Journal: World Journal of Gastroenterology

Article Title: FBP1 as a key regulator of focal adhesion kinase-mediated hepatic stellate cell activation: Multi-omics and experimental validation

doi: 10.3748/wjg.v31.i28.107361

Figure Lengend Snippet: In mouse models of liver fibrosis, focal adhesion kinase inhibition ameliorates hepatic fibrosis. A: Liver tissues from mice in each group were stained with hematoxylin and eosin (HE) to observe inflammatory cell infiltration, cellular morphology, and lobular architecture; Masson’s trichrome staining and Sirius red staining were performed to assess fiber deposition; B: Immunohistochemical analysis was conducted to detect the expression of fructose-1,6-bisphosphatase 1 (FBP1) protein in the livers of mice from each group, and the number of positive cells in each group was statistically analyzed. Scale bar: 200 μm, 50 μm; C: Western blot (WB) analysis was used to detect the expression of liver fibrosis marker alpha smooth muscle actin (α-SMA), as well as focal adhesion kinase (FAK) and phosphorylated FAK (p-FAK) proteins, with β-actin as the internal control; D: Grayscale value analysis was performed, and statistical significance relative to the normal group or carbon tetrachloride (CCl 4 ) group was assessed; E: WB analysis was employed to detect the protein expression of aerobic glycolysis enzymes, including lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), pyruvate kinase M2 (PKM2), and FBP1, with β-actin as the internal control; F: Grayscale value analysis was conducted, and statistical significance relative to the normal group or CCl 4 group was evaluated. All data are from three independent samples. Data are represented as the mean ± SD. a Not significant; b P < 0.01, c P < 0.001, d P < 0.0001.

Article Snippet: Recombinant FBP1 protein (HY- P70275 ) and PF562271 were obtained from MedChemExpress.

Techniques: Inhibition, Staining, Immunohistochemical staining, Expressing, Western Blot, Marker, Control

Journal: World Journal of Gastroenterology

Article Title: FBP1 as a key regulator of focal adhesion kinase-mediated hepatic stellate cell activation: Multi-omics and experimental validation

doi: 10.3748/wjg.v31.i28.107361

Figure Lengend Snippet: Focal adhesion kinase inhibitors can modulate the activation, migration, and aerobic glycolysis of hepatic stellate cells. A: After adding focal adhesion kinase (FAK) inhibitor at different concentrations (2.5 μM, 5 μM, 10 μM), proteins were extracted, and Western blot (WB) analysis was performed to detect the expression of liver fibrosis markers collagen type 1, alpha 1 (COL1A1) and alpha smooth muscle actin (α-SMA), as well as FAK and phosphorylated FAK (p-FAK) proteins, with β-actin serving as the internal control; B: Grayscale value analysis was conducted, and statistical significance relative to the control (dimethyl sulfoxide [DMSO]) group was assessed; C: WB analysis was employed to detect the protein expression of lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), pyruvate kinase M2 (PKM2), and fructose-1,6-bisphosphatase 1 (FBP1) in each group; D: Grayscale value analysis was performed for each proteome, and statistical significance relative to the control (DMSO) group was evaluated. All data are from three independent samples. Data are represented as the mean ± SD. a P < 0.05; b P < 0.01; c P < 0.001; d P < 0.0001; e Not significant.

Article Snippet: Recombinant FBP1 protein (HY- P70275 ) and PF562271 were obtained from MedChemExpress.

Techniques: Activation Assay, Migration, Western Blot, Expressing, Control

Journal: World Journal of Gastroenterology

Article Title: FBP1 as a key regulator of focal adhesion kinase-mediated hepatic stellate cell activation: Multi-omics and experimental validation

doi: 10.3748/wjg.v31.i28.107361

Figure Lengend Snippet: Fructose-1,6-bisphosphatase 1 recombinant protein (HY- P70275 ) inhibits the activation, migration, and aerobic glycolysis of hepatic stellate cells. A: At concentrations of 12.5 ng/mL, 25 ng/mL, 50 ng/mL, and 100 ng/mL of fructose-1,6-bisphosphatase 1 (FBP1) recombinant protein, Western blot (WB) analysis was conducted to detect the expression of FBP1, collagen type 1, alpha 1 (COL1A1), and alpha smooth muscle actin (α-SMA) proteins, which are markers of liver fibrosis, with β-actin serving as the internal control; B: Grayscale value statistics were performed for each protein group, and statistical significance relative to the control (dimethyl sulfoxide [DMSO]) group was evaluated; C: At the same concentrations of FBP1 recombinant protein, WB analysis was used to detect the expression of aerobic glycolysis enzymes, including lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), and pyruvate kinase M2 (PKM2), with β-actin as the internal control; D: Grayscale value analysis was conducted for each proteome, and statistical significance relative to the control (DMSO) group was evaluated; E: At a concentration of 100 ng/mL of FBP1 recombinant protein, the migration ability of LX-2 cells in each group was assessed using a wound healing assay; F: The cell migration area relative to the control (DMSO) group was evaluated; G: At a concentration of 100 ng/mL of FBP1 recombinant protein, the migration ability of LX-2 cells in each group was assessed using a Transwell migration assay, observed under a light microscope at 100 × magnification; H: The number of cells that migrated through the membrane of the Transwell chamber relative to the control (DMSO) group was assessed. All data are from three independent samples. Data are represented as the mean ± SD. a Not significant; b P < 0.01, c P < 0.001, d P < 0.0001.

Article Snippet: Recombinant FBP1 protein (HY- P70275 ) and PF562271 were obtained from MedChemExpress.

Techniques: Recombinant, Activation Assay, Migration, Western Blot, Expressing, Control, Concentration Assay, Wound Healing Assay, Transwell Migration Assay, Light Microscopy, Membrane

Journal: World Journal of Gastroenterology

Article Title: FBP1 as a key regulator of focal adhesion kinase-mediated hepatic stellate cell activation: Multi-omics and experimental validation

doi: 10.3748/wjg.v31.i28.107361

Figure Lengend Snippet: Decreased expression of fructose-1,6-bisphosphatase 1 in patients with liver fibrosis. A: Hepatic fructose-1,6-bisphosphatase 1 (FBP1) expression was examined by immunohistochemistry in patients with liver fibrosis and controls; B: Immunohistochemical staining was statistically analyzed to quantify the number of positive cells in each group; C: Expression of hepatic fibrosis marker alpha smooth muscle actin (α-SMA) and FBP1 was detected by Western blot analysis in patients with liver fibrosis and controls; D: Grayscale values of the respective protein bands were analyzed, and statistical significance relative to the normal group was assessed. All data are from three independent samples. Data are represented as the mean ± SD. a P < 0.05; b P < 0.001.

Article Snippet: Recombinant FBP1 protein (HY- P70275 ) and PF562271 were obtained from MedChemExpress.

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining, Staining, Marker, Western Blot

Journal: Animal Research and One Health

Article Title: Compensation Response to Hepatic Gluconeogenesis via β‐Hydroxybutyrylation of FBP1 and PCK1 in Dairy Cows

doi: 10.1002/aro2.70022

Figure Lengend Snippet: FIGURE 2 | FBP1 and PCK1 undergo Kbhb in a time‐ and dose‐dependent manner. (A) HEK293T cells transfected with Flag‐FBP1, Flag‐FBP2, and Flag‐PCK1 were treated with 10‐mM BHB for 24 h and Kbhb modification was detected. (B, C) Kbhb modification of Flag‐FBP1 (B) and Flag‐ PCK1 (C) in HEK293T cells treated with 10‐mM BHB for the indicated time. (D, E) Kbhb modification of Flag‐FBP1 (D) and Flag‐PCK1 (E) in HEK293T cells treated with indicated concentrations of BHB for 24 h.

Article Snippet: FBP1 (12842‐ 1‐AP), PCK1 (16754‐1‐AP), G6PC (66860‐1‐Ig), and β‐actin (66009‐1‐Ig) antibodies were obtained from Proteintech (Chicago, USA).

Techniques: Transfection, Modification

Journal: Animal Research and One Health

Article Title: Compensation Response to Hepatic Gluconeogenesis via β‐Hydroxybutyrylation of FBP1 and PCK1 in Dairy Cows

doi: 10.1002/aro2.70022

Figure Lengend Snippet: FIGURE 3 | BHB enhances glucose levels in bovine hepatocytes. (A) The bovine hepatocytes were treated with the indicated concentration of BHB, and the glucose concentrations were detected by kit. (B–D) The bovine hepatocytes were treated with indicated concentration of BHB, and endogenous expression of FBP1, PCK1, and G6PC genes was examined by RT‐qPCR. (E) The bovine hepatocytes were treated with indicated concentration of BHB, and the indicated proteins were detected by Western blot. (F–H) Quantitative data for the indicated protein level was presented. Statistical analyses were performed with the Student's t test and one‐way ANOVA. *p < 0.05, ***p < 0.001, ns stands for no significant change.

Article Snippet: FBP1 (12842‐ 1‐AP), PCK1 (16754‐1‐AP), G6PC (66860‐1‐Ig), and β‐actin (66009‐1‐Ig) antibodies were obtained from Proteintech (Chicago, USA).

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Animal Research and One Health

Article Title: Compensation Response to Hepatic Gluconeogenesis via β‐Hydroxybutyrylation of FBP1 and PCK1 in Dairy Cows

doi: 10.1002/aro2.70022

Figure Lengend Snippet: FIGURE 4 | BHB is transported into the cell via MCT1. (A, B) Kbhb modification of Flag‐FBP1 (A) and Flag‐PCK1 (B) in HEK293T cells treated with 10‐mM BHB in combination with 10‐μM AZD3965 for 24 h. (C, D) MCT1 knockdown by siRNA transfection. Flag‐FBP1 (C) and Flag‐PCK1 (D) Kbhb levels were analyzed by immunoblotting with indicated antibodies. (E) The bovine hepatocytes were treated with BHB and 10‐μM AZD3965, and the glucose concentrations were detected by kit. (F) The bovine hepatocytes were treated with BHB and MCT1 knockdown by siRNA, and the glucose concentrations were detected by kit. Statistical analyses were performed with the one‐way ANOVA. ***p < 0.001.

Article Snippet: FBP1 (12842‐ 1‐AP), PCK1 (16754‐1‐AP), G6PC (66860‐1‐Ig), and β‐actin (66009‐1‐Ig) antibodies were obtained from Proteintech (Chicago, USA).

Techniques: Modification, Knockdown, Transfection, Western Blot

Journal: Animal Research and One Health

Article Title: Compensation Response to Hepatic Gluconeogenesis via β‐Hydroxybutyrylation of FBP1 and PCK1 in Dairy Cows

doi: 10.1002/aro2.70022

Figure Lengend Snippet: FIGURE 5 | Kbhb of FBP1 and PCK1 is catalyzed by p300 and removed by HDAC. (A, B) P300 knockdown by siRNA in HEK293T cells. Flag‐FBP1 (A) and Flag‐PCK1 (B) Kbhb levels were analyzed by immunoblotting with indicated antibodies. (C, D) Immunoprecipitated FBP1 (C) and PCK1 (D) proteins were incubated with recombinant p300 protein and β‐hydroxybutyryl‐CoA (BHB‐CoA, 20 μM) for 1 h at 30°C, pH 8.0. (E, F) Kbhb modification of Flag‐FBP1 (E) and Flag‐PCK1 (F) in HEK293T cells treated with 10‐mM BHB in combination with NAM or TSA for 24 h. (G) The bovine hepatocytes with p300 knockdown were treated with 10‐mM BHB for 24 h and the glucose concentrations were detected by kit. (H) The bovine hepatocytes were treated with BHB and 50‐nM TSA, and the glucose concentrations were detected by kit. Statistical analyses were performed with the one‐way ANOVA. ***p < 0.001.

Article Snippet: FBP1 (12842‐ 1‐AP), PCK1 (16754‐1‐AP), G6PC (66860‐1‐Ig), and β‐actin (66009‐1‐Ig) antibodies were obtained from Proteintech (Chicago, USA).

Techniques: Knockdown, Western Blot, Immunoprecipitation, Incubation, Recombinant, Modification

Journal: Animal Research and One Health

Article Title: Compensation Response to Hepatic Gluconeogenesis via β‐Hydroxybutyrylation of FBP1 and PCK1 in Dairy Cows

doi: 10.1002/aro2.70022

Figure Lengend Snippet: FIGURE 6 | Kbhb of FBP1 and PCK1 promotes their activity. (A) The bovine hepatocytes were treated with 10‐mM BHB and CHX for the indicated time, and then Western blot was used to evaluate the levels of the indicated proteins. (B, C) The bovine hepatocytes were treated with 10‐mM BHB alone or in combination with 10‐μM AZD3965 for 24 h, and the activity of FBP1 (B) and PCK1 (C) was detected by ELISA kit. (D, E) The bovine hepatocytes were treated with 10‐mM BHB alone or in combination with MCT1 knockdown by siRNA, and the activity of FBP1 (D) and PCK1 (E) was detected by ELISA kit. (F, G) The bovine hepatocytes were treated with 10‐mM BHB alone or in combination with p300 knockdown by siRNA, and the activity of FBP1 (F) and PCK1 (G) was detected by ELISA kit. Statistical analyses were performed with the one‐ way ANOVA. ***p < 0.001.

Article Snippet: FBP1 (12842‐ 1‐AP), PCK1 (16754‐1‐AP), G6PC (66860‐1‐Ig), and β‐actin (66009‐1‐Ig) antibodies were obtained from Proteintech (Chicago, USA).

Techniques: Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Animal Research and One Health

Article Title: Compensation Response to Hepatic Gluconeogenesis via β‐Hydroxybutyrylation of FBP1 and PCK1 in Dairy Cows

doi: 10.1002/aro2.70022

Figure Lengend Snippet: FIGURE 7 | FBP1 and PCK1 Kbhb occur at lysine 43 and 191, respectively. (A) Schematic representation of experimental workflow for the identification of Kbhb‐containing protein substrates in HEK293T cells. (B, C) Illustration of PCK1 K191bhb (B) and FBP1 K43bhb (C) identified by mass spectrometry (MS). (D) Detecting FBP1 Kbhb in HEK293T cells transfected with FBP1 WT and K34R mutant plasmids and treated with 10‐mM BHB for 24 h. (E) Detecting PCK1 Kbhb in HEK293T cells transfected with PCK1 WT and K191R mutant plasmids and treated with 10‐ mM BHB for 24 h. (F, G) Immunoprecipitated FBP1 WT and K43R (F) and PCK1 WT and K191R (G) mutant plasmids were incubated with recombinant p300 protein and BHB‐CoA for 1 h at 30°C, pH 8.0, and the levels of Kbhb were detected by Western blot.

Article Snippet: FBP1 (12842‐ 1‐AP), PCK1 (16754‐1‐AP), G6PC (66860‐1‐Ig), and β‐actin (66009‐1‐Ig) antibodies were obtained from Proteintech (Chicago, USA).

Techniques: Mass Spectrometry, Transfection, Mutagenesis, Immunoprecipitation, Incubation, Recombinant, Western Blot

Journal: Animal Research and One Health

Article Title: Compensation Response to Hepatic Gluconeogenesis via β‐Hydroxybutyrylation of FBP1 and PCK1 in Dairy Cows

doi: 10.1002/aro2.70022

Figure Lengend Snippet: FIGURE 8 | BHB increases glucose levels and activity of FBP1 and PCK1 through K43bhb and K191bhb, respectively. (A, B) The bovine hepatocytes transfected with FBP1 WT or K43R and treated with indicated concentrations of BHB were utilized for glucose concentration (A) and Western blot (B). (C, D) The bovine hepatocytes transfected with PCK1 WT or K191R and treated with indicated concentrations of BHB were utilized for glucose concentration (C) and Western blot (D). (E, F) The bovine hepatocytes transfected with FBP1 WT or K43R and treated with indicated concentrations of BHB were utilized for the activity of FBP1 (E) and Western blot (F). (G, H) The bovine hepatocytes transfected with PCK1 WT or K191R and treated with indicated concentrations of BHB were utilized for the activity of PCK1 (G) and Western blot (H). (I, J) HEK293T cells transfected with Flag‐FBP1 and Flag‐PCK1 from humans and cows were treated with indicated concentrations of BHB for 24 h, and Kbhb modification was detected. Statistical analyses were performed with the Student's t test. *p < 0.05, ***p < 0.001, ns stands for no significant change.

Article Snippet: FBP1 (12842‐ 1‐AP), PCK1 (16754‐1‐AP), G6PC (66860‐1‐Ig), and β‐actin (66009‐1‐Ig) antibodies were obtained from Proteintech (Chicago, USA).

Techniques: Activity Assay, Transfection, Concentration Assay, Western Blot, Modification

Journal: Molecular Systems Biology

Article Title: High‐fat diet in a mouse insulin‐resistant model induces widespread rewiring of the phosphotyrosine signaling network

doi: 10.15252/msb.20198849

Figure Lengend Snippet:

Article Snippet: F16P1 , Cell Signaling Technology , 59172.

Techniques: Sequencing, BIA-KA, Membrane, Blocking Assay, Reverse Transcription, Isolation, SYBR Green Assay, Suspension, Software, Cell Culture

Journal: Journal of Virology

Article Title: FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53

doi: 10.1128/jvi.00729-15

Figure Lengend Snippet: Figure 1. FBP1 is specifically overexpressed in human HCC tumors with CHC background. (A). 677

Article Snippet: FBP1 shRNA lentiviral particles and P53 siRNA sense (GCA UGA ACC GGA 121 GGC CCA Utt) and antisense (5’-AUG GGC CUC CGG UUC AUG Ctt-3’) were purchased from Santa 122 Cruz Biotechnology Lentiviral particles expressing shRNAs targeting FBP1, p53, or BCCIP were 123 purchased from Santa Cruz Biotechnology (CA).

Techniques:

Journal: Journal of Virology

Article Title: FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53

doi: 10.1128/jvi.00729-15

Figure Lengend Snippet: Fig. 4. The downregulation of p53 and FBP1 have the opposite effect on p21-expression and HCV 716

Article Snippet: FBP1 shRNA lentiviral particles and P53 siRNA sense (GCA UGA ACC GGA 121 GGC CCA Utt) and antisense (5’-AUG GGC CUC CGG UUC AUG Ctt-3’) were purchased from Santa 122 Cruz Biotechnology Lentiviral particles expressing shRNAs targeting FBP1, p53, or BCCIP were 123 purchased from Santa Cruz Biotechnology (CA).

Techniques: Expressing

Journal: Journal of Virology

Article Title: FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53

doi: 10.1128/jvi.00729-15

Figure Lengend Snippet: Figure 7. FBP1 promotes HCV replication by regulating p53 and its regulatory proteins, TCTP, and 810

Article Snippet: FBP1 shRNA lentiviral particles and P53 siRNA sense (GCA UGA ACC GGA 121 GGC CCA Utt) and antisense (5’-AUG GGC CUC CGG UUC AUG Ctt-3’) were purchased from Santa 122 Cruz Biotechnology Lentiviral particles expressing shRNAs targeting FBP1, p53, or BCCIP were 123 purchased from Santa Cruz Biotechnology (CA).

Techniques:

Journal: Journal of Virology

Article Title: FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53

doi: 10.1128/jvi.00729-15

Figure Lengend Snippet: Fig. 8. The migration of FBP1-kd Huh7.5 cells is drastically reduced. 849

Article Snippet: FBP1 shRNA lentiviral particles and P53 siRNA sense (GCA UGA ACC GGA 121 GGC CCA Utt) and antisense (5’-AUG GGC CUC CGG UUC AUG Ctt-3’) were purchased from Santa 122 Cruz Biotechnology Lentiviral particles expressing shRNAs targeting FBP1, p53, or BCCIP were 123 purchased from Santa Cruz Biotechnology (CA).

Techniques: Migration

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Far Upstream Element-Binding Protein 1 Binds the 3′ Untranslated Region of PKD2 and Suppresses Its Translation

doi: 10.1681/ASN.2015070836

Figure Lengend Snippet: Effect of FUBP1 on PKD2 protein and mRNA levels. (A) Effect of FUBP1 overexpression on PKD2 in HeLa cells. Left panel shows representative WB data. Center panel shows quantified WB data averaged from n=3 independent experiments. Right panel shows PKD2 mRNA expression by RT-PCR. (B) Effect of FUBP1 siRNA KD on PKD2 expression in HEK cells. Left panel shows representative WB data. Center panel shows quantified WB data averaged from three independent experiments. Right panel shows PKD2 mRNA expression obtained by RT-PCR. (C) Effect of FUBP1 overexpression on activity of luciferase without (BI16) or with 3FI (BI16–3FI) as 3′UTR in HeLa cells. (D) Effect of FUBP1 siRNA KD on activity of luciferase without (BI16) or with 3FI (BI16–3FI) as 3′UTR in HEK cells. Data were obtained from n=3 independent experiments. *P<0.05; **P<0.01.

Article Snippet: KD of human FUBP1 by siRNA was performed using oligonucleotides at a final concentration of 20 nM with transfection reagent Lipofectamine 2000 according to the manufacturer's instructions. siRNA oligonucleotides were purchased from Santa Cruz Biotechnology (Cat no.: sc-43760) and control siRNA from Gene Pharma (Shanghai, China).

Techniques: Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Luciferase

Journal: BMC Cancer

Article Title: Fuse binding protein antagonizes the transcription activity of tumor suppressor protein p53

doi: 10.1186/1471-2407-14-925

Figure Lengend Snippet: FBP negatively affects p21 and BCCIP expression in Huh7 cells. (A) Stably FBP-knockdown (FBP-kd) Huh7 cells by expressing FBP1 targeting shRNA: Huh7 cells were transfected with lentivirus vector encoding FBP1 targeting shRNAs or with empty vector alone. Stable clones (NP-4, NP-5) were selected after several passages via puromycin selection and were confirmed for stable knockdown of FBP1 expression by Western blot analysis as compared to cells transformed with the vector alone (NP-2, NP-3). (B) . Expression of p53 and p21 in FBP-kd cells. Huh7 cells stably transduced with FBP1 shRNA (NP-4) or empty vector alone (NP-2) were grown for 48 h. Cell lysates were normalized with respect to protein and Western blotted for the expression of FBP1, p53, p21, and actin. Lane 1, control Huh7 cells; lane 2, FBP-kd Huh7 cells; lane 3, Huh7 cells transduced with a lentivirus vector only. (C) Construction of FBP1 expression clone resistant to FBP1-shRNA. The FBP1-shRNA targeted sequences spanning codons 248 to 254 and from 560 to 567 were subjected to point mutation in FBP1 expression clone (pCIA-CMV-FBP) without changing the amino acid sequence. (D) Transient expression of FBP1 in FBP-kd cells suppressed the expression of p21. FBP-kd cells were transfected with shRNA resistant FBP1 expression clone ((pCIA-CMV-FBP SHR ) or empty pCIA-CMV vector; 48-h later cells lysates were Western blotted for FBP1, p53 and p21 and Actin. Lane 1, control Huh7 cells, lane 2, Huh7 cells transduced with lentiviral vector alone; lane 3, FBP-kd cells; lane 4, FBP-kd cells transfected with empty vector; lane 5, FBP-kd cells transfected with shRNA resistant FBP1 expression clone. (E) Expression level of p21 in control and p53-kd, BCCIP-kd, and FBP-kd Huh7 cells. Lane1, control Huh7 cells; lane 2, lentivirus vector control; lane 3, p53-kd; lane 4, BCCIP-kd and lane 5, FBP-kd Huh7 cells. A colon cancer cell line, HCT116 without p53 (-/-) (lane 6) or with wild-type p53 (+/+) (lane 7) was used as controls.

Article Snippet: We generated stably transduced Huh7 cells with lentivirus vectors encoding shRNA targeting FBP1, p53, or BCCIP (Santa Cruz, CA) or with empty vector alone (SC-108080) following the manufacturer’s protocol.

Techniques: Expressing, Stable Transfection, Knockdown, shRNA, Transfection, Plasmid Preparation, Clone Assay, Selection, Western Blot, Transformation Assay, Transduction, Control, Mutagenesis, Sequencing

Journal: BMC Cancer

Article Title: Fuse binding protein antagonizes the transcription activity of tumor suppressor protein p53

doi: 10.1186/1471-2407-14-925

Figure Lengend Snippet: FBP interacts with p53 and its regulatory proteins, and regulates their expression. (A) Effect of radiation-induced stress on the expression of p53, p21, BCCIP and TCTP in control and FBP-kd Huh 7 cells. Cells grown for 48 h were irradiated with 3-Gy of γ-ray. The irradiated cells were grown for 0, 3, 6, and 12 h and examined for the expression of p53, p21, BCCIP and TCTP by Western blotting. Lanes 1-4, show results from cells grown for 0, 3, 6 and 12 h post-irradiation (B) Fold change in the mRNA level of FBP1, p53, p21, BCCIP and TCTP in irradiated control and FBP-kd Huh7 cells. Cells grown for 48 h were irradiated with 3-Gy of γ-ray and grown for 0, 3, 6, and 12 h. Fold changes in mRNA level of FBP1, p53, p21, BCCIP, and TCTP were determined by real-time quantitative PCR. Upper and lower panels show fold change in mRNA level in irradiated control and FBP-kd cells, respectively. Lanes 1-4, show results from cells grown for 0, 3, 6 and 12 h post irradiation. (C, D) Co-immunoprecipitation of FBP1, p53 and BBCIP. IP was done with either (C) anti-FBP1 antibody or (D) anti-BCCIP antibody, and the IP complexes were captured on protein A/G PLUS-agarose beads, resolved on 8% polyacrylamide SDS-PAGE gel and Western blotted for p53, BCCIP and TCTP and FBP1. (E) FBP, p53 and BCCIP form ternary complex in-vitro. We incubated 500 ng of each of the recombinant FBP, p53 and BCCIP at 4 o C for 30 min. The complex was then subjected to FBP-IP, p53-IP or BCCIP-IP. The IP complex was resolved by SDS-PAGE and Western blotted for FBP, p53 and BCCIP. Lanes 1-3, show ternary complex of FBP1-p53-BCCIP captured by immunoprecipitation of FBP, p53 and BCCIP. Lane 4-6 show the formation of binary complexes between FBP1-p53, p53-BCCIP and FBP1-BCCIP, respectively.

Article Snippet: We generated stably transduced Huh7 cells with lentivirus vectors encoding shRNA targeting FBP1, p53, or BCCIP (Santa Cruz, CA) or with empty vector alone (SC-108080) following the manufacturer’s protocol.

Techniques: Expressing, Control, Irradiation, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page, In Vitro, Incubation, Recombinant

Journal: BMC Cancer

Article Title: Fuse binding protein antagonizes the transcription activity of tumor suppressor protein p53

doi: 10.1186/1471-2407-14-925

Figure Lengend Snippet: The transactivation activity of p53 is activated in FBP-kd Huh7 cells. (A) Activation of p53 transcription activity in FBP-kd Huh7 cells. Reporter vectors were transfected to control Huh 7 cells or FBP-kd cells as described in the Methods. Forty-eight hours later, cells were irradiated with 4 Gy of γ-irradiation. Luciferase activities were measured 6-hour post-irradiation treatment. p53 reporter activity (left panel), p21 reporter activity (right panel). (B) Transient expression of FBP1 in FBP-kd cells suppresses p53 transcription activities. FBP-kd cells were transfected with shRNA resistant FBP1 expression clone (pCIA-CMV-FBP SHR ) along with reporter vectors; 48-h later cells were irradiated with 3 Gy of γ-irradiation. Luciferase activities were measured 6-hour post-irradiation treatment. p53 reporter activity (left panel), p21 reporter activity (right panel). (C) Relative p53 transcription activity in BCCIP-kd, p53-kd and FBP-kd cells. Huh7 cells knockdown for BCCIP; p53 and FBP1 were transfected with reporter vectors; 48-h later cells were irradiated with 3 Gy of γ-irradiation. Luciferase activities were measured 6-hour post-irradiation treatment. p53 reporter activity (left panel), p21 reporter activity (right panel).

Article Snippet: We generated stably transduced Huh7 cells with lentivirus vectors encoding shRNA targeting FBP1, p53, or BCCIP (Santa Cruz, CA) or with empty vector alone (SC-108080) following the manufacturer’s protocol.

Techniques: Activity Assay, Activation Assay, Transfection, Control, Irradiation, Luciferase, Expressing, shRNA, Knockdown