pn3 sp3fl  (Addgene inc)

Journal: Viruses

Article Title: Glucocorticoid Receptor (GR) and Specificity Protein 1 (Sp1) or Sp3 Transactivate the Bovine Alphaherpesvirus 1 (BoHV-1)-Infected Cell Protein 0 Early Promoter.

doi: 10.3390/v17020229

Figure Lengend Snippet: Figure 4. GR and Sp3 cooperatively transactivate the bICP0 E promoter. Neuro-2A cells were transfected with the designated EP-638 promoter constructs (0.5 µg DNA), plasmids that express GR and/or Sp3 proteins (1.0 µg DNA of each). Designated cultures were treated with 2% stripped FBS, and DEX (10 µM) was added to cultures. At 48 h after transfection, cells were harvested and protein lysate subjected to dual-luciferase assay. Promoter activity levels in the sample containing WT EP-638 co-transfected with only an empty vector and only treated with DMSO (no hormone) were normalized to a value of 1, with fold activation for other samples presented. The results are the average of 3 independent experiments and error bars denote the standard error. Two-way ANOVA test was used for analyzing the results. An asterisk denotes significant differences compared to respective construct alone (*, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001). A hash (#) denotes significant differences between indicated groups (#, p < 0.05 and ##, p < 0.01) in the transactivation of wt EP-638 co-transfected with GR and Sp3 relative to Neuro-2A cells transfected with mutant EP-638 constructs.

Article Snippet: The Sp1 (plasmid #27,264) and Sp3 (plasmid #24,541) expression vectors were purchased from Addgene.

Techniques: Transfection, Construct, Luciferase, Activity Assay, Plasmid Preparation, Activation Assay, Mutagenesis

Journal: Viruses

Article Title: Glucocorticoid Receptor (GR) and Specificity Protein 1 (Sp1) or Sp3 Transactivate the Bovine Alphaherpesvirus 1 (BoHV-1)-Infected Cell Protein 0 Early Promoter.

doi: 10.3390/v17020229

Figure Lengend Snippet: Figure 5. Sp3 increased the KLF4-mediated transactivation of the wt bICP0 E promoter. Neuro-2A cells were transfected with the designated EP-638 promoter constructs (0.5 µg DNA), KLF4 and/or Sp3 (1 µg DNA of each). The designated cultures were treated with 2% stripped FBS, and DEX (10 µM) was added to cultures. At 48 h after transfection, cells were harvested and protein lysate subjected to dual-luciferase assay. Promoter activity levels in the sample containing WT EP-638 co-transfected with only an empty vector and only treated with DMSO (no hormone) were normalized to a value of 1, with fold activation for other samples presented. The results are the average of 3 independent experiments and error bars denote the standard error. Two-way ANOVA test was used for analyzing the results. An asterisk denotes significant differences compared to the respective construct alone (*, p < 0.05; and ****, p < 0.0001). A hash denotes significant differences between indicated groups (#, p < 0.05 and ##, p < 0.01) in the transactivation of wt EP-638, co-transfected with a KLF4 and Sp3 relative to Neuro-2A cells transfected with mutant EP-638 constructs.

Article Snippet: The Sp1 (plasmid #27,264) and Sp3 (plasmid #24,541) expression vectors were purchased from Addgene.

Techniques: Transfection, Construct, Luciferase, Activity Assay, Plasmid Preparation, Activation Assay, Mutagenesis

Journal: Viruses

Article Title: Glucocorticoid Receptor (GR) and Specificity Protein 1 (Sp1) or Sp3 Transactivate the Bovine Alphaherpesvirus 1 (BoHV-1)-Infected Cell Protein 0 Early Promoter.

doi: 10.3390/v17020229

Figure Lengend Snippet: Figure 6. Analysis of cooperative interactions between GR and Sp3 or KLF4 and Sp3 of EP-638 promoter activity. (A) Neuro-2A cells were transfected with the EP-638 promoter construct (0.5 µg DNA), GR (1 µg DNA) and increasing concentrations of Sp3, as denoted. (B) Neuro-2A cells were transfected with the EP-638 promoter construct (0.5 µg DNA), KLF4 (1 µg DNA), and increasing concentrations of Sp3, as denoted. Promoter activity levels in the sample containing wt EP-638 co-transfected with only an empty vector were normalized to a value of 1, with fold activation for other samples presented. The results are the average of 3 independent experiments and error bars denote the standard error. Two-way ANOVA test was used for analyzing the results. An asterisk denotes significant differences compared to the respective construct alone (*, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001) or transactivation of wt EP-638 co-transfected with GR and Sp3 in Neuro-2A cells.

Article Snippet: The Sp1 (plasmid #27,264) and Sp3 (plasmid #24,541) expression vectors were purchased from Addgene.

Techniques: Activity Assay, Transfection, Construct, Plasmid Preparation, Activation Assay

Journal: Viruses

Article Title: Glucocorticoid Receptor (GR) and Specificity Protein 1 (Sp1) or Sp3 Transactivate the Bovine Alphaherpesvirus 1 (BoHV-1)-Infected Cell Protein 0 Early Promoter.

doi: 10.3390/v17020229

Figure Lengend Snippet: Figure 7. GR, Sp3, and/or KLF4 stimulate productive infection. (A) Neuro-2A cells were transfected with 1.0 µg BoHV-1 gCblue DNA and, where indicated, a plasmid expressing mouse GR protein (1.0 µg), Sp3 (1.0 µg), and/or KLF4 (1.0 µg). To maintain the same amount of DNA in each sample, an empty vector was included in the samples. After transfection, 2% stripped FBS was added to the medium. Designated cultures were then treated with water-soluble DEX (10 µM; Sigma) 24 h post- transfection. At 48 h after transfection, the number of β-Gal+ cells were counted. The value for the control (gCblue virus co-transfected with empty vector and then treated with PBS after transfection) was set to 1. (B) The results from DEX-treated cultures were compared to those from the control. The results are the average of 3 independent experiments and error bars denote the standard error. Two-way ANOVA test was used for analyzing the results. An asterisk denotes significant differences compared to the respective construct alone (*, p < 0.05).

Article Snippet: The Sp1 (plasmid #27,264) and Sp3 (plasmid #24,541) expression vectors were purchased from Addgene.

Techniques: Infection, Transfection, Plasmid Preparation, Expressing, Control, Virus, Construct

Journal: Development (Cambridge, England)

Article Title: Positive autoregulation of Sox17 is necessary for gallbladder and extrahepatic bile duct formation

doi: 10.1242/dev.203033

Figure Lengend Snippet: SOX17 + autoregulation and potential interactions with SP/KLF factors enhance Sox17 expression to levels necessary for GB formation. (A) Luciferase reporter assay testing WT (CR2F) and mutant (mut3, mut4, mut5) TSS2 promoter activity in mouse differentiated definitive endoderm with overexpression of SOX17 ( n =8, P <0.0001) or SOX9 ( n =8, P >0.9999) while using the pGL4 empty vector as a negative control (Mut3 with Sox17 overexpression, n =5, P <0.0001). Each dot represents one well of endoderm across at least three differentiation experiments. One-way ANOVA. (B) Luciferase reporter assay testing WT (CR2F) and mutant (mut4) TSS2 promoter activity in mouse differentiated definitive endoderm with overexpression of SP1 ( P <0.0001), SP2 ( P =0.8969) and SP3 ( P <0.0001). n =8. One-way ANOVA. Each dot represents one well of endoderm across at least three differentiation experiments. (C) Luciferase reporter assay testing WT (CR2F) and mutant (mut4) TSS2 promoter activity in mouse differentiated definitive endoderm with overexpression of SOX17+SP1 ( P =0.3388), SOX17+SP2 ( P =0.9931) and SOX17+SP3 ( P =0.0001). Each dot represents one well of endoderm across at least three differentiation experiments. n =8. One-way ANOVA. (D) Electrophoretic mobility shift assay (EMSA) demonstrates direct binding of SOX17 at m5 region within TSS2 promoter. The binding of SOX17 to WT and the mut4 mutant but not to the mut5 mutant probe generates shifted bands. SOX17 OE, Sox17 overexpression lysate from 293T cells; Com. probe, competing probe. Full image is in Fig. S5 . (E) EMSA comparing the binding of Sox17 at the m5 region in TSS2 and several binding sites that lie near genes that are differentially regulated in the GBs of Sox17 mut5/mut5 mice. While strong binding occurs on the optimized SOX17 binding motif, much weaker or no binding occurs at all other sites under the conditions of this experiment. Full EMSA image is in Fig. S5 . Bases marked in red deviate from the SOX17 consensus motif. (F) Within the Sox17 TSS2 promoter (blue highlight), SOX17 CUT&RUN data shows binding of SOX17 within Sox17 in the cXEN extra-embryonic endoderm stem cell line (GEO series GSE213661 , sample GSM6591690 ; ). Top panel viewing window, GRCm38/mm10: chr1:4,490,931-4,496,413. Bottom panel viewing window, GRCm38/mm10: chr1:4,493,565-4,493,823. (G) Model of the SOX17-SP interaction at CR2 which regulates TSS2 via a Sox17 + autoregulatory loop. In +/+ animals, SOX17 binds to m5 within the TSS2 promoter to positively regulate Sox17 expression. This interaction is also dependent on binding of an SP factor at m4. In Sox17 mut5/mut5 animals, the autoregulatory loop is broken as the SOX17 binding site is mutated, leading to a ∼35% reduction in Sox17 expression and a hypoplastic gallbladder (GB). Lastly, in the Δ50 bp deletion, three CREs (m3, m4 and m5) are disrupted. This deletion does not allow for SOX17 binding, thereby disrupting the positive autoregulatory loop. In addition, TF binding at m3 and m4 is also disrupted, leading to a ∼50% reduction in Sox17 expression and lack of GB organogenesis. Created in BioRender. Finnel, R. (2025) https://BioRender.com/e79s990 . Data are mean±s.e.m.

Article Snippet: Test vectors, pGL2 Firefly positive control vector, pRL-SV40 Renilla control vector, and overexpression plasmids for Sox17 (pRP[Exp]-mCherry-CAG>mSox17[NM_001289464.1]); Sox9 (pRP[Exp]-mCherry-CAG>mSox9[NM_011448.4]); Sp1 (pN3-Sp1FL, Addgene plasmid #24543 ); Sp2 (pN3-Sp2FL, Addgene plasmid # 107718 ); Sp3 (pN3-Sp3FL, Addgene #24541 ) (sequences found in ) were transfected to cell culture at day 6 of differentiation using the standard protocol of Xfect Transfection Reagent Kit (Takara Bio, 631318).

Techniques: Expressing, Luciferase, Reporter Assay, Mutagenesis, Activity Assay, Over Expression, Plasmid Preparation, Negative Control, Electrophoretic Mobility Shift Assay, Binding Assay