MSCs were plated into T25 plates. MSCs were irradiated with ELEKTA Synergy Linear Accelerator (Cravoley, UK) at 6 Gy (a dose rate of 350 cGy/min) over an appropriate field size when they were 80% confluent. Irradiated cells were allowed to recover for 12 h.
Isolation of CD133+ populations with flow cytometry
To isolate CD133+ cells from HCC cell lines Huh7 and PLC, both cells were suspended as single cell suspension, then labeled with PE-conjugated anti-CD133 antibody for 30 min, then CD133+ cells were isolated apart from CD133- cells by flow cytometry. They were named as Huh7-CD133 and PLC-CD133, respectively.
Transwell assay was used to perform co-culture assay. Huh7 and PLC were plated in 6-well plate at a density of 1Χ105, MSCs or IR-MSCs were seeded in transwell insert(0.4 μm pore), after co-culture for seven days, Huh7 and PLC were used for other experiments.
CD133 detection with flow cytometry
HCC cell lines Huh7 and PLC were co-cultured with IR-MSCs for 7 days, then the expression of CD133 was measured by flow cytometry. Cells were labeled with PE-conjugated anti-CD133 antibody, the percentage of CD133 + cancer cells was measured by MACSQuant Analyzer10 flow cytometric system (Miltenyi Biotech, Germany).
Colony formation assay
Huh7-CD133 and PLC-CD133 were co-cultured with IR-MSCs for 7 days, then were seeded into 6 well plate as 500 cells per well. 7 days later, cells were fixed and stained by 0.1% crystal violet solution. The number of colonies larger than 2 mm in diameter was counted.
Tumor formation in nude mice
Six-week-old male athymic BALB/c nu/nu mice were obtained from Shanghai Experimental Animal Center, Chinese Academy of Science. Mice were maintained under a pathogen-free condition and treated in accordance with the institutional animal welfare guidelines. For tumorigenicity assay, Huh7-CD133 and PLC-CD133 were co-cultured with IR-MSCs for 7 days. 1 × 106 cells in 100 μL were injected subcutaneously to the left back of mice. Mice were sacrificed 4 weeks after injection. Tumors were collected and volume and weight of tumors were calculated.
Total RNA was abstracted by Trizol assay and reverse-transcripted to cDNA by bestar qPCR RT kit. mRNA expression was detected by RT-PCR by using bestar real-time PCR master mix with an ABI Prism 7300 system. The primers are as follows: CD133, sense, AGTCGGAAACTGGCAGATAGC, antisense, GGTAGTGTTGTACTGGGCCAAT; Wnt3a, sense, AGCTACCCGATCTGGTGGTC, antisense, CAAACTCGATGTCCTCGCTAC; β-catenin, sense, AGCTTCCAGACACGCTATCAT, antisense, CGGTACAACGAGCTGTTTCTAC; GAPDH, sense, GGAGCGAGATCCCTCCAAAAT, antisense, GGCTGTTGTCATACTTCTCATGG. The primers for genes of Wnt family (Additional file 2: Table S1).
Total protein was acquired by RIPA and quantified by BCA assay. Proteins of the same mass were tested for expression of CD133 (64326, 1:1000,Cell signal technology) and Wnt3a (ab28472, 1:1000, abcam), β-catenin (ab6302, 1:1000, abcam), GAPDH(ab8245, 1:5000, abcam)by SDS-PAGE, GAPDH was used as control. The assay was performed according to protocols described before .
All the experiments were performed 4 times. Student T-test was done to analysis the difference between different groups. *P < 0.05 and **P < 0.01 represent a statistical difference. Data were expressed as the mean ± standard deviation (SD).