HEK293T, HCT116, MCF7, HeLa, HepG2, and H1299 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, HyClone) at 37 °C in 5% CO2 atmosphere. The HEK293T cells, MCF7, and H1299 cells were stored in our Lab. The HeLa (Cat. #3111C0001CCC000011) and HepG2 (Cat. #3111C0001CCC000035) cell lines were purchased from Chinese National Infrastructure of Cell Line Resource (Beijing, China). HCT116 cell line was a gift from Dr. Depei Liu (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Cat.# 3111C0001CCC000158). Before the experiments, the two cell lines were authenticated on cell micrograph compared to the cell lines on ATCC. HEK293T cells showed 90% transfect efficiency with GFP-tag plasmid. H1299 cells showed the lack of p53 protein expression by western blot assay. Mycoplasma contamination was detected by the EZ-PCR Mycoplasma Test Kit (Cat. #20–700-20), a PCR-based mycoplasma test kit, in cell cultures before the experiment. The kit includes a unique reaction mix that contains all the ingredients required for PCR: nucleotides, primers, Taq Polymerase and magnesium. After performing agarose gel electrophoresis, positive samples will yield a 270 bp fragment, but HEK293T and H1299 cell lines not.
We established two stably transfected clones of H1299 cells, in which Lin28B was knocked down by co-transduction of the cells with lentivirus encoding each of the shRNA specific for Lin28B, designated shLin28B-1, shLin28B-2, and shLuc was as a negative control. The shLin28B-1, shLin28B-2, or shLuc were cloned into the pLKO.1 vector. The shRNA sequences were as follows: shLin28B-1: GCAGGCATAATAAGCAAGTTA; shLin28B-2: GCCTTGAGTCAATACGGGTAA; shLuc: CGCTGAGTACTTCGAAATGTC.
Antibodies against β-actin (sc-47778), GAPDH (sc-166545), Myc (sc-789), Lamin B (sc-6216) and PCAF (sc-13,124) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to FLAG (F3165) and M2 (F2426) were purchased from Sigma (St. Louis, MO). Antibodies to Lin28B (4196) and acetyl lysine (9441) were purchased from Cell Signaling Technology (Beverly, MA).
Plasmids, constructs, and transfection
The FLAG-tagged and GFP-tagged Lin28B eukaryotic expression plasmids and GST-tagged Lin28B prokaryotic expression plasmids were constructed by cloning Lin28B into the pcDNA6-FLAG, pEGFP-C1-GFP and pGEX-4 T-1 vectors, respectively, using a PCR product from a cDNA library derived from HEK293T cells. pCX-FLAG-PCAF, pGEX-PCAF-HAT and pCX-FLAG-PCAF-∆HAT were as previously described . Truncated fragments of Lin28B were cloned using the PCR product of full-length GFP-tagged Lin28B. FLAG-Lin28B-∆CSD was constructed by deleting the CSD. Myc-tagged PCAF was cloned from FLAG-tagged PCAF into the pCMV-3tag7-Myc vector. The eukaryotic expression plasmid of PCAF containing the Cys574Ser mutation was generated by site-directed mutagenesis of Myc-PCAF. The cells were transfected with Vigofect reagent (Vigorous Biotech, Beijing, China) according to the manufacturer’s instructions. Assays were performed 3 times each in triplicate, and all results are shown as the mean ± SD.
Cells were cultured on glass cover slips and fixed in 4% paraformaldehyde at room temperature for 10 min, washed three times with PBS for 15 min, permeabilized with 0.25% Triton X-100 in PBS for 15 min at room temperature, washed three times as described above and blocked with 1% BSA blocking solution at 37 °C for 1 h. The cells were incubated with primary anti-Lin28B antibody and anti-PCAF antibody at 1:50 dilution, anti-Myc antibody at 1:100 dilution and anti-FLAG antibody at 1:500 dilution at 4 °C overnight and washed three times with PBS. After washing, the cells were incubated with FITC- or TRITC-conjugated secondary antibodies at a dilution of 1:200 at 37 °C for 1 h and then washed three times with PBS. The coverslips were stained with DAPI, mounted and examined with a laser scanning confocal microscope.
Co-immunoprecipitation (co-IP) and immunoblot analyses
Transiently transfected HEK293T cells were homogenized in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 50 mM NaF, 5 mM Sodiun Pyrophosphate, 50 mM Sodium β-glycerophosphate, 1 mM NaVO3, 1 mM DTT, 1 mM PMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin). Cell lysates were rotated at 4 °C for 30 min and centrifuged at 12,000 rpm for 15 min to remove insoluble material and incubated with anti-GFP beads or anti-FLAG M2 beads at 4 °C overnight. The collected beads were then washed three times and boiled in SDS gel-loading buffer for western blot analysis.
GST pull-down assay
The GST-tagged Lin28B fusion protein was expressed in Escherichia coli and purified using anti-GST beads. GST or GST-tagged Lin28B beads were individually incubated with cell lysates at 4 °C overnight. Finally, the beads were washed three times, and the bound proteins were analyzed by western blotting.
Quantitative real-time RT-PCR assays (RT-qPCR)
RT-qPCR assays were carried out as described previously [26, 27]. Specific primers for Lin28B (forward: AGCCCCTTGGATATTCCAGTC and reverse: AATGTGAATTCCACTGGTTCTCCT). The relative expression of let-7a-1 and let-7g was normalized to that of U6 snRNA using the comparative CT method according to the manufacturer’s instructions (Bio-Rad CFX Connect Real-Time System). The primer sequences used for let-7 are listed below (F: forward; R: reverse; RT: reverse transcription). Mature let-7a-1 (RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT, F: GCCGCTGAGGTAGTAGGTTGTA and R: GTGCAGGGTCCGAGGT), mature let-7g (RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTGACA, F: GCCGCTGAGGTAGTAGTTTGT and R: GTGCAGGGTCCGAGGT), mature mir-16-1 (RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGCCAA.
F: GCCGCTAGCAGCACGTAAATAT and R: GTGCAGGGTCCGAGGT) and U6 (RT: AAAATATGGAACGCTTCACGAATTTG, F: CTCGCTTCGGCAGCACA and R AACGCTTCACGAATTTGCGT) were used. The experiments were repeated at least three times with statistical analysis for individual experimental groups. All values were expressed as the mean ± SD.
In vitro acetyltransferase assay
In vitro acetyltransferase assays were performed as previously described and detected by using western blotting with anti-acetyl lysine antibody . Briefly, recombinant GST-Lin28B and GST-PCAF-HAT2 proteins were expressed in E. coli BL21 and purified using glutathione beads (Amersham Biosciences). For in vitro acetyltransferase assays, 1 μg of GST-PCAF-HAT2 and 5 μg of GST or GST-Lin28B were incubated in 25 μl of acetyltransferase assay buffer (50 mM Tris, pH 8, 10% glycerol, 10 mM butyric acid, 0.1 mM EDTA, 1 mM DTT, and 1 mM PMSF) with 20 μM acetyl CoA at 30 °C for 1 h. The reaction products were separated via 15% SDS-PAGE and analyzed by western blotting with anti-acetyl lysine antibody.
Preparation of cell fractions
The cell fractions were prepared as previously described , and the prepared cell fractions were separated with 10% SDS-PAGE and detected by western blotting.
Statistical analysis was performed using two-tailed Student’s t-test. All data were shown as mean with standard deviations (SD). Probabilities of P > 0.05 were considered as no significant (#), P ≦ 0.05 as significant (*) and P ≦ 0.01 as highly significant (**).