Facsverse system

Absolute cell counts were determined using the volumetric count tool of the BD FACSVerse™ System (BD Biosciences, USA). For evaluation of IL-6 expression, cells were fixed with 4% formaldehyde, permeabilized in 0.5% saponin and stained with anti-human IL-6 antibody (eBioscience Inc, USA) or isotype matched control. Cells were processed using the BD FACSVerse™ System (BD Biosciences). For every sample it was measured the geometric mean fluorescence intensity subtracted of the appropriate isotype control (ΔGeoMFI).

For cell proliferation assay, 3000 cells were seeded on 96-well plates and incubated for 24 h and treated with drugs at indicated concentrations for 3 days at 37°C. After drug treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well and incubated for 4 h at 37°C before media removal. DMSO was then added and shaken for 30 min at room temperature. Cell viability was determined by measuring the absorbance at 492 nm. Values represent the mean of 3 experiments (bars, ± SD).
For cell cycle analysis, cells were treated with PAC-320 for 48 h and fixed in 70% ethanol/30% PBS for 1 h at –20°C. After fixation, cells were washed once with PBS, resuspended, and incubated in propidium iodide (PI) buffer (60 μg/ml PI and 0.1 mg/ml RNase A) for 45 min at room temperature. Fluorescent-activated cell sorting (FACS) was conducted on at least 5,000 cells per condition using a BD FACSVerse system and BD FACSuite software. Cell cycle profiles were processed and analyzed using FlowJo version 6.4.7 (Tree Star, Ashland, OR, USA).
For apoptosis assay, cells were treated with PAC-320 for 48 h and harvested. Cells were stained as described in the Annexin V FITC Apoptosis Detection Kit (KeyGen Biotech) and data was acquired using a BD FACSVerse system and BD FACSuite software.

3000 of RRP15 stably overexpressed/knockdown cells or control cells were seeded on 96-well plates, or 1000 cells of HCT116 or SW620 were seeded on 96-well plates for 24 h and transfected with indicated siRNA. The cells proliferation was determined using CCK8 kit at indicated time according to the manufacturer’s protocol (Dojindo, Kyushu, Japan). Cell viability was determined by measuring the absorbance at 450 nm. For colony formation assay was performed in 6-wells dish in which 300 cells were seeded and cultured for 10 days. Colonies were counted manually after staining with 0.1% crystal violet.
For cell cycle analysis, stably transfected cells or cells transfected with siRNA were fixed in 70% ethanol/30% PBS for overnight at −20 °C. The cells were subsequently washed once with PBS, and incubated in propidium iodide (PI) buffer (60 g/ml PI and 0.1 mg/ml RNase A) for 45 min at room temperature. Cells were analyzed by Fluorescent-activated cell sorting (FACS) (BD FACSVerse system, New Jersey, USA) and at least 10,000 cells per condition were collected. Cell cycle profiles were processed and analyzed using FlowJo version 6.4.7.
For apoptosis assay, stably transfected cells or cells transfected with siRNA were harvested, and then stained as described in the Annexin V FITC Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China). Data were acquired using a BD FACSVerse system.