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: Experimental and Therapeutic Medicine

Article Title: HBxAg promotes HBV replication and EGFR activation in human placental trophoblasts

doi: 10.3892/etm.2021.10645

Figure Lengend Snippet: (A) Dual-luciferase reporter gene assay of HBx activity on the EGFR promoter. HBx + E, co-transfection of pGFP-HBx and pGL3-EGFR promoter plasmids; control, co-transfection of pGFP empty vector and pGL3-EGFR promoter plasmids; NC, co-transfection of pGFP-HBx and pGL3-basic empty vector. Rate, firefly luciferase and internal reference Renilla luciferase ratio. ** P<0.01 vs. control. (B) Western blot analysis of EGFR/PI3K/p-AKT expression. Protein fold change is displayed as the ratio of the EGFR/PI3K/p-Akt/Akt protein band to the β-actin protein band; p-AKT/AKT, the ratio of p-AKT protein band to the AKT protein band. * P<0.05, ** P<0.01 vs. NC. EGFR, EGFR overexpressing cells; NC, negative control; sh, short hairpin; HBx, hepatitis B virus X; p-, phosphorylated.

Article Snippet: Cells were seeded into 24-well plates at 3x10 5 cells/well and incubated for 16-18 h. pGL3-EGFR promoter luciferase expression vector (BK328 pGL3-basic-EGFR; Umibio (Shanghai) Co. Ltd.) and the internal reference vector pRL-TK (Promega Corporation) were co-transfected with X-tremeGENE HP (Roche Diagnostics) at room temperature for 20 min when cell densities reached 50-60% confluence, at a ratio of 30:1 (expression vector 0.3 µg/well and internal reference 0.01 µg/well). pGL3-EGFR promoter luciferase expression vector and pGFP-HBx (Addgene, Inc.) were co-transfected as the experimental group, pGL3-EGFR promoter luciferase expression vector and pGFP empty vector (Addgene, Inc.) were co-transfected as the control group, pGL3-Basic and pGFP-HBx were co-transfected as a negative control (NC) and pGL3-Control was used as a positive control (Promega Corporation).

Techniques: Luciferase, Reporter Gene Assay, Activity Assay, Cotransfection, Plasmid Preparation, Western Blot, Expressing, Negative Control

Journal: Cell Death and Differentiation

Article Title: PAK5 promotes the migration and invasion of cervical cancer cells by phosphorylating SATB1

doi: 10.1038/s41418-018-0178-4

Figure Lengend Snippet: PAK5-mediated phosphorylation promotes EMT in cervical cancer cells. a, b Effects of SATB1 and SATB1 Ser47A on the migration and invasion ability in HeLa and SiHa cells. Scale bar, 100μm for a and b. c HeLa and SiHa cells were transfected with pGL3-E, together with the indicated expression plasmids for Dual luciferase reporter assays. d Effects of SATB1 knockdown on EMT markers were analyzed by western blotting in HeLa cells overexpressing PAK5. e Effects of SATB1 overexpression on EMT markers in HeLa cells silencing PAK5. Data are showed as mean ± SD for three independent experiments. **, P < 0.01; ***, P < 0.001. pGL3-E pGL3-E-cadherin promoter, siCtrl siRNA control, siPAK5 siRNA PAK5, ns not significant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

Article Snippet: Briefly, E-cadherin promoter was subcloned into pGL3-Basic luciferase expression vector (Genepharma).

Techniques: Migration, Transfection, Expressing, Luciferase, Western Blot, Over Expression