Anti klf4

Antibodies used in this research are as follows: anti-OCT4 (sc-9081), anti-KLF4 (sc-20691), anti-GLIS1 (sc-67584), anti-c-MYC (sc-42), TRA-1-60 (sc-21705), SSEA1 (sc-21702), SSEA4 (sc-21704), anti-mouse (sc-2005), anti-rabbit (sc-2004) and anti-goat (sc-2020) from Santa Cruz; anti-SOX2 (AF2018) and anti-NANOG (AF1997) from R&D Systems; anti-LIN28A (#46020) from Abcam; anti-TRA-1-81 (09–0011) from Stemgent; Alexa Fluor 488 anti-mouse (A11029), Alexa Fluor 488 anti-rabbit (A11034) and Alexa Fluor 488 anti-goat (A11055) for immunostaining of iPS clones from Life Technologies. IRDye800 anti-mouse (610-132-121) for Odyssey imaging of iPSC colonies from Rockland.

The procedure of IHC and ICC staining was shown in the previous studies [52 (link)]. LGR6 staining was evaluated blindly and independently by two pathologists. The score for staining intensity (0 = no staining, 1 = light brown, 2 = brown, and 3 = dark brown) and staining frequency (0 = 0–10%, 1 = 11–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100%) were multiplied to obtain the immunoreactivity score (IRS). The IRS 0–3 was deemed negative, 4–6 weak positive, and >6 strong positive. Two different pathologists evaluated all the specimens in a blinded manner. The antibodies used were as follows: anti-LGR6 (#MAB8458, R&D Systems, USA), anti-β-catenin (#sc-7963, Santa Cruz, USA), anti-TCF7L2 (#sc-166699, Santa Cruz, USA), anti-c-Myc (#10828-1-AP, Proteintech, China), OCT4 (#sc-5279, Santa Cruz, USA), anti-SOX2 (#3579, Cell Signaling Technology, USA), anti-KLF4 (#sc-20691, Santa Cruz, USA), anti-ALDH1A1 (#sc-374149, Santa Cruz, USA), anti-LGR5 (#PAB2591, Abnova, Taiwan), anti-LGR4 (#sc-390630, Santa Cruz, USA).

In this retrospective study, we included 141 samples from patients who received curative treatment at Shanghai General Hospital. All patients met the following inclusion criteria: 1) available baseline data and histologic material, 2) HCC diagnosed by a physician based on unambiguous histologic characteristics (Sun et al., 2011 (link)). Age, gender, hepatitis B virus surface antigens (HBsAg), liver cirrhosis, α-fetoprotein (AFP), Child-Pugh grade, tumor size, tumor number, tumor differentiation grade, tumor distribution, and pathological Tumor Node Metastasis stage (TNM stage) were all systematically recorded. All patients provided informed consent, and the academic study was conducted in accordance with ethical standards.
The primary antibody was anti-KLF4 (1:500, Santa Cruz Biotechnology). The IHC procedures were performed carried out precisely as described previously (Sun et al., 2016 (link); Sun et al., 2017 (link)). KLF4 staining was classified into three categories based on the intensity of the staining and the percentage of positive cells: weak/negative, moderate, and strong (Wei et al., 2005 (link)). In addition, in order to differentiate between low and high KLF4 expression, negative/weak expression was deemed low, whereas moderate/strong expression was deemed high.

Cells were lysed in RIPA buffer (0.1% SDS, 50mM Tris-HCl pH8.0, 150mM NaCl, 0.5mM EDTA, and 1% NP-40) supplemented with a cocktail of protease inhibitors (Sigma). Cellular extracts were quantified for protein using BCA assay kit (Thermo Scientific Pierce, Rockford, IL), subjected to SDS-PAGE and electrotransferred to polyvinylidine difluoride membrane. For immunoblotting assays, anti-Nox4 [20 (link)], anti-Oct4 (H-134; Santa Cruz Biotechnology), anti-Sox2 (Millipore), anti-Klf4 (Santa Cruz Biotechnology), anti-c-Myc (Cell Signaling Technology, Danvers, MA), anti-β-Actin (Santa Cruz Biotechnology), anti-p-AKT (Cell Signaling Technology), anti-AKT (Cell Signaling Technology), anti-p-Erk-1,2 (Cell Signaling Technology), anti-Erk-1,2 (Cell Signaling Technology) and anti-α-tubulin (Sigma) antibodies were used. A horseradish peroxidase-conjugated secondary antibody (Invitrogen) was applied prior to visualization by chemiluminescence (Amersham Imager 680; GE Healthcare Life Sciences, Chicago, USA).

ChIP assays were performed using reagents obtained from Cell Signaling Technology (SimpleChIP^® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003) according to the manufacturer's instructions. Briefly, 4 × 10⁷ cells were fixed with 37% formaldehyde for 10 min at room temperature and were neutralized by the addition of glycine for 5 min at room temperature. The cells were then lysed and chromatin was harvested and fragmented using sonication and enzymatic digestion. The chromatin was then subjected to immunoprecipitation using an anti-KLF4 (Santa Cruz Biotech, sc-20691), anti-histone 3 and normal rabbit-IgG antibody. ChIP-enriched DNA was quantified using real-time PCR, and the primer sets were designed as follows: hTERT: 5'-CGGACCACCTCGCCTTACA-3'(sense) and 5'-CTGGGCTCCTTCCCTCATCG-3' (anti-sense). The PCR products were loaded onto 2% agarose gels and visualized with ultraviolet light.