Lin28b

The expression of Lin28A and Lin28B at protein level in colon cancer tissues was detected by immunohistochemistry as previously described [6 ]. Briefly, 5-μm-thick colon cancer tissue sections were prepared. After deparaffinization, rehydration and blocking endogenous peroxidase, the antigen was retrieved by microwave treatment. The sections were blocked by using 5% bovine serum albumin and then incubated with primary antibodies against Lin28B (Abcam, Cambridge, MA, USA) or Lin28A (Abcam) at 4°C overnight. After incubation with secondary antibody labeled with streptavidin-biotin peroxidase for 1h at room temperature, DAB substrate (ZSGB Bio, Beijing, China) was applied for staining.
The result was evaluated by a pathologist who was blinded to the clinical information. The staining score was given by allying intensity with extent. Staining intensity was quantified as follows: negative (0), weak (1), moderate (2), or strong (3). Staining extent was scored according to the percentage of positive cells: none (0), <25% (1), 25-50% (2), 50-75% (3), or >75% (4). The final score was calculated as, the intensity score × the extent score.

Cells in culture flasks or plates were lysed in ice-cold buffer containing protease inhibitor cocktail (Roche). Equal amounts of protein samples were loaded and separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore) followed by 5% non-fat milk or 3% BSA blocking. These blots were incubated at 4 °C overnight with primary antibodies against Bmi1, p16, cleaved-PARP, Sox2, Nanog, Oct4 (Cell signaling, 1:1000 dilution), E-cadherin, N-cadherin and Vimentin (BD Biosciences, 1:2000 dilution), Lin28B (Abcam, 1:1000 dilution), ubiquitinated histone 2A and total histone 2A (Millipore, 1:1000 dilution), and GAPDH (Santa Cruz, 1:1000 dilution) followed by incubations with the corresponding secondary antibodies. The relative levels of each protein were quantified with Quantity One software (Bio-Rad). The protein–protein interaction was determined by protein immunoprecipitation using Pierce™Co-Immunoprecipitation Kit (ThermoFisher) and performed according to the manufacturer’s protocol. The antibodies for IP and following western blot were anti-Bmi1 (Cell signaling, #6964) and anti-ubiquitin (Cell signaling, #3933, 1:1000).

Immunohistochemical staining using a primary antibody (LIN28B, Abcam) was performed on sections of placental villi. According to the instructions for the SP Rabbit and Mouse HRP Kit (DAB) (CWBIO, China), biotinylated anti-rabbit/mouse universal second antibody was combined with the first specific antibody, and the second antibody was labeled with biotin combined with the streptavidin-labeled peroxidase (HRP). A streptavidin complex was thereby achieved by labeling with antigen-specific first antibody-biotinylated second antibody-HRP. Blank control: the primary antibody was replaced with PBS, other steps were as previously mentioned (including endogenous peroxidase blocking and serum blocking). We used ImageJ software to carry out a sample image for immunohistochemistry and made quantitative comparisons using digital image analysis.

The cultured cells were collected and lysed with enhanced radioimmunoprecipitation assay lysis buffer (Boster Biological Technology, Wuhan, P.R. China) containing protease inhibitors. The bicinchoninic acid kit (Boster Biological Technology) was utilized to determine the protein concentration. Proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the separated proteins were electro-transferred to polyvinylidene fluoride membrane (Immobilon P, Millipore, Billerica, MA, USA), and the membrane was treated with 5% skimmed milk at ambient temperature for 2 h to block non-specific binding. The membrane was incubated with primary antibody at 4°C overnight and then with horseradish peroxidase conjugated secondary antibody at 37°C for 1 h. Enhanced chemiluminescence reagent (Thermo Fisher Scientific) was utilized to visualize the immunoreactive bands, followed by imaging using ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA). ImageJ analysis software was utilized to quantify the gray value of protein bands. Antibodies included SIRT6 (A7416, 1:2,000, ABclonal, Woburn, MA, USA), NRP-1 (A19087, 1:2,000, ABclonal), GAPDH (AC033, 1:50,000, ABclonal), Lin28B (ab191881, 1:2,000, abcam), rabbit secondary antibody (AS014, 1:10,000, ABclonal), and murine secondary antibody (AS003, 1:10,000, ABclonal).

The RIP assay was carried out as per the instructions of the RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA). Transfected TNBC cells were subjected to lysis in RIP buffer. Next, magnetic beads were conjugated with ELAVL1 (Abcam) antibody, LIN28B (Abcam) antibody or immunoglobulin G (IgG) (Abcam) antibody, followed by treatment with cell lysates. After the beads was washed, the RNA of the mixture were isolated for the detection of RT-qPCR. The assay was repeated at least three times.

All Western blots were probed with specific antibodies directed against β-Actin, RBPJ, LIN28B (Abcam, Cambridge, MA, United States), RTA (Abbiotec, Escondido, CA, United States), LANA (MBL Life Science, Japan), and p65 (CST Biotechnology, Santa Clara, CA, United States). β-Actin or GAPDH was used as the internal loading control.