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: PLoS ONE

Article Title: ABCG1 rs57137919G>A Polymorphism Is Functionally Associated with Varying Gene Expression and Apoptosis of Macrophages

doi: 10.1371/journal.pone.0097044

Figure Lengend Snippet: (a) Schematic diagram for constructing wild-type or mutant promoter at the ABCG1 rs57137919G>A polymorphism site. Two constructs were subcloned into pGL3-basic plasmid vectors with firefly luciferase reporter gene. pG, -367G (open bar); pA, -367A (grey bar). Luciferase activity assays for two ABCG1 promoter constructs in HEK293T cells (b), THP-1 cells (c), and HepG2 cells (d) are shown. Luciferase activities were measured with the dual-luciferase reporter assay system and normalized to Renilla luciferase activity 24 hours after transfection in the presence or absence of TO901317. The luciferase activity was a corrected relative value. Data are shown as mean ± SD of four independent experiments in triplicate. P <0.01 vs. pG in HEK293 cells, THP-1 cells, and HepG2 cells under both basal state and LXR agonist stimulation.

Article Snippet: The pGL3-basic empty vector was used as a negative control and the pGL3-control vector (Promega) was used as a positive control for the luciferase assay.

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