lv nep ys teto fuw ng2 p2a egfp t2a puro plasmid  (Addgene inc)

Journal: Frontiers in Immunology

Article Title: Gene therapy targeting inflammatory pericytes corrects angiopathy during diabetic wound healing

doi: 10.3389/fimmu.2022.960925

Figure Lengend Snippet: OXR1 relieves the oxidative stress, reduces the apoptosis, and recovers the growth potential in diabetic pericytes. (A) Schematic of an adenovirus carrying OXR1 under a pericyte-specific neuron glia antigen-2 (NG2) promoter (AV-NG2p-OXR1) and a control adenovirus carrying a scramble sequence (SCR) under the NG2 promoter (AV-NG2p-SCR). Both viruses also carried a blue fluorescent protein (BFP) as a reporter connected with the transgene with an IRES to be controlled together under the NG2 promoter. (B) Pericytes from DS (PC-DS) transduced by both viruses in culture. (C) CCK-8 assay for PC-NS transduced with either virus. (D, E) An annexin V apoptosis assay by representative flowcharts (D) and by quantification (E) . (F) RT-qPCR for apoptosis-associated protein Caspase 3, Caspase 9, and Cytochrome 9. (G) NOS activity. * p < 0.05. NS, no significance. N = 5. Scale bars are 10 µm.

Article Snippet: A backbone plasmid (Ng2 promoter-CRE-IRES-RFP) was purchased from Applied Biological Materials Inc. (000844A, Richmond, BC V6V 2J5, Canada) and then modified.

Techniques: Sequencing, CCK-8 Assay, Transduction, Apoptosis Assay, Quantitative RT-PCR, Activity Assay

Journal: Oncotarget

Article Title: The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

doi: 10.18632/oncotarget.3716

Figure Lengend Snippet: Figure 1: Immunohistochemical and western blotting analysis validates NG2 upregulation in human DIPG. (a) In human tissue, diffuse expression of NG2 was observed in DIPG tumor localized to membrane (inset). (b) Adjacent healthy brainstem tissue. Scale bar: 100 µM. (c) Graphical representation of western blotting data of NG2 expression in 22 DIPG tumor and 16 adjacent normal specimens. GAPDH was used for protein loading normalization. Average NG2 expression was plotted after normalizing NG2 levels to GAPDH in each sample. P < 0.05 was considered significant. (d) Average of normalized NG2 expression levels in NG2 western blot samples with histone 3.1 K27M mutation or histone 3.3 K27M mutation. P < 0.05 was considered significant.

Article Snippet: After 72 h, cell lysates Oncotarget12152www.impactjournals.com/oncotarget were prepared and luciferase expression was determined using luciferase assay kit according to the manufacturer’s protocol (Promega). nG2 knockdown with shrnA and mir129-2 PDGFB mouse neurospheres and adherent mouse tumor cells were transfected with NG2 specific shRNA (Origene) or control shRNA or miR129-2 plasmids or empty vector using XtremeGene HP transfection reagent, according to the manufacturer’s protocol (Roche).

Techniques: Immunohistochemical staining, Western Blot, Expressing, Membrane, Mutagenesis

Journal: Oncotarget

Article Title: The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

doi: 10.18632/oncotarget.3716

Figure Lengend Snippet: Figure 2: NG2 overexpression in PDGFB and PDGFB/H3.3 K27M/p53-/- mouse models. (a) In mouse, expression of NG2 in PDGFB mouse was limited to brainstem tumor (dotted area). (b) Sporadic expression of NG2 across brainstem and cerebellum of normal mouse. (c) Injection of NSG SCID mouse with mouse DIPG cells that are H3.3.K27 mutant and have PDGFB overexpression and p53 deletion resulted in overexpression of NG2 (dotted area). (d) In the adjacent normal brain tissue of NSG SCID mouse injected with PDGFB/ H3.3.K27M/p53-/- cells, NG2 overexpression was not detected. Insets are 40 × magnifications of the corresponding panel. Scale bar: 200 µM.

Article Snippet: After 72 h, cell lysates Oncotarget12152www.impactjournals.com/oncotarget were prepared and luciferase expression was determined using luciferase assay kit according to the manufacturer’s protocol (Promega). nG2 knockdown with shrnA and mir129-2 PDGFB mouse neurospheres and adherent mouse tumor cells were transfected with NG2 specific shRNA (Origene) or control shRNA or miR129-2 plasmids or empty vector using XtremeGene HP transfection reagent, according to the manufacturer’s protocol (Roche).

Techniques: Over Expression, Expressing, Injection, Mutagenesis

Journal: Oncotarget

Article Title: The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

doi: 10.18632/oncotarget.3716

Figure Lengend Snippet: Figure 3: NG2 upregulation in DIPG is partially due to hypermethylation and downregulation of its regulatory microRNA, miR129-2. (a) Luciferase vector with 3’-UTR sequence of NG2 cloned downstream of the firefly luciferase gene (Luc- 3’UTR) and plasmid vector with miR129-2 sequence were used. PDGFB mouse neurospheres were transfected in triplicates with luciferase and miR129-2 vectors, as shown, and luciferase expression was determined. Co-expression of Luc-3’UTR of NG2 and miR129-2 resulted in significant downregulation of luciferase expression as compared to cells transfected with luc-3’UTR and empty vector. P < 0.05 was considered significant. (b) PDGFB mouse neurospheres were transfected with miR129-2 or NG2-shRNA plasmids. Upregulation of either miR129-2 or NG2-shRNA resulted in NG2 downregulation in vitro when compared to empty vector or control shRNA treated cells. (c) Human DIPG cells, SF8628, were transduced with GFP expressing pCDH-control or pCDH-miR129-2 lentivirus. Immunofluorescence analysis by confocal microscopy revealed upregulation of miR129-2 (indicated by GFP) resulted in NG2 (red) downregulation in vitro when compared to control transduced cells. DAPI was used to stain nuclei. Scale bar = 5 µm. (d) Average expression of miR129-2 as assessed by RT-PCR (*miR129) or illumina expression chip assay (miR129), miR129 hypermethylation (miR129 me), and NG2 mRNA levels in human DIPG compared to adjacent normal. miR129-2 is shown to be downregulated in DIPG as assessed by RT-PCR and illumina chip assays. Hypermethylation of miR129-2 at four or more CpG sites corresponding to miR129-2 promoter were detected in 57% of DIPGs. As expected, downregulation of miR129-2 due to hypermethylation resulted in upregulation of NG2 mRNA as detected by NG2 mRNA using illumina platform. (e) To reverse miR129-2 hypermethylation, PDGFB mouse neurospheres were treated with 5-Azacytidine, a DNA methyl-transferase inhibitor drug. NG2 expression decreased in mouse tumor cells when treated with 10-30 µM 5-Azacytidine as compared to untreated cells. NG2 expression in each sample was normalized to corresponding GAPDH signal and average NG2 expression from three experiments was plotted.

Article Snippet: After 72 h, cell lysates Oncotarget12152www.impactjournals.com/oncotarget were prepared and luciferase expression was determined using luciferase assay kit according to the manufacturer’s protocol (Promega). nG2 knockdown with shrnA and mir129-2 PDGFB mouse neurospheres and adherent mouse tumor cells were transfected with NG2 specific shRNA (Origene) or control shRNA or miR129-2 plasmids or empty vector using XtremeGene HP transfection reagent, according to the manufacturer’s protocol (Roche).

Techniques: Luciferase, Plasmid Preparation, Sequencing, Clone Assay, Transfection, Expressing, shRNA, In Vitro, Control, Transduction, Immunofluorescence, Confocal Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

doi: 10.18632/oncotarget.3716

Figure Lengend Snippet: Figure 4: Mouse and human DIPG neurospheres exhibit symmetric NG2 expression in vitro and are immature stem cells co-expressing oligodendrocyte (Olig2) and astrocyte (GFAP) markers. (a) PDGFB mouse tumor neurospheres (Mouse) and human primary DIPG cells (SF8628 and SUDIPGVI) were grown and stained using NG2 antibody (red) and DAPI (blue) nuclear staining. NG2 is equally expressed in dividing cells suggesting that its expression is defective (symmetric) in mitotic cells. Scale bar = 5 µm. (b) PDGFB mouse tumor (Mouse) and SF8628 human DIPG primary cells were expanded in culture and immunostained for DAPI (nuclear), oligodendrocyte marker (Olig2, green), NG2 (red), and astrocyte marker (GFAP, magenta). Confocal microscopy was used to assess expression of above-mentioned proteins in mitotic cells. Scale bar = 5 µM

Article Snippet: After 72 h, cell lysates Oncotarget12152www.impactjournals.com/oncotarget were prepared and luciferase expression was determined using luciferase assay kit according to the manufacturer’s protocol (Promega). nG2 knockdown with shrnA and mir129-2 PDGFB mouse neurospheres and adherent mouse tumor cells were transfected with NG2 specific shRNA (Origene) or control shRNA or miR129-2 plasmids or empty vector using XtremeGene HP transfection reagent, according to the manufacturer’s protocol (Roche).

Techniques: Expressing, In Vitro, Staining, Marker, Confocal Microscopy

Journal: Oncotarget

Article Title: The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

doi: 10.18632/oncotarget.3716

Figure Lengend Snippet: Figure 5: Orthotopic injection of NG2 expressing cells causes aggressive brainstem tumors in vivo resulting in a robust murine model of DIPG. (a) NG2 expressing neurospheres were isolated from PDGFB mouse pontine tumor and expanded in culture and stained for NG2 (red) and DAPI (blue) (b and c). Neurospheres were then injected into the brainstems of two-day-old (P2) mice. Whole brain was obtained at 3 weeks post injection and subjected to H&E staining (d-g). Injected cells show an infiltrative pattern of growth (g, arrow) at the site of injection. H&E staining also showed a large infiltrating tumor within pons (region p in d). 10 × magnifications (e-f) revealed the highly cellular tumor within the pons (e) that exhibits characteristics of DIPGs including tumor vascularity (f, arrows). Injected cells were infiltrated through the pons and away from the injection site (g, arrow). This pontine tumor is positive for the astrocytic marker GFAP (h), proliferation marker Ki67 (i), PDGFRβ (j), PDGFRα (k), and NG2 (l). Scale bar = 100 µM

Article Snippet: After 72 h, cell lysates Oncotarget12152www.impactjournals.com/oncotarget were prepared and luciferase expression was determined using luciferase assay kit according to the manufacturer’s protocol (Promega). nG2 knockdown with shrnA and mir129-2 PDGFB mouse neurospheres and adherent mouse tumor cells were transfected with NG2 specific shRNA (Origene) or control shRNA or miR129-2 plasmids or empty vector using XtremeGene HP transfection reagent, according to the manufacturer’s protocol (Roche).

Techniques: Injection, Expressing, In Vivo, Isolation, Staining, Marker

Journal: Oncotarget

Article Title: The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

doi: 10.18632/oncotarget.3716

Figure Lengend Snippet: Figure 6: miR129-2 targets and downregulates NG2 in vivo. Brain sections from NG2-dsRed mice were analyzed 2 weeks after the intracranial injections with control lentiviral vector on left side and lentiviral vector harboring miR129-2 on right side. Detection of the green signal (GFP) indicates in vivo transduction of either control or miR129-2. Area enclosed by dotted line in 10 × image (Scale bar: 100 µM) represents the site of injection. 40 × magnification images (Scale bar: 10 µM) of boxed area in merged images are shown to demonstrate the downregulation of NG2 (red) in miR129-2 (green) transduced cells (arrows) while control transduced cells are expressing normal levels of NG2.

Article Snippet: After 72 h, cell lysates Oncotarget12152www.impactjournals.com/oncotarget were prepared and luciferase expression was determined using luciferase assay kit according to the manufacturer’s protocol (Promega). nG2 knockdown with shrnA and mir129-2 PDGFB mouse neurospheres and adherent mouse tumor cells were transfected with NG2 specific shRNA (Origene) or control shRNA or miR129-2 plasmids or empty vector using XtremeGene HP transfection reagent, according to the manufacturer’s protocol (Roche).

Techniques: In Vivo, Control, Plasmid Preparation, Transduction, Injection, Expressing