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49 protocols using Cisplatin

XTT viability assays were performed as described previously [26 (link)]. Briefly, 1–3 × 103 cells were plated onto 96‐well plates before treatment with cis‐diamminedichloroplatinum‐II (Cisplatin; Accord Healthcare, London, UK) for up to 96 h. Regarding the effect of conditioned media on Cisplatin sensitivity, GCT cells were pretreated with CM from TM cells for 24 h before Cisplatin treatment. Every day viability was screened by adding 50 μL 2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐5‐[(phenylamino)carbonyl]‐2H‐tetrazolium (XTT; 1 mg· mL−1;neoLab Migge GmbH, Heidelberg, Germany) and 0.5 μL N‐methyl dibenzopyrazine methyl sulfate (PMS; 1.25 mm; Sigma‐Aldrich) and measuring absorbance 4 h later in a UV/VIS spectrometer (450 nm vs. 650 nm, iMark Microplate Absorbance Reader, BioRad, Feldkirchen, Germany). Each time point/concentration was measured in quadruplicates. LD50 doses were calculated using graphpad prism software version 8 (GraphPad Software, San Diego, CA, USA).
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On the first day, 5x103 B16-F1 cells were seeded per well on a 96-well plate and left for one day in an incubator (Kambič, Slovenia) at 37°C and humidified 5% CO2. On the second day (24 h after cell seeding), the 3.3 mM stock cisplatin (Accord HealthCare, Poland) was diluted in 0.9% NaCl (physiological solution) to obtain the 10x higher concentration of cisplatin than desired with the cells (1, 10, 100, 330 μM). Diluted cisplatin was then mixed with the DMEM in ratio 1:9 and cells were incubated in DMEM with cisplatin for 10 min, 1 h, 24 h or 48 h. After the indicated time, DMEM with cisplatin was substituted with DMEM only. On the fourth day (72 h after cell seeding), the MTS survival assay was performed as described in the subsection Cell survival following electroporation only.
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3

Establishing Cisplatin-Resistant Bladder Cancer Cell Lines

The human UC cell lines (UCCs) RT-112 and T-24 were obtained from the DSMZ (ACC 418, ACC 376), whereas 253J and J82 cell lines were kindly provided by J. Fogh (New York, USA). Parental UCCs and their long-term cisplatin-treated sublines were grown in DMEM GlutaMAX-I (Gibco, Darmstadt, Germany) containing 10% heat-inactivated fetal bovine serum. Long-term cisplatin-treated sublines (LTTs) were established by continuously escalating cisplatin (Accord Healthcare, Muenchen, Germany) dosages over several months until stable resistant cell lines could be maintained at doses of 50 μM (RT-112-LTT), 3.3 μM (J82-LTT), 6.6 μM (253J-LTT), and 23 μM (T-24-LTT) as described previously5 (link).
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4

Combination Cytotoxicity Assay with Cisplatin and Erlotinib

Cisplatin (Accord Healthcare) was purchased from the University Pharmacy, University of Pecs, Hungary). Erlotinib was purchased from Selleckem (Houston, TX, USA). Drugs were added to cells at final concentration of 30 nM Cisplatin, and various concentrations (1 nM, 10 nM, 100 nM and 1 μM) of erlotinib for 48 h. The choice of erlotinib optimal concentration was determined using a cell viability assay. Recombinant human IL-6 and IL-8 was purchased from R&D Systems (Minneapolis, MN, USA) and used at a final concentration of 100 ng/ml for 48 h.
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Cisplatin (Accord Health care Inc., stock 1 mg/ml) and etoposide (Nova Plus, stock 20 mg/ml) were from the pharmacy at Children’s Hospital Los Angeles. GRK2 siRNA (pool of 4 GRK2-specific siRNAs, cat#sc-29337) was from Santa Cruz Biotechnology. Non-silencing negative control siRNA (cat#1027281) was from Qiagen, AllStars. FAM-labeled siRNA negative control (cat#AM4620) was from Life Technologies. Other reagents are listed in specific procedures. All other reagents are from Sigma-Aldrich Co unless otherwise specified.
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U-2OS cells were seeded into 6-well plates (#92006, TPP, Trasadingen, Switzerland) at a density of 1 × 103 cells/mL. After 24 h the cells were treated with 10 μM PJ34 (P4365, Merck, Darmstadt, Germany) or 10 μM Olaparib (S1060, Selleckchem, Houston, TX, USA) for 30 minutes, and then with 5 μg/mL cisplatin (Accord Healthcare Inc., Durham, UK). Cells were incubated for 6 days and then counted manually after staining with 0.5% crystal violet dissolved in 20% ethanol [69 (link)].
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Mice were implanted with PDX tumours as described above. When tumours demonstrated sustained growth, mice were randomized into vehicle control or treatment groups (n = 3–7 mice/group). Cisplatin (Accord Healthcare, London, UK) (1 mg/kg or 4 mg/kg) or vehicle (saline) was administered weekly via intraperitoneal injection. AZD8055 (Axon MedChem, Groningen, Netherlands) (2.7 mg/kg or 10 mg/kg in 10% DMSO, 40% Polyethylene glycol 300 (Sigma)) or vehicle was administered daily via intraperitoneal injection. RG7388 (Selleckchem, Munich, Germany) (50 mg/kg or 75 mg/kg in 2% hydroxypropylcellulose (Sigma), 0.2% Tween-80 (Sigma)) or vehicle was administered daily via oral gavage. Tumour growth was assessed 3 times a week using caliper measurements. All mice were sacrificed after 21 days of treatment. For future analysis the tumours were resected and paraffin embedded. Tumour volumes are plotted as mean ± SEM for all groups. Two-way ANOVA with Dunnett post hoc test was used to determine the significance of all pair-wise comparisons using GraphPad Prism.
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Chemotherapeutic agents, such as cisplatin (Accord Healthcare, Milan, Italy), paclitaxel (Accord Healthcare, Milan, Italy), and docetaxel (Hospira, Naples, Italy), were dissolved in 0.9% NaCl and added to the culture medium at the concentrations indicated in Table 1. Curcumin from Curcuma longa (≥95% purity), was purchased from Sigma-Aldrich (St. Louis, MO, USA). A 10 mM stock solution was prepared in dimethylsulfoxide (DMSO, MP Biomedicals, Santa Ana, CA, USA) and stored at +4 °C.
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Tumor samples were collected in customized breast medium, after which slices were generated using a Leica VT 1200S Vibratome as described previously [2 (link)]. Metastatic BC biopsies were embedded in 4% low melting agarose in PBS at 37 °C under a shallow angle before slicing. Slicing resulted in 3–5 slices from a biopsy (300 µM thick and several mm in length). Culturing was performed at 37 °C in a 5% CO2 atmosphere under constant rotation at 60 rpm using a Stuart SSM1 mini orbital shaker.
For the development of the drug sensitivity assay, tissue slices were cultured for three days with a constant concentration of cisplatin (Accord Healthcare, Utrecht, The Netherlands) or docetaxel (Biovision, ITK Diagnostics, Uithoorn, The Netherlands). Proliferating cells for all conditions were labeled by adding 30 μM 5-Ethynyl deoxyuridine (EdU) (Invitrogen, Carlsbad, CA, USA) to the culture media during the last 2 h, before fixation for cisplatin treatment and during the last hour before fixation for docetaxel treatment. Tumor slices were fixed in 10% neutral buffered formalin for at least 24 h at room temperature. Subsequently, tumor slices were embedded in paraffin and 4 μm sections were generated.
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Decarbazine (DTIC) (Lipomed GmbH) and cisplatin (Accord Healthcare) were used at indicated concentrations, during the indicated times. Dimethyl sulfoxide (DMSO) was used as a control. Whole cell protein extracts were prepared by total cell lysis and immunoblot was performed as described previously in reference4 (link). Antibodies used for Western blotting were: B7-H3 (1:1000, AF1027, R&D), phosoho-p38 (#9211), p38 (#8690), GAPDH (#5174) (1:1000, Cell Signaling) and α-tubulin (1:50000, CP06, Millipore). Protein concentrations from total cell lysates were measured using Pierce® BCA Protein Assay Kit (Thermo Scientific). Immunoblot expression levels were quantified using ImageJ. Uncropped blots are provided in the Supplementary Information.
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