Pfkfb3

Cells were lysed by sonication in anti-pY lysis buffer (50 mM Tris pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, 1 mM NaF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 mM Na₃VO₄, and 1 mM PMSF). Protein concentration was determined by Bicinchoninic acid protein (BCA) assay (Thermo Fisher Scientific). 25 μg of protein per sample were boiled in 6x Lammaeli buffer (BIORAD) for 5 minutes and separated on 4-20% gradient SDS-PAGE gels (BIORAD). Samples were then transferred to PVDF membranes for 2 hours in 3% MeOH Tris-Glycine Transfer buffer (BIORAD). Western blots were blocked in 5% non-fat dry milk in Tris-buffered Saline with 1% Tween-20 (TBST). Blots were probed with the following antibodies in 5% BSA/TBST overnight at 4°C: Glut-1, CPT1α (1:1000; Abcam), HK2, PFKFB3, LDHA (1:1000; Cell Signaling), and β-actin as a loading control (1: 10,000; Sigma- Aldrich). Membranes were washed with TBST and incubated with HRP-conjugated goat anti-rabbit or rabbit anti-mouse (Jackson ImmunoResearch 1:10,000) secondary antibodies for 2 hrs in 5% non- fat milk/TBST at room temperature. Chemiluminescence was detected using SuperSignal West Pico PLUS Chemiluminescence solution (Thermo Fisher Scientific) and the iBright FL1500 imaging system (Invitrogen).

The paraffin-embedded sections were deparaffinized and hydrated with a series of xylene and different concentration of ethyl alcohol. The sections were incubated with 3% H₂O₂ for 30 min and then were antigen retrieved by microwave. The primary RORα (1:100 dilution), G6PD (1:1000 dilution), PFKFB3 (1:100 dilution), Ki-67 (Rabbit, 12202 T, 1:400 dilution, Cell Signaling Technology, USA) and Proliferating Cell Nuclear Antigen (PCNA, Rabbit, 13110 T, 1:4000 dilution, Cell Signaling Technology, USA) antibodies were employed to incubate for 2 h at room temperature, respectively. Subsequently, the sections were incubated with the conjugated second antibody for 30 min at room temperature. The DAB kit (ZSGB-BIO, OriGene Technologies, Beijing, China) and 20% hematoxylin were utilized to staining at room temperature. The calculation of relative protein expression levels, and definition of low and high expression levels were mentioned in our previous studies [18 (link)].

Dulbecco’s modified Eagle’s medium (DMEM), DMEM/F12, fetal bovine serum (FBS), penicillin and streptomycin were obtained from GIBCO (Carlsbad, CA). 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15; Selleck, Houston, TX), dimethylsulfoxide (DMSO; Sigma-Aldrich, St Louis, MO), Gelatin, propidium iodide (PI) and Ribonuclease A (Sigma-Aldrich, St Louis, MO), Mounting Medium with DAPI (Zhongshan, Beijing, China) were purchased. Matrigel^TM matrix (BD Biosciences, San Jose, CA), transwell Boyden chamber system and 6-well Ultralow Adherence plates (Corning life Sciences, Wilkes Barre, PA), 0.5% Triton X-100 (MP Biomedical, Solon, OH) were also used. Epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from Peprotech (Rocky Hill, NJ). Antibodies including PFKFB3, phosphor-Rb, cyclinD1, cleaved-caspase3 (Cell Signaling, Danvers, MA), Bcl2 (Abcam, Cambridge, UK) and β-Actin (Santa Cruz, CA) were purchased. All other chemicals were classified as analytical grade reagents.

Tissue microarrays of triplicate 1-mm diameter cores were prepared from paraffin blocks using a manual tissue arrayer (Beecher Scientific, Estigen, Estonia) as previously described.^{16 (link)} “CLL-cell rich” cores with >80% CD79a⁺ cells were used for analysis. Proliferating cells were detected based on their expression of Ki67 (Agilent). The following other antibodies were used: anti-MYC was from Abcam; anti-hexokinase 2 was from Sigma; anti-GLUT3 was from Novus; anti-pyruvate kinase M1, pyruvate kinase M2, and PFKFB3 were from Cell Signaling Technology; anti-NDUFS3, TOMM20, and succinate dehydrogenase complex subunit A (SDHA) were from Abcam. The primary antibody reaction was detected using a peroxidase-labeled system (Super Sensitive Polymer-HRP IHC Detection System; BioGenex, Fremont, USA). Heat induced epitope retrieval (citrate) was used for all antibodies. The slides were scanned with the Pannoramic 250 Flash II system. Immunostaining was quantified by computerized image analysis using the DensitoQuant tool in CaseViewer (3DHistTech, Budapest, Hungary).

The indicated cells were washed twice with ice-cold PBS and lysed in a radioimmunoprecipitation assay buffer (RIPA). In total, 30–100 μg of protein per sample was separated on a 10% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Hybond^® P, GE Healthcare) at 4 °C for 1.5 h. Blots were incubated for 1 h with 5% nonfat dry milk to block nonspecific binding sites, and then they were incubated overnight at 4 °C with polyclonal/monoclonal antibodies specific to AGO2 (Cat# ab32381), NOTCH1 (Cat# ab52627), VEGFA (Cat# ab46154) (all from Abcam) or PLK1 (Cat# 4513), PFKFB3 (Cat# 13123), SNAIL (Cat# 3895) (all from Cell Signaling Technology). The immunoreactivity was detected using a peroxidase-conjugated antibody and was visualized by an ECL Plus Western Blotting Detection System (GE Healthcare). The blots were stripped before reprobing with an antibody against actin (Abcam) or GAPDH (Santa Cruz Biotechnology).

The cells were lysed using RIPA buffer and protein content was measured with a BCA protein assay kit (Abcam), resolved by 10% SDS-PAGE gels and then electro-transferred onto nitrocellulose membranes. Then primary antibodies (Cell Signaling Technology, USA) were added to the membrane and incubated for 1 h at room temperature. The PFKFB3 (#13123) and ENO2 (#24330) were purchased from Cell Signaling Technology and diluted at 1:1000. Protein bands were visualized by electrochemiluminescence and protein expression was normalized using β-actin as a loading control.