ZR-75-1, MCF7, and T-47D cells were seeded in media containing FBS. Eight hours after seeding, the cells were switched to CTS and incubated for 16 hours before addition of hormone treatment. Dihydrotestosterone (A8380, Merck) was diluted in 99.5% ethanol. Hydroxyflutamide (H4166, Merck) was diluted in DMSO. dihydrotestosterone and hydroxyflutamide were added to CTS containing media for a final ethanol and DMSO concentration of 0.1% (v/v) 5-10 minutes before addition to the cells, and the medium was replaced every 24 hours. Final dihydrotestosterone concentration was 10nM, and the final hydroxyflutamide concentration was 10μM. Cells were harvested 48 hours or 7 days after the start of steroid treatment. All treatments were done in triplicates and repeated three times.
A8380
Manufactured by Merck Group
Sourced in United States
The A8380 is a laboratory equipment product from Merck Group. It is a versatile device designed for general laboratory applications. The core function of the A8380 is to perform tasks related to sample preparation, processing, and analysis. Specific details about its intended use or performance characteristics are not available in this unbiased and factual description.
Automatically generated - may contain errors
Lab products found in correlation
E8875, by Merck Group (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Clean blot ip detection reagent, by Thermo Fisher Scientific (1 mentions)
Enzalutamide, by Merck Group (1 mentions)
C9481, by Merck Group (1 mentions)
P8074, by Merck Group (1 mentions)
Bp 110r, by Boston BioProducts (1 mentions)
Ptc 209, by MedChemExpress (1 mentions)
Sa001 500, by GenDEPOT (1 mentions)
Sa002 500, by GenDEPOT (1 mentions)
5α androstan 17β ol 3 one, by Merck Group (1 mentions)
Benzyl benzoate b6630, by Merck Group (1 mentions)
S3547, by Merck Group (1 mentions)
A6885, by Merck Group (1 mentions)
F9397, by Merck Group (1 mentions)
Forma series 2 water jacket co2 incubator, by Thermo Fisher Scientific (1 mentions)
Gfp ax10 cam 105, by Zeiss (1 mentions)
Sml1524, by Merck Group (1 mentions)
C57bl 6n, by Orient Bio (1 mentions)
B9061, by Merck Group (1 mentions)
11 protocols using a8380
1
Hormone Treatment of Breast Cancer Cells
2
Hormone-Responsive Breast Cancer Cell Lines
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ZR-75-1, MCF7, T-47D, and SK-BR-3 cells were seeded in media containing FBS. Eight hours after seeding, the cells were switched to charcoal treated serum (CTS) and incubated for 16 hours before addition of hormone treatment. Estradiol (E8875, Merck) and dihydrotestosterone (A8380, Merck) were diluted in 99.5% ethanol and added to CTS containing media for a final ethanol concentration of 0.1% (v/v) 5-10 minutes before addition to the cells, and the medium was replaced every 24 hours. Final estradiol concentration was 5nM or 10nM and dihydrotestosterone concentration was 10nM. Cells were harvested after 24, 48 hours or 7 days of steroid treatment. All treatments were done in triplicates and repeated three to six times.
3
Prostate Cell Line Treatments and Analyses
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LNCaP prostate cancer cells (ATCC # CRL-1740) and PrEC normal prostate epithelial cells (Cambrex Bio Science Cat. No. CC-2555: PrEC1 tissue acquisition no. #13683) were cultured, as described previously5 (link). VCaP (ATCC # CRL-2876) prostate cancer cells were cultured, as described previously47 (link). All cell lines were authenticated by STR profiling (CellBank Australia, Westmead, NSW, Australia) and cultured for <6 months after authentication. Mycoplasma contamination testing was routinely performed in the cell lines while in culture (MycoAlert™ Mycoplasma Detection Kit, Lonza).
LNCaP or VCaP cells were seeded at 60–70% confluence and starved with phenol red-free RPMI Medium 1640 containing 5% charcoal-dextran stripped Fetal Bovine Serum (FBS) (LNCaP) or DMEM containing 2.5% charcoal-stripped FBS (VCaP) for 72 h prior to a 2 h, 4 h, or 16 h (LNCaP) or just 16 h (VCaP) treatment with 10 nM 5-α-dihydrotestosterone (DHT; Sigma Aldrich #A8380) or ethanol-treated control. LNCaP cells were treated with Anacardic Acid (6-pentadecylsalicylic acid, AA; Calbiochem) at 90 µM for 48 h. The untreated controls were mock treated with the equivalent amount of the vehicle used to dissolve the drugs, 100% DMSO.
LNCaP or VCaP cells were seeded at 60–70% confluence and starved with phenol red-free RPMI Medium 1640 containing 5% charcoal-dextran stripped Fetal Bovine Serum (FBS) (LNCaP) or DMEM containing 2.5% charcoal-stripped FBS (VCaP) for 72 h prior to a 2 h, 4 h, or 16 h (LNCaP) or just 16 h (VCaP) treatment with 10 nM 5-α-dihydrotestosterone (DHT; Sigma Aldrich #A8380) or ethanol-treated control. LNCaP cells were treated with Anacardic Acid (6-pentadecylsalicylic acid, AA; Calbiochem) at 90 µM for 48 h. The untreated controls were mock treated with the equivalent amount of the vehicle used to dissolve the drugs, 100% DMSO.
4
Enzalutamide and PTC Inhibitors in Prostate Cancer
5
Modulating sex hormones and autophagy
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Three‐month‐old sex‐matched and weight‐matched littermate mice were subcutaneously (s.c.) injected with Nal‐Lys gonadotropin releasing‐hormone antagonist (Antide) (bioWorld, Dublin, OH, USA) at a dose of 6.0 mg/kg body weight weekly at the indicated times. Antide was dissolved in 20% propylene glycol and 0.9% saline. The duration of Antide treatment is determined according to experimental purposes. i.e. To investigate whether sex hormones directly regulate autophagy activity, we treated 3‐month‐old GFP‐LC3 transgenic mice for 4 months in which autophagic activity was altered to evaluate early events, while a pool of MuSCs were relatively maintained.
For sex hormone replenishment experiments, 5α‐androstan‐17β‐ol‐3‐one C‐IIIN [dihydrotestosterone (DHT), A8380; Sigma‐Aldrich] was packed in 0.5‐mm length silica tubes (Dow Corning, Midland, MI, USA). Silica tubes containing DHT were s.c. implanted. Implants were replaced every month to maintain serum DHT levels.
For sex hormone replenishment experiments, 5α‐androstan‐17β‐ol‐3‐one C‐IIIN [dihydrotestosterone (DHT), A8380; Sigma‐Aldrich] was packed in 0.5‐mm length silica tubes (Dow Corning, Midland, MI, USA). Silica tubes containing DHT were s.c. implanted. Implants were replaced every month to maintain serum DHT levels.
6
PCOS Model via Prenatal Androgenization
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Females in the estrus phase, as determined by vaginal smears38 , were mated overnight. Wt females were mated with APNtg males and APNtg females were mated with wt males. Pregnancy was confirmed by a distinct weight gain. On GD 16.5 the groups were subdivided and assigned to receive a subcutaneous injection in the interscapular area of vehicle or 250 μg 5α-Androstan-17β-ol-3-one (dihydrotestosterone (DHT), A8380, Sigma-Aldrich, St. Louis, USA) dissolved in 2.5 μl benzyl benzoate (B6630, Sigma-Aldrich) and 47.5 μl sesame oil (S3547, Sigma-Aldrich) for three days. Prenatal androgenization with DHT induces a PCOS-like phenotype in the offspring18 (link),39 (link). APNtg and wt dams gave birth to both wt and APNtg offspring. Female offspring were weaned at 4 weeks of age and genotyped as previously described12 (link). The breeding scheme and study design are shown in Fig. 1 .
7
Endometrial Stromal Cell Culture and Treatment
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Primary endometrial stromal cells (ESCs) were isolated from endometrial biopsies as previously described [4 (link)], yielding a ~ 95% pure stromal cell culture [13 (link)]. ESCs (6 × 105 cells) were cultured in 6-well plates for 24 h prior to treatments. Confluent ESC monolayers were treated with cAMP (500 μM, A6885, Sigma, UK), DHT (10−8 M, A8380, Sigma), and DHT + cAMP (10−8 M + 500 μM) for 24 h, 48 h, and 72 h. RNA was extracted after treatments and cell morphology assessed throughout the duration of the experiment.
8
Microtissue Response to (Anti-)Androgens
9
Prostate Cancer Cell Line Characterization
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We analyzed five human prostate cancer cell lines, LNCaP, VCaP, 22RV1, DU145 and PC-3 cells and 293FT cells. LNCaP, VCaP, 22RV1 and PC-3 were obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cell lines were authenticated by short tandem repeat (STR) profiling and tested negative for mycoplasma contamination. Cell lines were grown in RPMI medium or high-glucose Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated FBS, 10 IU/ml penicillin, and 10 μg/ml streptomycin. Cells were cultured and stored according to the suppliers' instructions. All cells for this study were used within 6 months of resuscitation and cultured at 37 °C in 5% CO2 (Thermo, 3131). Hypoxic conditions (1% O2, 5% CO2 and 94% N2) were created in a Forma Series II Water Jacket CO2 incubator (model: 3131; Thermo Scientific). For DHT (A8380, Sigma) treatment, cells were preconditioned in hormone-free culture overnight and the final concentration of DHT was 10 nM .
10
Prostate Sphere-Formation Assay Protocol
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Prostate sphere-formation assays were performed following a previously described protocol54 (link). Briefly, 1 × 104OLFM4-wild or OLFM4-knockout GFP reporter RWPE1 cells were suspended in 50 µl growth medium and mixed with 50 µl Matrigel, then cultured in 12-well plates for up to 14 days. For cells treated with 100 nM DHT (Sigma-Aldrich, #A8380) or 0.1 to 1 μM (+)-JQ1 (Sigma-Aldrich, #SML1524), the treatment medium was replaced with fresh medium containing the treatment reagent every 2 days. Dimethyl sulfoxide (DMSO) was used as a vehicle control for all treatment reagents. Images of spheres were captured with an AX10 cam 503 mono or GFP AX10 Cam 105 Color with a ZEISS microscope (AX 10) and ZEISS software for different timepoints, and GFP-positive colonies larger than 50 µm in diameter were counted.
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