renilla luciferase reporter vectors  (Addgene inc)

Journal: bioRxiv

Article Title: CANVAS causing AAGGG repeat expansions cause tissue-specific reduction in RFC1 expression and increase sensitivity to DNA damage

doi: 10.1101/2025.11.18.688292

Figure Lengend Snippet: A. Circular dichroism spectra of 1 µM (A) 5’-(AAAAG)8-3’ and (B) 5’-(AAGGG)8-3’ measured in 10 mM lithium cacodylate buffer at pH 7.2, with 100 mM of KCl (black lines), NaCl (grey lines), or LiCl (light grey lines), maintained at 20°C. In 100 mM KCl 5’-(AAGGG) -3’ CD spectrum displayed a positive peak at 264 nm, a negative peak near 240 nm, and an additional positive peak around 210 nm, with a melting temperature (Tm) of 65.1 ± 0.6 °C, suggestive of parallel G4 ( ; ). In NaCl or LiCl, the structure shifted toward a less stable hybrid G4, with Tm values of ∼40°C and ∼45°C, respectively. In contrast 5’-(AAGGG) 8 -3’ CD spectrum displayed a minimum at ∼250 nm, a prominent positive peak near 220 nm, and a shoulder around 230 nm, a pattern characteristic of poly(dA)-rich sequences rather than G4s , with a Tm > 95°C under all tested ionic conditions. B. In vitro transcription assay of CTTTT- or CCCTT-containing vectors showing a repeat size- and motif-dependent reduction of RNA transcription. The transcriptional products were separated on a denaturing gel along with a ssRNA ladder. Full transcripts (arrows) were densitometrically quantified and are represented as single data points, mean and standard error. AU =arbitrary units. Data were analysed using one-way ANOVA followed by Dunnett’s T3 multiple comparisons post-hoc test. n=2 technical replicates. C. Schematic representation of Firefly luciferase reporter vector containing increased sizes of non-pathogenic CTTTT or pathogenic CCCTT repeat (left) and quantification of relative Firefly to Renilla luminescence of cells co-transfected with Firefly and Renilla luciferase vectors (right). RLU= relative luminescence units. Data are shown as single data points, mean and standard error. Data were analysed using one-way ANOVA followed by Dunnett’s T3 multiple comparisons post-hoc test. Experiments were performed in triplicate on four independent occasions. D. Schematic representation of pGint fluorescence reporter vector containing increased sizes of non-pathogenic CTTTT or pathogenic CCCTT repeat (left) and relative EGFP to mCherry fluorescence quantification of HEK cells co-transfected with pGint and mCherry vectors (right). Data are shown as single data points, mean and standard error. Data were analysed using one-way ANOVA followed by Dunnett’s T3 multiple comparisons post-hoc test. Experiments were performed in triplicates or quadruplicates on three independent occasions.

Article Snippet: 50ng Firefly Luciferase plasmids were co-transfected in a 1:1 ratio with a control reporter vector constitutively expressing Renilla Luciferase (Origene), using Lipofectamine TM 3000 Transfection Reagent (ThermoFisher Scientific).

Techniques: Circular Dichroism, In Vitro, Transcription Assay, Luciferase, Plasmid Preparation, Transfection, Fluorescence

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