Journal: Frontiers in Cardiovascular Medicine

Article Title: Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia

doi: 10.3389/fcvm.2021.745810

Figure Lengend Snippet: Pfkfb3 inactivation in macrophages suppresses LPS-induced inflammation via the NF-κB signaling pathway. (A–E) Representative Western blot results (A) of phosphorylated and total level of Erk, Jnk, p38 and p65 in Pfkfb3 WT and Pfkfb3 ΔMϕ BMDMs treated with LPS (100 ng/mL) at the indicated time points, and relative ratio of p-Erk/Erk (B) , p-Jnk/Jnk (C) , p-p38/p38 (D) and p-p65/p65 (E) were quantitated by densitometric analysis of the corresponding Western blots ( n = 4). (F,G) qPCR analysis of the mRNA levels of Il1b (F) and Il6 (G) in BMDMs treated with LPS (100 ng/mL) for 4 h, with or without AZ26 (10 μM) pretreatment for 30 min ( n = 4). (H) Representative Western blot results (left) of p-p65, p65 and Actb in BMDMs treated with LPS (100 ng/mL) for 5 min, with or without AZ26 (10 μM) pretreatment for 30 min, and relative ratio of p-p65/p65 (right) was quantitated by densitometric analysis of the corresponding Western blots ( n = 4). All data are represented as mean ± SEM, ** P < 0.01 and *** P < 0.001 for indicated comparisons (unpaired two-tailed Student's t test).

Article Snippet: In some experiments, cells were incubated with 100 ng/mL LPS (L3137, Sigma-Aldrich, St. Louis, MO, USA) or 10 μm AZ26 (HY-101971, MedChemExpress, Monmouth Junction, NJ, USA) at various time points as indicated.

Techniques: Western Blot, Two Tailed Test

Journal: Pharmaceutical research

Article Title: Liposomes co-Loaded with 6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3 (PFKFB3) shRNA Plasmid and Docetaxel for the Treatment of non-small Cell Lung Cancer.

doi: 10.1007/s11095-017-2244-x

Figure Lengend Snippet: Fig. 4 Impact of PSH-L at different doses of pshPFKFB3 on PFKFB3 mRNA expression in A549 cells. Bar graph depicts real-time RT-PCR expression of PFKFB3 as compared to untreated control cells. The results show mean of three independent experiments with SEM. NC-pDNA-L liposomes with 100 ng of NC-pDNA, LF2K100 = Lipofectamine-2000 complexed with pshPFKFB3 100 ng, PSH-L20, 40, 60 and 100 = PSH-L with 20 ng, 40 ng, 60 ng and 100 ng of pshPFKFB3.

Article Snippet: Q-PCR primers for mouse PFKFB3 were purchased from Origene (Rockville, MD).

Techniques: Expressing, Quantitative RT-PCR, Control, Liposomes

Journal: Pharmaceutical research

Article Title: Liposomes co-Loaded with 6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3 (PFKFB3) shRNA Plasmid and Docetaxel for the Treatment of non-small Cell Lung Cancer.

doi: 10.1007/s11095-017-2244-x

Figure Lengend Snippet: Fig. 8 A. Western blot densitometric analysis of PFKFB3, various cell survival markers, apoptotic markers and stress related markers. The results show mean of three independent experiments. * indicates p < 0.05 compared with untreated controls; ** indicates p < 0.01 compared to untreated controls.

Article Snippet: Q-PCR primers for mouse PFKFB3 were purchased from Origene (Rockville, MD).

Techniques: Western Blot

Journal: Nature Communications

Article Title: BRAF activation by metabolic stress promotes glycolysis sensitizing NRAS Q61 -mutated melanomas to targeted therapy

doi: 10.1038/s41467-022-34907-0

Figure Lengend Snippet: a Spectral counts) of the identified proteins in the PFKFB2 protein complexes upon the indicated treatments in NRAS Q61 - and BRAF V600E -mutant melanoma cells ( n = 3 biologically independent experiments). Proteins already described to interact with PFKFB2 (black); new proteins described in this work (blue). b Representative immunoblot showing the amounts of the indicated proteins after isolation of His-tagged PFKFB2 complexes in NRAS Q61 and BRAF V600E mutant melanoma cells. Numbers show the fold induction of the indicated ratio ( n = 3 biologically independent experiments). c Graphs showing the fold change differences with respect to the control (spectral peak areas) of the indicated phosphorylated/unphosphorylated ratio peptides in NRAS Q61 and BRAF V600E mutant melanoma cells upon the indicated treatments. Each condition represents three biologically independent experiments ( n = 3) that were pooled before the mass spectrometry analysis. d Immunoblot showing the binding of PFKFB2 to BRAF and p-BRAF S445 in NRAS Q61 mutant melanoma cells under 1 h GS; n = 3 independent experiments per treatment). e Representative in vitro interaction assay of GST-BRAF and recombinant PFKFB2 (rPFKFB2). Immunoblots show the amounts of the identified proteins after GST pull-down ( n = 3 independent samples). GST protein was used as a control. f Representative in vitro kinase assay ( n = 3 independent samples) using rPFKFB2, GST-ARAF, GST-BRAF, and GST-CRAF. Ponceau S staining shows rPFKFB2. g Immunoblot showing the amount of the identified proteins after isolation of the protein complexes as indicated in the scheme. Total lysate 5% (T.L.), 4 h GS, and pull-down (P.D.) ( n = 2 independent biological experiments). h Representative in vitro interaction assay of rPFKFB2, rPFKFB3 and GST-BRAF. The immunoblots show the amount of the identified proteins after the PFKFB2/PFKFB3 immunoprecipitation or GST pull-down ( n = 3). Isotype IgG and GST were used as controls. i Immunoblot showing the amount of PFKFB3 expressed in melanoma cell lines, including patient-derived cell lines. ( n = 2 independent biological experiments) . j Immunofluorescence showing the expression of PFKFB3 in human melanoma samples. Bars represent 400 µm. ( n = 7 NRAS Q61 -mutated and n = 8 BRAF V600E -mutated). The graph shows the H-score of the evaluated samples (mean ± SD; p value, unpaired two-sided t test). k Immunoblot showing the activation of ERK1/2 upon 4 h GS and/or sorafenib treatment in PFKFB2 or PFKFB3 knockdown NRAS Q61 and BRAF V600E mutant melanoma cells ( n = 2 biologically independent experiments). C Control, GS glucose starvation; Sor. Sorafenib; Scrbl. scrambled siRNA.

Article Snippet: In vitro dimerization experiments were performed using PFKFB2 (Origene), PFKFB3 (Origene) and GST-BRAF (Millipore) recombinant proteins.

Techniques: Mutagenesis, Western Blot, Isolation, Control, Mass Spectrometry, Binding Assay, In Vitro, Recombinant, Kinase Assay, Staining, Immunoprecipitation, Derivative Assay, Immunofluorescence, Expressing, Activation Assay, Knockdown

Journal: eLife

Article Title: The injured sciatic nerve atlas (iSNAT), insights into the cellular and molecular basis of neural tissue degeneration and regeneration

doi: 10.7554/eLife.80881

Figure Lengend Snippet:

Article Snippet: Antibody , PFKfb3 (rabbit monoclonal) , Cell Signaling Technology , 13123 , 1:300.

Techniques: Control, Staining, Ointment, Cell Culture, Electron Microscopy, Protease Inhibitor, DC Protein Assay, Microscopy, Homogenization, Western Blot, Protein Concentration, Cell Isolation, Membrane, FACS, Cell Counting, Sequencing, Imaging, Enzyme-linked Immunosorbent Assay, Software, Sterility, RNAscope, Multiplex Assay, Hybridization, SYBR Green Assay, Real-time Polymerase Chain Reaction, Tissue Homogenization, Nucleic Acid Concentration, Blocking Assay

Journal: bioRxiv

Article Title: Glucose-Fueled Histone Modifications Drive HIV-1 Latency Reversal at Hypoxia

doi: 10.1101/2025.09.14.676169

Figure Lengend Snippet: (a) Reactivation of latent HIV-1 NL4-3 in an in vitro primary T CM model of HIV latency established from 4 independent donor cells was measured by HIV p24 expression when latency reversal is stimulated by PKC agonist, ingenol-3,20-dibenzaote, in the absence or presence of glycolytic inhibitor, 2-deoxyglucose (2-DG, 5mM) as 2h pretreatment under normoxic (21% O 2 ) conditions. Statistical significance was measured using paired t-test; *p<0.05. (b) Immunoblots of indicated glycolytic enzymes [hexokinase 2 (HK2), lactate dehydrogenase (LDHA) and fructose-2,6-biphosphatase 3 (PFKFB3)] assessed in whole-cell lysates of uninfected primary CD4 T cells either untreated or treated with a panel of PKC agonists [PEP005 (100nM), ingenol-3,20-dibenzoate (IDB, 100nM), PMA (100nM), prostratin (100nM)] or HDAC inhibitors [RMD (10nM), SAHA (2.5mM), mocetinostat (10mM), MS275 (10mM)] in 5mM glucose media for 48h under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as loading control. Immunoblots are representative of 3 replicates in independent donors. (c) Mean fluorescence intensity (MFI) of fluorescent glucose analog, 2-NBDG (400mM) in uninfected primary CD4 T cells either untreated or stimulated with PKC agonist, PEP005 (100nM) or HDAC inhibitor, RMD (10nM) for 48h under normoxic or hypoxic conditions to determine effects of LRAs on glucose uptake. Means and SDs of triplicate samples are shown. Statistical significance was determined using paired t-test, comparing each treatment to mock in each case; *p<0.05, ns = non-significant. (d & e) Global metabolomic analysis tracking the flux of (d) lactate and glucose ions and (e) indicated glycolytic intermediates using isotopic 13 C in uninfected primary CD4 T cells that were either untreated or pretreated with glycolytic inhibitor, 2-deoxyglucose, 2-DG (5mM) for 2h under hypoxic (1% O 2 ) conditions, then stimulated for 24h with PKC agonist, PEP005 (100nM) or HDAC inhibitor, RMD (10nM). Means and SDs of triplicate samples are shown. Statistical significance was measured using paired t-test; *p<0.05, ns = non-significant. In (e), heat map represents relative concentrations of each metabolite to untreated sample. Significant increases of p<0.05 are depicted in red and significant decreases of p<0.05 are depicted in blue.

Article Snippet: The following antibodies were used: HIF-1α (Proteintech, #20960-1-AP), Phospho GLUT-1 S226 (Sigma-Aldrich, #ABN991), GLUT-1 (EMD Millipore Corp, #07-1401), GLUT-3 (Proteintech, #20403-1-AP), HK2 (Cell Signaling Technology, #2867T), PFKFB3 (BETHYL, #A304-249A-T), LDHA (Cell Signaling Technology; #3582S), Phospho S6 S240/244 (Cell Signaling Technology; #5364T), S6 (Cell Signaling Technology; #2217S), Phospho NF-κB p65 (Ser536) (Cell Signaling Technology, #3033), NF-κB p65 (Santa Cruz, #sc-372) H3K9ac (Cell Signaling Technology, #9649T), H3K27ac (Cell Signaling Technology; #8173T), pan lactyl lysine (PTM; #1401), H3K9lac (PTM; #1419RM), H4K8lac (PTM; #1415), pan H3 (Cell Signaling Technology; #4499T) and histone H3 (96C10) (cell Signaling Technology, #3638), GAPDH-HRP (Cell Signaling Technology; #8884S) and anti-rabbit IgG-HRP (Cell Signaling Technology, #7074S and Thermo Fischer Invitrogen, #G21234).

Techniques: In Vitro, Expressing, Western Blot, Control, Fluorescence

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 1 Alisertib inhibits the proliferation, migration, and invasion of ATC cells in vitro and vivo. A, C CCK-8 assays showed that Alisertib (5 μmol and 10 μmol) treatment significantly suppressed cell proliferation in 8305 C (A) and CAL-62 (C) cells in a dose-dependent pattern. B, D After treatment with the indicated concentration of Alisertib for 2 days, the migration/invasion assay was performed in 8305 C (B) and CAL-62 (D) cells. Representative images are shown. The above data are presented as the mean ± SD of three independent experiments. E Five representative tumors from NSG mice injected with CAL-62 are shown, n = 5 mice per group. The subcutaneous xenografts were dissected and are shown on day 13. F, G Tumor volumes were measured with growth curves (F) and tumor weights (G) in the control group (DMSO) and Alisertib group (30 mg/kg). H Representative IHC images and p-Aurora-A and Ki-67 staining in xenograft tumor specimens of control and Alisertib groups are shown. p-Aurora-A and Ki-67 expression were significantly decreased after treatment with Alisertib. Scale bars, 50 μm. The data are presented as the mean ± SD. All *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: Migration, In Vitro, CCK-8 Assay, Concentration Assay, Invasion Assay, Injection, Control, Staining, Expressing

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 2 Aurora-A promotes the proliferation, migration and invasion of TC cells, and its expression is correlated to the poor prognosis of TC. A Representative IHC images of low expression and high expression of Aurora-A were shown. Scare bar is 50 µm. B Varying expression levels of Aurora-A in 20 normal samples, 126 DTC samples and 29 ATC samples. **P < 0.01, ***P < 0.001, compared with control normal samples. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with DTC samples. C Kaplan–Meier survival analysis showed that high Aurora-A expression correlated with short survival time in ATC patients. D Aurora-A expression in TPC-1 cells was detected by western blotting in the Aurora-A knockdown group. E CCK-8 assays showed that Aurora-A downregulation significantly enhanced cell proliferation in TPC-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. F, G Representative images of transwell assay of migration (F) and invasion (G) of TPC-1 cells with or without Aurora-A knockdown. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. H Aurora-A expression in KTC-1 cells was detected by western blotting in Aurora-A overexpressed group. I CCK-8 assays showed that Aurora-A upregulation significantly enhanced cell proliferation in KTC-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. J, K Representative images of transwell assay of migration (J) and invasion (K) of KTC-1 cells with or without Aurora-A overexpression. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. Above data are presented as the mean ± SD of three independent experiments. L Representative bioluminescent images and statistical analysis of the two groups on days 35 after tail vein injection (n = 5 per group) M Representative H&E staining of metastatic tumors in the lungs and livers. N Representative immunohistochemical staining for Ki-67 and p-Aurora-A. ***P < 0.001.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: Migration, Expressing, Control, Western Blot, Knockdown, CCK-8 Assay, Transwell Assay, Over Expression, Injection, Staining, Immunohistochemical staining

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 3 Multiomics and bioinformatics analyses indicated Aurora-A regulates the glycolysis in TC cells. A The Venn plot of proteome and RNA-seq revealed 5309 genes had significant alterations at the mRNA and protein levels after Aurora-A knockdown. B KEGG analysis of 5309 genes showed significant alterations in both proteome and RNA-seq. Glycolysis pathway can be remarkably enriched. C Analysis of TCGA database revealed Aurora-A was positively correlated with the glycolysis pathway. D Volcano plot highlighting phosphorylation changes of proteins. 318 proteins with 423 sites were identified with greater than 1.5-fold change in Aurora-A knockdown relative to control TPC-1 cells and plotted in red (upregulation) or blue (downregulation), respectively. E KEGG analysis of the phosphorylation-changed proteins after Aurora-A knockdown in TPC-1 cells. The glycolysis pathway was significantly enriched.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: RNA Sequencing, Knockdown, Phospho-proteomics, Control

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 4 Aurora-A-mediated PFKFB3 phosphorylation regulates the glycolysis of TC cells. A The interaction between Aurora-A and PFKFB3 was verified by an IP assay in TPC-1 cells. B Interaction between Aurora-A and PFKFB3 is examined by GST pull-down. C Phosphorylation level on PFKFB3 was determined by Anti-PFKFB3 (phospho S461) antibody after in vitro kinase assay. D The expression of p-PFKFB3/PFKFB3 in KTC- 1 cells with Aurora-A overexpression and Alisertib-treated 8305 C and CAL-62 cells was analyzed by western blotting. E Comparable level of glycolysis, glycolytic reserve and capacity of 8305 C, TPC-1, and KTC-1 cells measured by Seahorse XF Extracellular Flux analysis. ECAR = extracellular acidification rate. F The ADP/ATP ratio was measured in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3, in TPC-1 cells with Aurora-A knockdown and in 8305 C cells with Alisertib treatment, respectively. G The expression of p-Aurora-A, Aurora-A, p-ERK, ERK, p-AKT, AKT in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3. H, I The proliferation, migration and invasion ability in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3 was analyzed by CCK-8 assay (H) and transwell of migration and invasion assay (I), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with Aurora-A upregulation group. J Western blotting analyzed the expression of p- Aurora-A, Aurora-A, p-ERK, ERK, p-AKT, and AKT in 8305 C cells with Alisertib treatment or combined with exogenous ATP (2 mM). K CCK-8 assays showed that Alisertib (10 μmol) significantly inhibited cell proliferation in 8305 C, which can be remarkably restored by exogenous ATP (2 mM) supplementation. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the Alisertib-treated group. L Transwell of migration and invasion assay detected the migration and invasion ability of 8305 C cells pretreated with Alisertib for 2 days or combined with exogenous ATP (2 mM). *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the Alisertib-treated group. The data are presented as the mean ± SD of three independent experiments.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Expressing, Over Expression, Western Blot, Knockdown, Migration, CCK-8 Assay, Invasion Assay, Control

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 5 Targeting Aurora-A improves the efficacy of Sorafenib treatment in TC cells in vitro. A, D Western blotting detected p-ERK, ERK, p- AKT, and AKT expression in 8305 C (A) and TPC-1 (D) cells, respectively, exposed to Alisertib (5 μmol), Sorafenib (1 μmol) and their combination for 48 h. B, E The CCK-8 assay was performed in 8305 C (B) and TPC-1 (E) cells exposed to Alisertib (5 μmol), Sorafenib (1 μmol) and both drugs for 96 h. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the combination group. C, F Transwell of migration and invasion assay detected the migration and invasion ability of 8305 C (C) and TPC-1 (F) cells pretreated with Alisertib (5 μmol), Sorafenib (1μmol) or both drugs for 2 days. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the combination group. The data are presented as the mean ± SD of three independent experiments.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: In Vitro, Western Blot, Expressing, CCK-8 Assay, Control, Migration, Invasion Assay

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 6 The combination of Alisertib and Sorafenib is a promising therapeutic approach for advanced TC in vivo. A Representative images of subcutaneous xenografts in NSG mice injected 8305 C cells treated with Alisertib (30 mg/kg), Sorafenib (30 mg/kg) or both drugs. n = 5 mice per group. The subcutaneous xenografts were dissected on day 13. B, C Tumor volumes were measured with tumor weights (B) and growth curves (C) in each group. D The expression levels of Ki67 and p-Aurora-A in xenografts of each group were assessed by immunohistochemical staining. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the combination group. The data are presented as the mean ± SD.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: In Vivo, Injection, Expressing, Immunohistochemical staining, Staining, Control