firefly luciferase reporter vectors  (Genechem)

Journal: Advanced Science

Article Title: Detachment‐Induced FAK‐STAT3‐NNMT Inhibits CTCs Anoikis to Promote Breast Cancer Metastasis by Enhancing Fatty Acid Oxidation

doi: 10.1002/advs.202522837

Figure Lengend Snippet: Detachment‐induced NNMT is upregulated by the FAK‐STAT3 axis. (A) Representative image of the positive correlation between FAK and STAT3 in breast cancer and pan‐cancer according to the GEPIA 2 database analysis. (B) Representative results of STAT3, P‐STAT3, FAK, and P‐FAK proteins by WB in the detached BT‐549, MCF‐7, and MDA‐MB‐231 cells. (C,D) The protein and mRNA levels of NNMT and P‐STAT3 after treating detached cells with FAK inhibitor PF‐562271 (10 µ m ) and P‐STAT3 inhibitor C188‐9 (10 µ m ) for 48 h by WB and QPCR. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (E,F) The protein and mRNA levels of NNMT and P‐STAT3 after treating detached cells with FAK inhibitor PF‐562271 (10 µ m for 48 h) and P‐STAT3 activator (1 µ m for 24 h) by WB and QPCR. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (G,H) ChIP assay using P‐STAT3 antibodies in MDA‐MB‐231 cells cultured under suspension conditions was performed, followed by PCR and qPCR, which showed P‐STAT3 enrichment at the NNMT promoter vs. IgG control. ** p < 0.01. I: Dual‐luciferase reporter assays showed that co‐transfection with the full‐length NNMT promoter and a STAT3‐expressing plasmid significantly enhanced luciferase activity, whereas this enhancement was abolished when a truncated NNMT promoter was used. Data were presented as mean ± SEM. **** p < 0.0001.

Article Snippet: All plasmids used in this assay, including the firefly luciferase reporter vectors (e.g., pGL4‐basic containing the wild‐type or truncated NNMT promoter), the STAT3 expression plasmid, and the Renilla luciferase internal control plasmid (pRL‐TK), were custom‐synthesized by GeneChem Co., Ltd. (Shanghai, China).

Techniques: Cell Culture, Suspension, Control, Luciferase, Cotransfection, Expressing, Plasmid Preparation, Activity Assay

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: 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: pCMV-Luc Firefly Luciferase reporter vector (TR30004) plasmid was purchased from Origene while pGint plasmid was a kind gift from Dr. Mariano Garcia-Blanco (Addgene plasmid # 24217).

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