Cell viability assay

The cells were seeded into 96-well culture plates at a density of 1.5×10⁴ cells/well in a 100 µL medium and were treated with different concentrations (15–60 µM) of luteolin for 24, 48, and 72 hours (Figure 1). As for the rescue test (Figure 2), after transfecting miR-301-MIMIC for 24 hours, the cells were treated with 30 µM (the representable value near to half maximal inhibitory concentration [IC50]) luteolin for 24 hours. Viable cells were evaluated using the CCK-8 assay, according to the manufacturer’s instructions. Briefly, the CCK-8 solution (50 µL/well) added to the cells in 24-well plates was incubated at 37°C for 4 hours, and the optical density of each well was read at 490 nm using a microplate reader (ELX800; BioTek Instruments Inc., Winooski, VT, USA). The viability was assessed with the following equation:

Luteolin regulates the cell proliferation, apoptosis, and chemosensitivity in PCa cells.

Notes: (A) After exposure to various concentrations (15–60 µM) of luteolin for 24 hours, viability of PC3 and LNCaP cells was determined using the CCK-8 assay. The viability ratio decreased in a dose-dependent manner (*P<0.01 compared with the untreated control group). (B) PC3 and LNCaP cells were treated with 15–60 µM luteolin for 24 hours and stained with Annexin V and PI. The apoptosis ratio was detected by FCM. The numbers in the first quadrant were the living cell ratio.

Abbreviations: Con, control; CCK-8, Cell Counting Kit-8; PCa, prostate cancer; PI, propidium iodide; FCM, flow cytometry.

Functional screening of miR-301 target sites using luciferase reporter assay.

Notes: (A) The selection criteria of the miRNA targets were based on their common detection in the target prediction online databases (TargetScan5.1 http://www.targetscan.org), as well as the full complementarity between the seed region of miR-301 and the 3′-UTR of DEDD2. (B) HEK-293T cells were cotransfected with miR-301-MIMIC, psi-Check2, WT-psi-Check2, or MUT-psi-Check2 of DEDD2 gene. The luciferase activity levels were measured 48 hours after transfection. The results from at least three independent experiments are presented as the mean ± SE. In this panel, the luciferase assay results show the regulation of DEDD2 by miR-301 (*P<0.01 compared with the control). (C) The DEDD2 mRNA expression levels in PC3 and LNCaP cells transfected with miR-301-MIMIC or miR-301-AMO were determined by qRT-PCR (*P<0.01 compared with the control). (D) The DEDD2 protein expression levels in PC3 and LNCaP cells transfected with miR-301-MIMIC or miR-301-AMO were examined by Western blot analysis. (E) After exposure to IC50 luteolin, the mRNA levels of DEDD2 in both PC3 and LNCaP cells were found to be increased >2.5 times with qRT-PCR. (F) The protein levels of DEDD2 in PC3 and LNCaP cells had the same tendency toward mRNA.

Abbreviations: Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DEDD2, death effector domain containing 2; AMO, anti-miRNA oligonucleotide; Lut, luteolin; IC50, half maximal inhibitory concentration; miRNA, microRNA; MUT, mutate; NC, negative control; PCa, prostate cancer; pMIR-Report, untreated control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SE, standard error; UTR, untranslated region; WT, wild type.