 nm using a microplate spectrophotometer.
    2.3.2.5. Enzyme-linked immunosorbent assays for secreted proteins. Confluent cultures of MCF7 cells were plated in 6 well plates, treated with vehicle or 24R,25(OH)2D3 for 15 min as described above, aspirated, and then incubated in complete media. After 24 h, conditioned media were collected in 5 mL polypropylene tubes, frozen at -80 °C and lyophilized (Labconco, Kansas City, Missouri, Freezone Freeze Dryer #7740020). Lyophilized samples were reconstituted in 200 μL of filtered 1XPBS containing 1% BSA and assayed for OPG and RANKL using sandwich ELISAs (R&D Systems, Human TRANCE/RANK L/TNFSF11 DuoSet ELISA #DY626, Human Osteoprotegerin/ TNFRSF11B DuoSet ELISA #DY805). Cell monolayers were lysed in 1 mL 0.5% Triton™ X-100. Cell lysates were assayed for total DNA content (Promega, QuantiFluor dsDNA, #E6150). OPG and RANKL content are expressed as a ratio of OPG/RANKL.
    2.3.2.6. Matrix metalloproteinase 1. Collagenase (MMP1) content in the cultures was measured using an MMP1 assay kit (SensoLyte® Plus 520 MMP-1 Assay Kit *Fluorimetric and Enhanced Selectivity*, Anaspec #AS-72012). Cells were plated in 24 well plates and treated with vehicle or 24R,25(OH)2D3 as described above. At harvest conditioned media were collected; cell monolayers were lysed in 500 μL 0.05% Triton-X-100 and total DNA content measured. Total MMP1 was measured in the media and normalized to total DNA content. Data are presented as a ratio of ng MMP1/μg DNA.
    2.3.2.7. Wound closure assay. A wound closure assay was used to demonstrate metastasis. Confluent cultures of MCF7 cells were treated for 15 min with media containing vehicle or 24R,25(OH)2D3. Another set of cultures was treated with 10−7 M 17β-estradiol as a positive control. Following treatment, a scratch was drawn from one end of the well to the other using a sterile 1 mL pipette tip. The loosened cells were aspirated along with the treatment media, and fresh media added to the cultures. A phase-contrast microscope was used to take pictures of each well at 0, 3, 6, 9, 12, and 24 h after the scratch was made. Wound area was calculated using Adobe Photoshop™ 2 software, normalized to the original wound closure area, and presented as percent wound closure. Data presented are from one of two repeated experiments.