Cell counter

For 2D culture, irradiated cells were washed with PBS buffer, trypsinized and counted using a cell counter (Coulter) after irradiation. An appropriate number of cells were plated into each 60-mm dish to produce colonies. For 3D culture, the irradiated and control cells were first recovered from Matrigel by using Recovery Solution (BD) on ice for 30 min, and then trypsinized and resuspended in medium. An appropriate number of cells were plated into each 60-mm dish to produce colonies. After incubating for 10 days, the cells were fixed with 10 mL fresh Carnoy's fluid, stained with 0.5% crystal violet for 20 min. The number of colonies with greater than 50 cells were counted as survivors. Plating efficiencies (PE) were calculated as follows: numbers of colonies formed/numbers of cells plated. Surviving fractions were calculated as follows: PE (irradiated)/PE (unirradiated). The parameters of the survival curve, such as the α and β values, were obtained from the survival fraction (SF) data by curve fitting using the nonlinear model as follows: SF = (1 + exp(−α*(D - β)))⁻¹. Where D is the radiation dose delivered to the cells. All experiments were performed in triplicate. The experiment was at least repeated for three times independently.

1 × 10⁵ B16F10 cells, either untreated or pretreated for 24 h with 1 μM of H1152, were cultivated in vitro. B16F10 cells were counted after 2, 4, 6, and 8 days of culture with Cell Counter (Coulter) to evaluate their in vitro proliferation, which allows evaluating the toxicity of the H1152 treatment.

VCaP, LNCaP, and HEK293 cells were trypsinized and washed twice with PBS. VCaP cells were counted on a Coulter cell counter and aliquoted in appropriate quantities. Cells were pelleted by centrifugation and pellets were lysed in Mammalian Protein Extraction Reagent (M-PER; Thermo Scientific). Following pre-clearing by centrifugation (13,000 × g for 10 min), protein concentrations of cell lysates were determined by using Protein Assay Reagent (Bio-Rad, Hercules, CA). LNCaP and HEK293 cell lysates were spiked with purified recombinant ERG protein at the indicated concentrations, and then both sets of lysates were separated on NuPAGE Bis-Tris (4-12%) gels (Life Technologies) and transferred onto PVDF membranes. Membranes were blocked in Blocking Buffer (LI-COR, Lincoln, NE) and incubated with specific antibodies against ERG (ERG MAb 9FY; Biocare Medical Inc., Concord, CA), and GAPDH (Santa Cruz biotechnology, Santa Cruz, CA). Membranes were washed in Tris-Buffered Saline + Tween 20 (TBST) before incubation with appropriate secondary antibodies (goat anti-Mouse IRDye 800CW or goat anti-Rabbit IRDye 680CW, LI-COR). Signals of proteins detected were visualized and quantitatively measured using the Odyssey infra-red imaging scanner and software (LI-COR).

Rats were euthanized at 8 h after the LPS challenge, and bronchoalveolar lavage fluid (BALF) was collected as previously described [9 (link),10 (link)]. Briefly, the right main bronchus was ligated, and a catheter was inserted into the trachea. Double-lavage procedures were performed using 0.5 mL of normal saline to pass through the catheter twice. The BALF samples were kept on ice prior to cell counting. Total cell counts in the BALF were determined with a cell counter (Coulter Inc., Miami, FL, USA).

Cultured cells were detached using trypsin (0.05%)—EDTA (0.01%) after massive amplification in multi-layer flasks (875 cm² Cell Culture Multi-Flask, Corning, Corning, NY, USA). Suspended cells were counted using a Coulter Cell Counter and viability was assessed using trypan blue. Cells were centrifuged for 10 min at 300× g and resuspended at a density of 1 × 10⁷ cells per 200 µL in serum-free fibroblast culture media for HeLa cells and fibroblasts, or in serum-free keratinocyte medium for keratinocytes. Mice were anesthetized using isoflurane inhalation and 200 µL of the cell suspension was injected subcutaneously, within an hour after cell detachment from the culture flasks. Permanent skin tattoos were done prior to cell injection to track the injection site. At least 10 mice were injected in each group.

Fasted blood samples were taken from six healthy volunteers aged 60–73 years, in sodium heparin vacutainer tubes (Greiner Bio-One Limited, Gloucestershire, United Kingdom). The study was conducted according to guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Ethics Committee of the University of Reading. The ethics approval number was UREC 14/05. Written informed consent forms were obtained from all subjects. Blood was layered over an equal volume of lympholyte (Cedarlane Laboratories Limited, Burlington, Ontario, Canada) and centrifuged at 930 xg for 15 min at room temperature. Peripheral blood mononuclear cells (PBMCs) were harvested from the interface, washed once with PBS, and then resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (containing glutamine, Autogen Bioclear Ltd., Wiltshire, UK). These steps were repeated to achieve low contamination of erythrocyte. The pellet was finally resuspended in RPMI 1640 medium and cell numbers counted using trypan blue and a cell counter (Coulter, Fullerton, CA, USA). Cells were adjusted to the required concentration.