pgl3-enhancer vector  (Panomics Inc)

Journal: bioRxiv

Article Title: Addressing anemia severity in antimony-resistant Leishmania donovani infection at the nexus of oxidative outburst and iron transit

doi: 10.1101/2024.03.04.583250

Figure Lengend Snippet: (A.i.) Giemsa-stained images of LD-S, LD-R, in murine peritoneal MФs with or without the presence of suboptimal dose of SbV (1.52µg/ml). In some experimental condition LD infection was performed in the presence of N-acetyl-L-cysteine (NAC), while in some experimental conditions, LDs are pre-treated with H 2 O 2 + 6-AN. All the infections were performed for 24hrs (A.ii.) Bar graph showing amastigotes/100MФs in all the experimental sets mentioned in A.i. (B.) Western blot of whole cell lysate showing significant high expression of Heme-oxygenase 1 (HO1) in LD-R infected MФs while no significant change in expression level was observed for Ferritin (FTH1) at 4hrs pi. β-actin is used as the positive control. (C.) Schematic representation of HO-1 promoter region with p50 and c-Rel binding sites. Site A (−4/−635) was demarcated as green, and Site B (−636/−1377) was demarcated as red. (D.i.) Western blot of nuclear fraction showing significant high expression of p50 and c-Rel in LD-R infected MФs 4hrs pi with Histone (H3) as loading control. (D.ii.) Confocal images representing the localization of p50 and c-Rel, in MФs, with DAPI representing the nucleus. The right-most panel shows the RGB-profile plot with grey regions demarcating the region where p50 and cRel are colocalized with DAPI. p50 and c-Rel were found to be localized in the nucleus of LD-R-infected MФs. One representative small nucleus of LD has been marked in ( * ) to show the infected MФs. (E.) Fold luciferase activity of RAW264.7 cell lysate transfected either with HO1+PGL3 promoter, or Site A ( −/− ), Site B( −/− ) deleted constructs followed by infection with LD-S and LD-R for 4hrs. PGL3 enhancer empty vector is used for normalization for each transfected set. Each experiment was performed in 3 biological replicates and graphical data are represented as Mean with SEM. P ≤ 0.05 is marked as *, P ≤ 0.01 is marked as **, P ≤ 0.001 is marked as ***, and P ≤ 0.0001 is marked as ****.

Article Snippet: Murine HO-1 promoters (−1385/+137), 1522bp using primers 5’-AAGGTACCTGAGGCTGGAGAGATGGCC-3’ and 3’-TAAAAGCTTCACCGGACTGGGCTAGTTCAG-5’ were PCR amplified and cloned in promoterless PGL3 enhancer empty vector (Promega, E1771) at the upstream of luciferase gene.

Techniques: Staining, Infection, Western Blot, Expressing, Positive Control, Binding Assay, Luciferase, Activity Assay, Transfection, Construct, Plasmid Preparation

Journal: Cell Death Discovery

Article Title: Notch1 promotes resistance to cisplatin by up-regulating Ecto-5′-nucleotidase (CD73) in triple-negative breast cancer cells

doi: 10.1038/s41420-023-01487-x

Figure Lengend Snippet: A Graphic representation of the Notch CSL binding site region in the CD73 proximal promoter and Negative control region. B The recruitment of Notch1 to the CD73 promoter in MDA-MB-231 cells was assessed using ChIP assays. PCR products were detected in the presence of anti-Notch1 primary antibody. C Schematic illustration of the establishment of CD73 promoter luciferase reporter (pGL3-CD73pro-WT) and individual Notch CSL binding site deletion mutant (pGL3-CD73pro-MT) vector. D Dual-Luciferase assays were performed in MDA-MB-231 cell by co-transfection with LV201-SH, LV201-N1ICD, pGL3-CD73pro-WT or pGL3-CD73pro-MT and luciferase activity was normalized to the Renilla minimal. E Notch1 was silenced in MDA-MB-231 cells by siRNA and then co-transfected with the pGL3-CD73pro-WT or pGL3-CD73pro-MT. Dual-Luciferase values were used to indicate promoter activity. * p < 0.05, ** p < 0.01, and *** p < 0.001 (Student’s t test) as compared with control cells. Data were presented as the mean ± SEM. ( n = 3).

Article Snippet: The CD73 promoter region (−1428 upstream of transcriptional start site and extending to 209 bp) was cloned into the SacI/Smal sites of pGL3-Enhancer vector (Panomics, Fremont, CA, USA) shows the primers used to amplify the target sequence, with one primer containing the CSL binding site (pGL3-CD73pro-WT) and a mutant deleting the CSL binding site (pGL3-CD73pro-MT).

Techniques: Binding Assay, Negative Control, Luciferase, Mutagenesis, Plasmid Preparation, Cotransfection, Activity Assay, Transfection