human inpp4b cdna sequence  (Addgene inc)

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Primer sequences for the genes analyzed by real-time PCR analysis. FW: forward primer; RV: reverse primer.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: INPP4B expression in different retinoblastoma cell lines and primary RB patient tumors. The INPP4B expression in the healthy human retina (hRet) and chemosensitive as well as etoposide resistant (Etop) Y79 and RB355 retinoblastoma cell lines as revealed by quantitative real-time PCR (a) and western blot analyses (b). ß -Actin served as a loading control in (c) quantification of INPP4B protein expression in chemosensitive compared to etoposide resistant (Etop) Y79 and RB355 retinoblastoma cell lines. (d) INPP4B expression levels in primary RB patient tumors after treatment with chemotherapeutics ( n = 10) and without treatment ( n = 11). INPP4B expression values relative to expression in the healthy human retina and normalized to the expression of ribosomal 18S, used as an internal control. All experiments were performed at least in triplicate, and results were depicted with a standard error of the mean (SEM). A student's t -test was used to calculate statistical differences (ns p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001) between control and experimental groups.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Verification of an efficient INPP4B overexpression (OE) in etoposide resistant Y79 and RB355 cells after lentiviral transduction as revealed by quantitative real-time PCR (a) and western blot analysis (b, c). ß -actin served as a loading control and micro manager 1.4 software was used to calculate the relative intensity ratios. All experiments were performed at least in triplicates and results depicted with a standard error of the mean (SEM). A student's t -test was used to calculate statistical differences ( ∗ p < 0.05; ∗∗∗ p < 0.001) between control and experimental groups.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Over Expression, Transduction, Real-time Polymerase Chain Reaction, Western Blot, Control, Software

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Effects of INPP4B overexpression in etoposide resistant (Etop) Y79 and RB355 cells. INPP4B overexpression (OE) significantly reduces cell growth of Y79-Etop (a) and RB355-Etop (b) cells with significant reduced cell viability for both RB cell lines and reduced proliferation level for RB355-Etop cells compared to control cells (ctr), as revealed by growth curves (a, b), WST-1 assays (c), and BrdU stains (d). All experiments were performed at least in triplicate, and results depicted with a standard error of the mean (SEM). A student's t -test was used to calculate statistical differences (ns p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001) between control and experimental groups.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Over Expression, Control

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Effects of INPP4B overexpression on apoptosis levels in etoposide-resistant Y79 and RB355 cells. INPP4B overexpression (OE) increases the caspase-mediated apoptosis levels of etoposide-resistant (Etop) Y79 and RB355 cells compared to control cells (ctr) as revealed by DAPI cell counts (a) and caspase 3/7 assays (b). (c) Exemplary immunocytochemical stains of INPP4B overexpressing etoposide resistant Y79 and RB355 and control cells with an active, cleaved caspase-3 antibody (red fluorescence) and DAPI (blue fluorescence) counterstaining. White arrowheads indicate capase-3 positive cells. Magnification: 200x. All experiments were performed at least in triplicate and results were depicted with a standard error of the mean (SEM). A student's t -test was used to calculate statistical differences ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001) between control and experimental groups.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Over Expression, Control, Fluorescence

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Effect of INPP4B overexpression on contact-independent growth of etoposide resistant Y79 and RB355 cells as revealed by soft agarose assays. Both RB cell lines display significantly reduced colony numbers (a), and RB355-Etop colonies were significantly smaller (b). (c) Representative photographs of colonies formed in soft agarose. All experiments were performed at least in triplicate and results depicted with a standard error of the mean (SEM). A student's t -test was used to calculate statistical differences (ns p > 0.05; ∗∗∗ p < 0.001) between the control and experimental groups.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Over Expression, Control

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Impact of lentiviral INPP4B upregulation in etoposide resistant RB (etop) cells on tumor formation in CAM assays. (a) The left photo panel depicts CAM tumors in ovo , and the right panel measuring (in cm) of excised tumors. In vivo CAM assays revealed that tumors forming from INPP4B overexpressing (OE), etoposide resistant (Etop) Y79, and RB355 cells were smaller than those developing from control cells (ctr). (b–d) Graphical evaluation of CAM tumor weight (b), size (c), and tumor formation capacity (d). A student's t -test was used to calculate statistical differences (ns p > 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001) between control and experimental groups.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: In Ovo, In Vivo, Control

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Depiction of the migratory potential of GFP-labelled, etoposide resistant (Etop) INPP4B overexpressing (OE) Y79 and RB355 cells and control cells (ctr) after injection into a CAM vein. (a) Representative CAM whole mounts stained for CAM vessels with an antidesmin antibody (red fluorescence). Extravasated GFP-labelled INPP4B overexpressing, etoposide resistant Y79 and RB355 cells are displayed in green. Magnification: 200x. (b) Quantification of GFP-positive (GFP+) punches of the lower CAM. All experiments were performed at least in triplicate and the results were depicted with a standard error of the mean (SEM). A student's t -test was used to calculate statistical differences (ns p > 0.05; ∗∗ p < 0.01) between control and experimental groups.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Control, Injection, Staining, Fluorescence

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Western blot analyses of AKT/p-AKT and SGK3/p-SGK3 expression levels after INPP4B overexpression in etoposide resistant (Etop) RB cell lines. Representative western blots depicting AKT/p-AKT (a) and SGK3/p-SGK3 expression levels (b) after INPP4B overexpression (OE) in etoposide-resistant Y79 and RB355 cells. ß -actin served as a loading control and micro manager 1.4 software was used to calculate the relative intensity ratios.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Western Blot, Expressing, Over Expression, Control, Software

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Volcano plots of differentially expressed genes (DEGs) after INPP4B overexpression in etoposide (Etop) resistant Y79 (a) and RB355 (b) cells compared to control cells. Downregulated DEGs are depicted in green, upregulated DEGs are depicted in red; nonregulated genes are depicted in blue. The red circle indicates INPP4B expression.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Over Expression, Control, Expressing

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: UMAP analysis and heatmap of the responsive gene selection (RGS) after INPP4B overexpression in etoposide (Etop) resistant RB cells and control cells. (a) UMAP analysis of three biological replicates of etoposide resistant Y79-Etop and RB355-Etop cells. (b) Heatmap of significantly up- and downregulated RGSs with a minimum fold chance of 1.5 after INPP4B overexpression in Y79-Etop (b) and RB355-Etop (c) cells.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Selection, Over Expression, Control

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Responsive gene selection (RGS) after INPP4B overexpression in etoposide-resistant Y79 cells. Genes with positive fold change (FC) values represent upregulated genes after INPP4B overexpression, while negative FC values indicate downregulated genes.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Selection, Over Expression, Activity Assay, Protein-Protein interactions, Activation Assay, Membrane, Chemotaxis Assay, Protein Binding, Binding Assay, Migration, RNA Binding Assay, Marker, Biomarker Discovery

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Responsive gene selection (RGS) after INPP4B overexpression in etoposide resistant RB355 cells. Genes with positive fold change (FC) value represent upregulated genes after INPP4B overexpression, while negative FC values indicate downregulated genes.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Selection, Over Expression, Migration, Activity Assay, Protein-Protein interactions, Ubiquitin Proteomics, Binding Assay, RNA Binding Assay, Transduction, Membrane

Journal: Journal of Oncology

Article Title: Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma

doi: 10.1155/2023/2270097

Figure Lengend Snippet: Validation of differentially expressed genes identified by RNAseq analysis after INPP4B overexpression in Y79-Etop (a) and RB355-Etop (b) RB cells. Quantitative real-time PCR verified the significant upregulation of CAPG , HCST , IFIT2 , PRAMEF27, CABP1 , and TNFSF4 (red bars) after INPP4B overexpression. Downregulation of the genes HYAL3, GALP, EMP1, PTAFR , and PLK5 did not reach significance (green bars). All experiments were performed at least in triplicates and results depicted with a standard error of the mean (SEM). A student's t -test was used to calculate statistical differences ( ∗ p value <0.05) between control and experimental groups.

Article Snippet: To generate the INPP4B overexpression vector (pLenti_CMV_INPP4B), the human INPP4B cDNA sequence was cut from the pEAK-Flag/INPP4B plasmid (#24324, Addgene, Watertown, MA, USA; [ ]) via XhoI and NotI fast digest restriction enzymes (Thermo Scientific, Oberhausen, Germany) and ligated into the XhoI and NotI digested pENTR4 vector (#17424; Addgene, Watertown, MA, USA; [ ]).

Techniques: Biomarker Discovery, Over Expression, Real-time Polymerase Chain Reaction, Control

Journal: Cancers

Article Title: Endosomal mTORC2 Is Required for Phosphoinositide-Dependent AKT Activation in Platelet-Derived Growth Factor-Stimulated Glioma Cells

doi: 10.3390/cancers13102405

Figure Lengend Snippet: Activation of AKT by mTORC2 is mediated by early endosomal PtdIns(3,4)P 2 : ( A ) schematic representation of the heterodimerization of mCherry-FKBP-MTM1 (3-phosphatase), mCherry-FKBP-SJ2-Sac1 (4-phosphatase), or CFP- FKBP-Inp54p (5-phosphatase) to early endosomal membrane-anchored iRFP-FRB-Rab5, following addition of rapamycin; ( B ) representative confocal images of U87MG cells coexpressing iRFP-FRB-Rab5 and mCherry-FKBP-MTM1 (top, red), mCherry-FKBP-SJ2-4Ptase (middle, red), or CFP-FKBP-Inp54p (bottom, red) in the presence of rapamycin (40 nM) during PDGF activation for 5 min. Cells were fixed and stained with antibodies to pAKT(Ser473) (green). Scale bars, 20 μm; insets, 2 μm; ( C , D ) the relative fluorescence intensity ratios of pAKT(Ser473) to targeted phosphatase, respectively, were evaluated at the early endosomes in the presence of rapamycin (40 nM) during PDGF–BB activation for 5 min ( C ). The relative fluorescence intensity ratios of total pAKT(Thr308) to the targeted phosphatase were also measured in the presence of rapamycin (40 nM) during PDGF–BB activation for 5 min ( D ). Data are presented as the mean ± SEM from three independent experiments ( n = 11–17 cells examined for each experiment), and p -values were calculated using Student’s two-tailed t -test. * p < 0.05, ** p < 0.01, N.S., not significant; ( E ) representative confocal images of U87MG cells coexpressing iRFP-FRB-Rab5 and mCherry-FKBP-MTM1 (top, red), mCherry-FKBP-INPP4B-4Ptase (middle, red), or CFP-FKBP-Inp54p (bottom, red) in the presence of rapamycin (40 nM) during PDGF–BB activation for 5 min. Cells were fixed and stained with antibodies to PtdIns(3,4)P 2 (green). Arrowheads indicate PtdIns(3,4)P 2 in targeted 5-phosphatase. Scale bars, 10 μm; insets, 1 μm; ( F ) the relative fluorescence intensity ratios of PtdIns(3,4)P 2 to targeted phosphatase, respectively, were evaluated at the early endosomes in the presence of rapamycin (40 nM) during PDGF–BB activation for 5 min. Data are presented as the mean ± SEM from three independent experiments ( n = 10–11 cells examined for each experiment), and p -values were calculated using Student’s two-tailed t -test. * p < 0.05, ** p < 0.01.

Article Snippet: The mCherry-FKBP-INPP4B plasmid was generated using PCR amplification from the pEAK-Flag/INPP4B plasmid containing full-length INPP4B cDNA (24324; Addgene, USA) and cloning into the mCherry-FKBP plasmid.

Techniques: Activation Assay, Membrane, Staining, Fluorescence, Two Tailed Test