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: Technology in Cancer Research & Treatment

Article Title: MiR-32 Inhibits Proliferation and Metastasis by Targeting EZH2 in Glioma

doi: 10.1177/1533033819854132

Figure Lengend Snippet: MiR-32 suppressed enhancer of zeste homolog 2 (EZH2) expression by targeting its 3′-untranslated region (UTR). A, The binding sites of miR-32 on EZH2 3′-UTR. B and C, Luciferase reporter assay with the pGL3-EZH2-3′-UTR-WT or pGL3-EZH2-3′-UTR-Mut were cotransfected with miR-32 mimics/inhibitor/NC. D, EZH2 expression in cells transfected with miR-32 mimic/inhibitor/NC. *** P < .001 and ** P < .01.

Article Snippet: The wild type (WT) and mutant type (Mut) 3′-UTR of EZH2 were cloned into pGL3-REPORT luciferase vector (Ambion; Thermo fisher Scientific, Inc., California).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Transfection

Journal:

Article Title: Mutation of hepatocyte nuclear factor-1? inhibits Pkhd1 gene expression and produces renal cysts in mice

doi: 10.1172/JCI200420083

Figure Lengend Snippet: DNase hypersensitive site mapping and deletion analysis of the mouse Pkhd1 promoter. (A) Structure of the 5′ end of the Pkhd1 gene. Boxes indicate exons. Bent arrow indicates the transcription initiation site at +1. Bar indicates the 3′ probe used for indirect end labeling. Vertical arrows indicate hypersensitive sites. (B) Southern blot of genomic DNA from mIMCD-3 cells (right) and 10T1/2 cells (left) after digestion with graded concentrations of DNase I. Open arrow indicates the parental 8.1-kb EcoRI fragment. Closed arrows indicate sub-bands corresponding to hypersensitive sites located at the positions indicated on the right. (C) Northern blot showing endogenous expression of Pkhd1 (upper panel) and HNF-1β (middle panel) in mIMCD-3 cells (lane 2) and absence of expression in 10T1/2 cells (lane 1). Lower panel shows expression of GAPDH as a loading control. (D) Deletion analysis of the Pkhd1 promoter. Left panel shows plasmids containing fragments of the Pkhd1 promoter linked to a promoterless luciferase (Luc) reporter gene. Bent arrow indicates the transcription initiation site at +1, gray boxes indicate exons, and black boxes indicate the consensus HNF-1 site. Right panel shows luciferase activity in transfected mIMCD-3 cells (white bars) and 10T1/2 cells (gray bars). Data are presented as mean ± SE of six to nine independent transfections. *P < 0.05 compared with empty pGL3-Basic.

Article Snippet: These results indicate that the consensus HNF-1 site is required for the activity of the Pkhd1 promoter in transfected mIMCD-3 cells. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 Mutational analysis of the Pkhd1 promoter and binding of HNF-1. (A) Luciferase activity in mIMCD-3 cells transfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector).

Techniques: End Labeling, Southern Blot, Northern Blot, Expressing, Luciferase, Activity Assay, Transfection

Journal:

Article Title: Mutation of hepatocyte nuclear factor-1? inhibits Pkhd1 gene expression and produces renal cysts in mice

doi: 10.1172/JCI200420083

Figure Lengend Snippet: Mutational analysis of the Pkhd1 promoter and binding of HNF-1. (A) Luciferase activity in mIMCD-3 cells transfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector). Data are presented as mean ± SE of nine independent transfections. *P < 0.01 compared with WT promoter. (B) EMSA performed with a 44-bp DNA fragment containing the consensus HNF-1 site and reticulocyte lysates programmed with HNF-1α (lanes 2–7), HNF-1β (lanes 9–14), or unprogrammed lysates (lanes 1 and 8). Binding reactions were performed in the presence of anti–HNF-1α Ab (lane 3), anti–HNF-1β Ab (lane 10), or 100-fold excess unlabeled competitor (Comp.) DNA fragment (lanes 4–7, 11–14). (C) EMSA performed using the 44-bp DNA fragment and nuclear (Nuc) extracts from mIMCD-3 cells (lanes 2–8) or no protein (lane 1). Binding reactions were performed in the presence of anti–HNF-1β Ab (lane 3), irrelevant Ab (lanes 4), or 100-fold excess unlabeled DNA fragment (lanes 5–8). In B and C, arrows indicate retarded band, and arrowheads indicate supershifted band. †Complex that does not contain HNF-1β. (D) Luciferase activity in HeLa cells cotransfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector) and expression plasmids encoding HNF-1α, HNF-1β, or empty pcDNA3. Data are presented as mean ±SE of six independent transfections. *P < 0.01 compared with cells cotransfected with empty expression plasmid.

Article Snippet: These results indicate that the consensus HNF-1 site is required for the activity of the Pkhd1 promoter in transfected mIMCD-3 cells. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 Mutational analysis of the Pkhd1 promoter and binding of HNF-1. (A) Luciferase activity in mIMCD-3 cells transfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing