Cck 8 assay kit

OCI-LY3 and OCI-LY10 cells were kindly provided by professor Wenbin Qian of Zhejiang University (Zhejiang, China). Z-VAD-FMK and SB203580 were obtained from Selleck Chemicals (Houston, TX). CCK-8 Assay kit was purchased from Meilunbio (Dalian, China). N-acetylcysteine (NAC) and glutathione (GSH) were obtained from Abcam (Cambridge, MA). The Cell Apoptosis detection kit and Cell Cycle Staining Kit were purchased from MultiSciences (Hangzhou, China). Primary antibodies, including anti-cleaved-PARP (ab191217), anti-cleaved-Caspase 3(ab32042), anti-Bax (ab32503), anti-Bad(ab32445), anti-Bcl2(ab32124), anti-β-Actin(ab179467), anti-p21(ab109520), anti-p27(ab32034), anti-p53 (ab179477), anti-p38(ab170099), anti-chk1(ab40866), anti-p-chk2, and anti-γ-H2AX, were purchased from abcam (Cambridge, MA). Antibodies including anti-p-p38(9215S), anti-chk2(3440S), anti-p-chk1(2348P) were purchased from Cell Signaling Technology (Massachusetts, USA). Secondary antibodies, including goat anti-rabbit and goat anti-mouse IgG-HRP were obtained from Beyotome (Shanghai, China).

Two CRC cell lines (HCT116 and HT29) were bought from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 µg/mL of penicillin- streptomycin. Oxaliplatin (Qilu Pharmaceutical Co., Ltd in Jinan, China) was dissolved in DMSO at a concentration of 30 mol/L and the cells were treated by Oxaliplatin for 24 h. CCK8 assay kit (Meilun Co.,Ltd, Nanjing, China’s) was used to detect the vitality of the cells according to the manufacturer’s instructions. Briefly, 2 × 10³ of cells were plated in 96 well plates. After treatment with DMOS or Oxaliplatin, cells were incubated in 10% CCK8 reagent for 2 h. The OD value was measured at 450 nm with TECAN Infinite 200 PRO Plate reader. Total RNA was extracted using the TRIzol chemical. The primary strand DNA was created using the PrimeScript RT chemical agent Kit and gDNA tool (Invitrogen; Thermo Fisher Scientific, Inc.). Step-by-step RT-PCR procedures were carried out in accordance with the manufacturer’s instructions. The expression of lncRNAs was measured using the 2^−ΔΔCT method. In Supplemental Tables 1, the primers for the seven lncRNAs used in the RT-PCR test are listed.

The CCK-8 assay kit (MA0218, Meilunbio, Dalian, China) was used for accessing the proliferation. The PCa cells were seeded onto 96-well plates with a density of 5000 cells per well. Then the cells were incubated for 24, 48, 72, or 96h. Next, the complete medium containing 10% CCK-8 assay kit was added into each well. The plates needed to incubated for about 1h. Finally, the absorbance was determined with a multiplate reader at 450 nm.

The antibodies to CISD2 (60758 S), IRP2 (37135S), and HIF-1α (#36169) were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies to Transferrin Receptor (sc-32272) were obtained from Santa Cruz (Dallas, TX, USA). The antibodies to β-Actin (ab8226) were obtained from Abcam (Cambridge, MA, USA). CCK-8 Assay Kit was obtained from Meilunbio (Dalian, China). C11-BODIPY (581/591), deferoxamine (DFO), Rhodamine B-[(1, 10-phenanthroline-5-yl)-aminocarbonyl] benzyl ester (RPA), DCF-DA, MitoTracker, and MitoSOX were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The BeyoClick™ EdU Cell Proliferation Kit and ATP detection kit purchased from Beyotime (Shanghai, China), Annexin V-FITC/PI apoptosis detection kit was purchased from TransGen Biotech (Beijing, China).

Cells were inoculated in a 96-well plate (5.0×10³ cells/well). Cell viability was determined using a CCK-8 assay kit (Meilunbio, MA0218-1, China). Each well was incubated with 100 μl of medium and 10 μl of CCK-8 reagent for no more than 4 h. The absorbance of each well was determined at 450 nm.