Systematic analysis of bicistronic reporter assay data

NRF2, a crucial antioxidant transcription factor in ovarian cancer (OC), is closely associated with CEBPB activation. However, the regulatory mechanism of NRF2 by CEBPB in OC remains poorly understood. In this study, we systematically evaluated the malignant behavior of SKOV3 and A2780 cells through comprehensive approaches, including CCK-8 kits, clone formation assays, and flow cytometry analysis. Cellular antioxidant capacity was quantitatively assessed using the DCFH-DA and total-antioxidant capacity (T-AOC) assays. Molecular mechanisms were investigated through multiple experimental approaches: the interaction between NRF2 and the DUSP1 promoter was examined using dual fluorescence reporter assays, while the activation status of CEBPB, NRF2, DUSP1, antioxidant proteins, and MAPK pathway components was analyzed via immunofluorescence and western blotting. Our findings demonstrate that CEBPB overexpression significantly enhanced cellular proliferation, clone formation, cell cycle progression, and antioxidant capacity, while simultaneously reducing apoptosis rates and reactive oxygen species (ROS) levels. Conversely, CEBPB knockdown or NRF2 inhibition produced opposing effects. These results establish that CEBPB-mediated NRF2 activation promotes OC cell proliferation and antioxidant defense mechanisms. Mechanistically, we identified that NRF2 directly binds to the DUSP1 promoter, as confirmed by dual-luciferase reporter assays. NRF2 activation led to upregulation of DUSP1 and phosphorylated ERK1/2 levels, while downregulating JNK and p38 phosphorylation. These findings were further validated in vivo, confirming that CEBPB activates NRF2 to regulate the MAPK pathway through DUSP1, thereby promoting OC cell proliferation and antioxidant capacity. In conclusion, our study reveals a novel regulatory axis in which CEBPB activates NRF2 to regulate the MAPK pathway via DUSP1, driving malignant progression and enhancing antioxidant activity in OC.

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

CRYAA activates the SIRT1-pi3K/AKT signaling pathway by suppressing mir-155-5p to protect the RPE.

BackgroundAdvances in mass spectrometry-based proteomics have enabled the incorporation of proteomic data into systems approaches to biology. However, development of analytical methods has lagged behind. Here we describe an empirical Bayes framework for quantitative proteomics data analysis. The method provides a statistical description of each experiment, including the number of proteins that differ in abundance between 2 samples, the experiment's statistical power to detect them, and the false-positive probability of each protein.Methodology/Principal FindingsWe analyzed 2 types of mass spectrometric experiments. First, we showed that the method identified the protein targets of small-molecules in affinity purification experiments with high precision. Second, we re-analyzed a mass spectrometric data set designed to identify proteins regulated by microRNAs. Our results were supported by sequence analysis of the 3′ UTR regions of predicted target genes, and we found that the previously reported conclusion that a large fraction of the proteome is regulated by microRNAs was not supported by our statistical analysis of the data.Conclusions/SignificanceOur results highlight the importance of rigorous statistical analysis of proteomic data, and the method described here provides a statistical framework to robustly and reliably interpret such data.

In recent years, many tools have been developed for targeted treatments of cancer or rare diseases including nucleic acid (NA)-based therapeutics. One of the developmental caveats is the assessment of their functionality in vivo. We therefore developed a sensitive dual fluorescence-based FluoroDetect assay for the testing of small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and RNA-binding compounds. For a proof of principle, we inserted the noncoding HSat3 RNA as a prime target into the 3'UTR of an mCherry fluorescence protein. The FluoroDetect assay was designed to be adapted by the In-Fusion cloning with any wished target site. The assay can be used as a quick transient model for short-term experiments or as a lentiviral reporter for long-term experiments. Readout of the assay is possible with fluorescence microscopy, plate reading, or flow cytometry, and can be used to measure the cellular distribution of NA therapeutics, siRNAs, and ASOs. Thus, the FluoroDetect assay is a tool to screen NA drug candidates and facilitates the optimization and quantification of NA delivery in NA-based therapies.

BackgroundSilver pomfret (Pampus argenteus) is one of the most widely distributed and economically important pelagic fish species. However, an unique morphological feature of P. argenteus is the loss of pelvic fins, which can increase the energy requirement during food capture to some extent and is therefore not conducive to artificial culture. Tbx4/5 genes are highly conserved regulatory factors that regulate limb development in vertebrates and are in turn regulated by microRNAs (miRNAs). However, the miRNAs that directly regulate the Tbx4/5 genes in P. argenteus remain to be elucidated.MethodsThe Tbx4/5 genes of P. argenteus were first cloned, and the small RNA transcriptomes were sequenced by high-throughput sequencing during the critical period of the fin development at days 1, 7, and 13 of hatching. The miRNAs regulating the Tbx4/5 genes of P. argenteus were subsequently predicted by bioinformatics analysis, and the related miRNAs were verified in vitro using a dual fluorescence reporter system.ResultsA total of 662 miRNAs were identified, of which 257 were known miRNAs and 405 were novel miRNAs were identified. Compared to day 1, 182 miRNAs were differentially expressed (DE) on day 7, of which 77 and 105 miRNAs were downregulated and upregulated, respectively, while 278 miRNAs were DE on day 13, of which 136 and 142 miRNAs were downregulated and upregulated, respectively. Compared to day 13, four miRNAs were DE on day 7, of which three miRNAs were downregulated and one miRNA was upregulated. The results of hierarchical clustering of the miRNAs revealed that the DE genes were inversely expressed between days 1 and 7, and between days 1 and 13 of larval development, indicating that the larvae were in the peak stage of differentiation. However, the number of DE genes between days 7 and 13 of larval development was relatively small, suggesting the initiation of development. The potential target genes of the DE miRNAs were subsequently predicted, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of target genes were performed. The results suggested that the DE miRNAs were involved in growth, development, and signal transduction pathways, of which the Wnt and Fgfs signaling pathways are known to play important roles in the growth and development of fins. The results of dual fluorescence reporter assays demonstrated that miR-102, miR-301c, and miR-589 had a significant negative regulatory effect on the 3′-UTR of the Tbx4 gene, while miR-187, miR-201, miR-219, and miR-460 had a significant negative regulatory effect on the 3′-UTR of the Tbx5 gene. Altogether, the findings indicated that miRNAs play an important role in regulating the growth and development of pelvic fins in P. argenteus. This study provides a reference for elucidating the interactions between the miRNAs and target genes of P. argenteus in future studies.

Screening of novel nuclear receptor agonists by a convenient reporter gene assay system using green fluorescent protein derivatives

miR-210 is closely related to the occurrence of pancreatic cancer. In addition, Runx3 is a tumor suppressor gene and inhibits tumorigenesis. However, miR-210?s effect on Runx3 level is unclear. Therefore, Our study explored miR-210?s effect on the proliferation and migration of pancreatic cancer cells. PANC-1 cell line was transfected with miR-210 Mimics or miR-210 Mimic+pFBD-Runx3 plasmids followed by analysis of miR-210 and Runx3 level by qRT-PCR, targeting relationship by the dual fluorescein reporter assay, Runx3 and Tubulin protein expression by Western blot, cell proliferation by MTT assay and cell migration by Transwell assay. Compared with normal tissue, miR-210 was significantly upregulated in pancreatic cancer tissue (P < 0.01), while Runx3 mRNA was significantly downregulated (P < 0.01). Runx3 was a target protein of miR-210. miR-210 Mimics transfection significantly reduced Runx3 level and increased cell proliferation, which was significantly reduced in the miR-210 Mimic+pFBD-Runx3 group. miR-210 Mimics significantly promote cell migration and the addition of Runx3 prevented miR-210 Mimics-induced cell migration. miR-210 binds to the 3′-UTR region of Runx3 mRNA, reduces Runx3 expression, and promotes cell proliferation and migration. Increased Runx3 can inhibit miR-210?s effect on pancreatic cancer cells.

G protein-coupled receptors (GPCRs) are the largest class of proteins in the human body and are considered to be excellent targets for drug discovery. However, the vast majority of GPCRs are understudied. Using a large-scale TaqMan array screen on both male and female C57BL6 murine livers, we identified GPR17 as a highly expressed orphan receptor within the liver (ΔCt = 20.23, normalized to GAPDH). Hepatic expression of Gpr17 was confirmed using reverse transcriptase polymerase chain reaction (RT-PCR) and was also identified in murine whole kidneys from both males and females. While GPR17 has known roles in oligodendrocyte maturation and myelination in the central nervous system, its functions within the liver and kidney remain unknown. To investigate its physiological functions in respective tissues, we started by uncovering GPR17’s localization pattern, ligand profile, and signaling mechanism. Using custom RNAScope in-situ hybridization probes, we found Gpr17 mRNA expression within hepatocytes and cortical nephron segments of the kidney. RNAScope colocalization studies using lotus tetragonolobus lectin (LTL) as a proximal tubule marker revealed robust cortical Gpr17 mRNA expression within both LTL+ and LTL- nephron segments. Additionally, ongoing subcellular localization experiments using Alpha mouse liver 12 (AML12) and Madine-Darby canine kidney (MDCK) cell lines will shed light on GPR17 tissue-specific expression within subcellular departments. To screen for potential ligands, we cloned Gpr17 into flag-tagged PME18s and Presto-Tango vectors. Live-cell surface labeling of HEK293T expressing the GPR17 constructs confirmed surface expression facilitating ligand screening. GPR17 downstream signaling has yet to be studied. Thus, we used the PRESTO-TANGO assay, an exogenous chemical ligand screening assay that relies on a luminescence readout to indicate -arrestin recruitment and receptor activation. A large, non-biased chemical screen of over 500 physiological compounds was tested for activation of GPR17. To date, we identified seven exogenous activators of GPR17. Additionally, literature suggests that synthetic MDL29,951 (MDL) and endogenous oxysterols function as GPR17 ligands. To determine GPR17 signaling mechanisms, we employed Dual Luciferase cAMP-based reporter assays. On their own, GPR17 ligands did not induce a rise in intracellular cAMP. However, when cAMP levels were elevated via forskolin treatment alongside ligand administration, GPR17 activation resulted in a significant reduction of intracellular cAMP, suggesting that GPR17 signaling is coupled to the Gi pathway. Results confirmed MDL and select oxysterol ligand behavior and indicated the utilization of a Gi signaling mechanism by hepatic and renal GPR17. Ongoing bioluminescence resonance energy transfer studies using compact ONE vector G-protein Optical biosensors are aimed at confirming GPR17’s coupling to the Gi pathway. NIH This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Intergenic region between TATA-box binding protein and proteasome subunit C3 genes of Medaka function as the bidirectional promoter in vitro and in vivo

The rational drug design combining the bioassay identified a novel selective androgen receptor (AR) degrader for both AR and AR-VRs and illustrated the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment.

The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough. Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes. We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels. Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein.

A greater understanding of the regulatory mechanisms that govern γ-globin expression in humans, especially the switching from γ- to β-globin, which occurs after birth, would help to identify new therapeutic targets for patients with β-hemoglobinopathy. To further elucidate the mechanisms involved in γ-globin expression, a novel fluorescent-based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183-kb intact human β-globin locus under the control of (G)γ- or (A)γ-globin promoter and β-globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage-specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)-mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a γ-globin-linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with γ-globin gene suppression.

Regarding the statistical analysis of the quantitative data obtained in control and dosage groups in toxicity studies, we tried to simplify the decision tree method. In a new decision tree presented in this article, one-way analysis of variance and Kruskal-Wallis nonparametric analysis of variance are excluded from the traditional decision tree: (1) Bartlett's test is used as a test for the equality of k variances: (2) Then, if the k sampled populations have equal variances (p > 0.05 by the Bartlett's test), Dunnett's multiple comparison test is performed: otherwise, Steel's test is used. This new method, which increases the power in some conditions, may serve as an alternative to the traditional decision tree method.

Drill string depth is a fundamental measurement performed by surface sensor systems. However, the depth reading on an electronic drilling recorder (EDR) often diverges from the pipe tally and requires periodic adjustment to remain aligned with the pipe tally total. In this paper, we investigate what causes depth errors and quantify the error magnitude in land-based wells. The main results show that the depth adjustment workflow varies significantly from driller-to-driller, and workflow errors are a major contributor to total depth error. The investigation is performed with two main efforts. First, depth error is characterized by analyzing the corrections made to EDR depth in 29 onshore wells. Scatterplot analysis reveals that errors can be broadly classified into 3 main types: offset errors, accumulation errors, and workflow errors. Workflow errors can be further categorized into 7 subtypes. Statistical analysis of correction data shows that 75% of depth corrections are caused by offset and accumulation errors. However, workflow errors, such as human error and casing error, account for over 50% of depth corrections when measured on a per-footage basis. In the second effort, a user experience study is conducted to understand the depth correction workflow currently used by drillers. Interviews with 24 rig personnel reveal that workflows are not entirely consistent from driller-to-driller. Drillers tend to adopt the depth calibration methodologies taught by their mentors. A more consistent workflow is possible with increased training and a best practice guideline.