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

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: Signal Transduction and Targeted Therapy

Article Title: Piezo1 activation suppresses bone marrow adipogenesis to prevent osteoporosis by inhibiting a mechanoinflammatory autocrine loop

doi: 10.1038/s41392-025-02455-w

Figure Lengend Snippet: Piezo1 suppresses Lcn2 expression and secretion via the inhibition of Ccl2-evoked NF-κB activation. BMMSCs were isolated from femurs and tibias of 10-week-old male PDGFRα-Piezo1 KO mice and WT controls. a Gene expression of Ccr2 in BMMSCs, as determined by real-time PCR. b–g Recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) was added to the adipogenic and osteogenic induction medium during BMMSC differentiation. The expression levels of osteogenic genes ( b–d ) and adipogenic genes ( e–g ) were determined via real-time qPCR. h–n WT and KO BMMSCs were treated with rmCcl2 (100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) for 24 h. h Sonicated BMMSCs were subjected to chromatin immunoprecipitation (ChIP) using an anti-p65 antibody. The ChIP DNA samples were then subjected to qPCR amplification using specific primers against the promoter region of Lcn2, as shown in supplementary Fig. . Representative gel images of end-point ChIP-qPCR products are shown. i Quantitative results are shown as the percentage of input DNA: % of Input = 2^(CT Input −CT IP )/dilution factor × 100%. j Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Lcn2 promoter (Lcn2-WT) or the mutated Lcn2 promoter with mutations in the NF-κB binding motif (Lcn2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. k Schematic diagram illustrating the actions of rmCcl2, the CCR2 antagonist, and the NF-κB-specific inhibitor QNZ on NF-κB translocation and Lcn2 expression; created with BioRender ( https://BioRender.com ). l ELISA analysis of total p65 in the nucleus and cytoplasm of BMMSCs. m Representative immunofluorescence images of intracellular p65 localization. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. n P65 nuclear translocation was quantified by ImageJ software via Pearson’s correlation coefficient (PCC) between the p65 immunofluorescence signal and DAPI staining across segmented nuclear regions. o , p WT and KO BMMSCs were treated with the NF-κB-specific inhibitor QNZ (10 nM) for another 2 h after 24-h treatment of rmCcl2 (100 ng/mL) and INCB3344 (10 nM). o The mRNA level of Lcn2 . p Lcn2 levels in the culture medium were determined by ELISA. q Illustration of how the AP-1 inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific inhibitor T-5224 (10 μM) for 24 h. r Sonicated BMMSCs were subjected to ChIP assay using an anti-c-Jun antibody. Representative gel images of end-point ChIP-qPCR products are shown. s Quantitative results are shown as the percentage of input DNA. t The mRNA level of Ccl2 . u Ccl2 levels in the culture medium were determined by ELISA. v Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Ccl2 promoter (Ccl2-WT) or the mutated Ccl2 promoter with mutations in the c-Jun binding motif (Ccl2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. n = 5 for each group. The data are presented as the means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001. See also supplementary Figs. –

Article Snippet: The luciferase reporter assay was performed by transfecting 0.2 μg of firefly and Renilla luciferase reporter vectors (Addgene, #128046) driven by either the wild-type mouse Lcn2 or Ccl2 promoter (−1999/+1) or their respective mutants (in which the NF-κB or c-Jun DNA binding site was mutated) (Azenta) into BMMSCs seeded in 96-well plates using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, #L3000001).

Techniques: Expressing, Inhibition, Activation Assay, Isolation, Gene Expression, Real-time Polymerase Chain Reaction, Recombinant, Sonication, Chromatin Immunoprecipitation, Amplification, ChIP-qPCR, Luciferase, Reporter Assay, Transfection, Construct, Binding Assay, Sequencing, Activity Assay, Translocation Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Software

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Expression of Ca v 1.3 calcium channel in the human and mouse colon: posttranscriptional inhibition by IFNγ

doi: 10.1152/ajpgi.00394.2016

Figure Lengend Snippet: Posttranscriptional mechanism of reduced Cav1.3 mRNA expression in HCT116 cells treated with IFNγ. A: HCT116 cells were treated with ActD to inhibit de novo transcription and treated with 100 U/ml of IFNγ for 24 h. *Differences between IFNγ and control (P < 0.05). B: 4 deletion constructs of the cloned murine Cav1.3 gene promoter in Firefly luciferase (Luc) reporter vector pGL-basic (Promega), or promoterless vector, were cotransfected with Renilla luciferase vector pRL-null into HCT116 cells and treated with control medium or with 100 U/ml of IFNγ for 24 h. All tested promoter constructs were functional, but no significant effects of IFNγ were detected.

Article Snippet: All reporter constructs were created in pGL3-basic vector with Firefly luciferase as a reporter (Promega, Madison, WI) and sequenced on both strands to confirm fidelity.

Techniques: Expressing, Control, Construct, Clone Assay, Luciferase, Plasmid Preparation, Functional Assay

Journal: The Journal of Experimental Biology

Article Title: Circadian coupling of mitochondria in a deep-diving mammal

doi: 10.1242/jeb.246990

Figure Lengend Snippet: Clock genes expression profile in synchronized hooded seals fibroblasts and tissues. (A) mRNA expression of core clock genes arntl (blue line) and per2 (black line) in hooded seal fibroblasts synchronized by temperature cycle (°C), as represented in the upper panel. Arntl and per2 mRNAs show a 24-h period and are in antiphase. P -values and phases were calculated through JTK cycle analysis ( n =4). (B) mRNA expression of core clock genes arntl (blue line) and per2 (black line) in hooded seal fibroblasts synchronized by temperature cycling and then left at constant temperature (upper panel). Arntl and per2 mRNAs maintain a 24-h period and their antiphasic relationship also in constant conditions. P -values and phases were calculated through JTK cycle analysis ( n =4). (C) Photon multiplier tube recordings of hooded seal skin fibroblasts transfected with arntl :luciferase (blue line) and per2 :luciferase (black line). Cells were synchronized with dexamethasone. (D) Arntl mRNA expression in hooded seal kidney and liver tissue, sampled in mid-light phase (ZT6) and mid-dark phase (ZT18). * P <0.05 (one-way ANOVA; n =3 except liver ZT18, where n =2). Data are expressed as means±s.e.m.

Article Snippet: The latter consisted of the pLV6 backbone and a mouse per2 promoter with adjacent luciferase sequence contained in the pGL3 basic E2 vector (Addgene plasmid 48747).

Techniques: Expressing, Transfection, Luciferase