Rs 2000 pro 225 x ray irradiator

For the cell viability assay, RM1 and hybrid cells (2000 cells/well) were seeded into 96-well plates and cultured at a 37 °C incubator for 24 h. For ferroptosis inducers, cells were treated with elastin (Selleck, 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM), RSL3 (MCE, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM), and FIN56 (Selleck, 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM) for 24 h, and the matched volume of DMSO was used as a control. For the chemotherapy drug, cells were treated with docetaxel (MCE, 0.5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM, 20 nM) for 48 h, with a matched volume of DMSO used as a control. For radiation sensitivity, the cells were irradiated with 6 Gy X-ray using an RS 2000 PRO 225 X-ray irradiator (Rad Source) with a single dose at 2 Gy/min, and cell viability was assessed at 0 h, 24 h, 48 h, and 72 h after irradiation. The cell viability was assayed with a Cell Count Assay Kit (Yeasen, 40203ES60) according to the manufacturer’s instructions and measured at wavelength 450 nm with a microplate reader (BioTek Synergy HTX) [23 (link)]. The cell viability was calculated and compared with the control for both cell lines.

HeLa or H1299 cells were lysed in immunoprecipitation (IP) buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) containing protease inhibitor cocktails. The cell lysates were incubated with mouse anti-Myc (CST, 2276) or rabbit anti-poly/mono-ADP Ribose (E6F6A) monoclonal antibody (CST, 83732) on a rotary shaker at 4 °C overnight. Mouse or rabbit IgG (Santa Cruz, sc-2025 or sc2027) was used as a negative control for detecting the pull-down specificity. Protein G beads (Santa Cruz, sc-2001) or protein A beads (Santa Cruz, sc-2002) were added and incubated at room temperature for 2 h. The agarose beads were collected by centrifugation, washed five times with IP buffer according to the manufacturer’s instructions, and eluted in SDS sample buffer for the subsequent Western blotting assay. Olaparib (Selleck, S1060) was purchased from Selleck Chemicals. The instrument used for X-ray irradiation was a RAD SOURCE RS 2000pro-225 X-RAY IRRADIATOR.

RM1 (5 × 10⁵) and hybrid tumor cells (5 × 10⁵) were suspended in 100 µL PBS. Tumor cells were injected subcutaneously into the flank of 6- to 8-week-old C57BL/6 male mice. For the bone metastasis model, the mice were injected with 1 × 10⁵ tumor cells through the caudal artery. When the tumor volume reached ~ 100 mm³ or there was significant bioluminescence in the bone area, the mice were randomly divided into different treatment groups, including erastin (20 mg/kg, dissolved in 5% DMSO + 40% PEG300 + 5% Tween 80 + 50% ddH₂O, i.p., daily), RSL3 (100 mg/kg, dissolved in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, i.p., biweekly), and docetaxel (12.5 mg/kg, dissolved in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, i.p., biweekly), with the matched volume of vehicle solution used as the negative control. For radiotherapy, the mice were anesthetized and received local ionizing radiation on tumors at 8 Gy using an RS 2000 PRO 225 X-ray irradiator (Rad Source) with a single dose at 2 Gy/min. Tumor volume and body weight were measured every 3 days, and the volume was calculated as follows: length × width²/2. Mice were euthanized when the tumor volume exceeded 2000 mm³.