firefly luciferase activity  (Promega)

Journal: bioRxiv

Article Title: Single-chain permuted proteins for dimerization-based control of protein activity and cellular processes

doi: 10.1101/2025.03.31.645681

Figure Lengend Snippet: (A) Switching of N and C terminal domains of the original protein sequence and the addition of the linker between domains renders the protein inactive and incapable of reconstitution without additional signals. To establish the PROPER system, the N and C terminal segments are switched, and linkers between protein domains and a dimerization domain are added. Following the signal for dimerization, the perTEVp monomers assemble a dimer resulting in an active enzyme. (B) Protein model of a perTEVp-CC dimer. (C) To engineer the perTEVp the wild-type protein was split after 118AA and three designs were developed, differing in the length of flexible linkers between the protease domains and between the protease and the dimerization domain. (D, E) To determine the activity of perTEVp the HEK293T cells were transiently transfected with the perTEVp and a cyclic luciferase reporter (CycLuc) with a TEV protease cleavage site. Designs without dimerization domains exhibited only minimal activity. (F-H) To determine whether the perTEVp can induce cell death, the HEK293T cells were transiently transfected with the perTEVp and a gasdermin D with a TEV protease cleavage site. To evaluate the extent of induced cell death the propidium iodide (PI) uptake and LDH assay were performed. (I) Induction of cell death by perTEVp designs with dimerization domains as demonstrated by confocal microscopy. Cells exhibiting typical pyroptotic morphology are shown. Dead cells were stained with 7-AAD (red) and nuclei were labeled with Hoechst 33342 (blue). Scale bars 50 μm. Plots show the means ± SEM of at least 9 replicates combined from at least three independent experiments. Conditions were compared using a one-way ANOVA with a Tukey’s multiple comparisons post-hoc test (**** P < 0.0001; *** P <0.001; ** P < 0.01; * P <0.05).

Article Snippet: To analyze Firefly luciferase activity the HEK293T cells or HeLa cells were lysed 48 hours after transfection using the passive lysis buffer (Biotium, USA).

Techniques: Sequencing, Activity Assay, Transfection, Luciferase, Lactate Dehydrogenase Assay, Confocal Microscopy, Staining, Labeling

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Modulation of RASD2 by miRNA ‐485‐5p Drives Thyroid Cancer Progression and Metastasis

doi: 10.1002/kjm2.70028

Figure Lengend Snippet: miR‐485‐5p regulation of RASD2 in thyroid cancer. (A) Venn diagram showing overlapping miRNA predictions from three databases (TargetScan, miRDB, StarBase). (B) Sequence alignment between miR‐485‐5p and RASD2 3′‐UTR showing predicted binding site. (C) Dual‐luciferase reporter assay results in IHH4 and TPC‐1 cells transfected with wild‐type or mutant RASD2 3′‐UTR constructs and miR‐485‐5p mimic or negative control. (D) Western blot analysis of RASD2 protein levels in cells transfected with miR‐485‐5p mimic or negative control. (E) TCGA database analysis of miR‐485‐5p expression in thyroid cancer vs. normal tissues. (F) miR‐485‐5p expression in paired tumor‐normal tissues by qRT‐PCR ( n = 96 pairs). (G) Correlation analysis between miR‐485‐5p and RASD2 expression levels in thyroid cancer tissues. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After 48 h, luciferase activities were measured using the Dual‐Luciferase Reporter Assay System (HY‐K1013, MedChemExpress).

Techniques: Sequencing, Binding Assay, Luciferase, Reporter Assay, Transfection, Mutagenesis, Construct, Negative Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Estrogen-induced FXR1 promotes endocrine resistance and bone metastasis in breast cancer via BCL2 and GPX4

doi: 10.3389/fcell.2025.1563353

Figure Lengend Snippet: FXR1 interacts with the GPX4 mRNA to regulate ferroptosis. (a, b) , Viability (a) and death (b) of FXR1 depleted MCF-7 cells were detected, treated with 10 µM erastin or 1 µM RSL3 combined with 2 µM FER1, 1 µM LIP1 or 5 µM DFO. (c) , Heat map analysis of RNA-seq data showing mRNA expression of ferroptosis-related genes in FXR1 depleted cells compared to control cells. (d, e) , qPCR (n = 3 biological replicates) and immunoblot analysis of GPX4 expression in FXR1 depleted MCF-7 (d) and T47D (e) cells. (f, g) , qPCR (n = 3 biological replicates) analyzed the interaction of FXR1 with GPX4 mRNA by RIP assays in MCF-7 (f) and T47D (g) cells. (h) , Schematic representation of luciferase reporter plasmids containing full-length and mutated GPX4-3′UTR (up). FXR1 recognition motif predicted by RBPsuite was shown (down). (i) , Luciferase reporter plasmids were co-transfected with FXR1 expression plasmid or vector control in HEK-293T cells, and luciferase activities were determined. (j, k) , FXR1 depleted MCF-7 (j) and T47D (k) cells were treated with actinomycin (d) . GPX4 mRNA were examined at the indicated time points. (l) , Schematic diagram of full-length and domain mutated of FXR1. (m, n) , Flag-FL, Flag-KH, or Flag-RGG were transfected in HEK-293T cells for RIP assays using Flag antibody. Immunoblot (m) and qPCR (n = 3 biological replicates) (n) were performed to analyze the association of different FXR1 domains with GPX4 mRNA. (o) , Immunoblot assessment of GPX4 levels in FXR1-depletion MCF-7 and T47D cells rescued with empty vector or GPX4 plasmid. (p, q) , Lipid peroxidation was assessed in FXR1 depleted MCF-7 (p) and T47D (q) cells rescued with empty vector or GPX4 plasmid. Results are shown as mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant (Unpaired two-tailed Student’s t test in (f, g, i) , two-way ANOVA test in (j, k) others one-way ANOVA test.).

Article Snippet: Subsequently, the sample supernatant was utilized to measure luciferase activity with Firefly luciferase activity normalized to Renilla luciferase activity (Beyotime).

Techniques: RNA Sequencing, Expressing, Control, Western Blot, Luciferase, Transfection, Plasmid Preparation, Two Tailed Test