搜索结果：Cell metabolism

CD320, also known as the transcobalamin receptor, is a key receptor that mediates the cellular uptake of circulating vitamin B12 bound to transcobalamin. In humans, CD320 abnormalities cause metabolic and neurological disorders. Previous biochemical studies indicate that the CD320 is a monomeric receptor. In this study, we conducted molecular and cellular experiments in human embryonic kidney 293 cells to examine biosynthesis and molecular forms of human CD320. We found that CD320 was present on the cell surface in an oligomeric form, which consisted of five homomers interconnected via disulfide bonds, possibly in one or both low-density lipoprotein receptor (LDLR)-like domains. Deletion of the LDLR-like domains prevented CD320 oligomerization and cell surface localization, thereby impairing cellular uptake of vitamin B12. By analyzing biochemical forms of CD320 in intracellular compartments, we showed that CD320 oligomerization occurred in the endoplasmic reticulum (ER) and that this process required the transmembrane domain but not N- or O-glycosylation of CD320. Moreover, we showed that the CD320 ΔE88 variant, identified in infants with abnormal vitamin B12 metabolism, did not prevent CD320 oligomerization but delayed CD320 trafficking out of the ER, resulting in low levels of CD320 oligomers on the cell surface. Together, our findings provide important insights into the biochemical nature and cellular mechanisms underlying the function of CD320 and associated pathologies in vitamin B12 metabolism.

Three-dimensional (3D) dynamic conditions are necessary for physiologically relevant mesenchymal stem cell (MSC) culture. Furthermore, bioreactor-based processes and hydrogel-encapsulation substantially improve niche standardization, expansion control, and process scalability. The integration of these technologies can overcome some challenges that prevent the clinical translation of MSCs. However, few studies have investigated the synergistic use of bioreactors and hydrogels, and none have explored the combination of high-throughput encapsulation platforms, such as microfluidics or millifluidics-generated microgels, with standard stirred-tank systems. In this study, we characterized the continuous culture of MSCs in a stirred-tank reactor, encapsulating the cells in gelatin methacryloyl (GelMA) microgels using a low-cost, user-friendly approach. The effects of seeding density, GelMA's degree of functionalization (DoF), bioreactor sampling protocol, and donor screening were assessed using cell metabolic activity, differentiation, proliferation, and extracellular vesicle (EV) secretion. Gentle dynamic culture enhanced MSCs' metabolic activity. However, cell proliferation was inhibited within the microgels, and no cell migration was observed on the hydrogel's surface. GelMA with a low DoF and high cell seeding density favored cell survival during culture, whereas pronounced donor-dependent differences were observed in cell proliferation and metabolism. The yield, size distribution, and protein content of MSC-EVs were affected by seeding density under dynamic 3D conditions. Furthermore, MSC differentiation led to readily measurable changes in the microgels. Our findings highlight the platform's potential for high throughput microtissue generation and efficient assessment of bioactive compounds.

It is known that altered amino acid metabolism can influence immunological responses, thus studying the molecular mechanisms underlying these changes requires analytical methods for reliably determining the quantities of individual analytes. To address the ongoing need for quantitative assessment of amino acid composition, a validated method for determining their quantities was proposed. This study presents the development and validation of a novel method for the quantitative analysis of selected amino acids using high-performance liquid chromatography coupled with a quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an electrospray ionization source. The method has been satisfactorily validated in terms of linearity, range, precision, accuracy, and stability. Its applicability to biological matrices was demonstrated by the successful quantification of amino acids in HaCaT cell lysates. Owing to the use of a robust, state-of-the-art analytical platform based on Q-TOF technology, which enables high-resolution mass detection, the method is readily adaptable for the inclusion of additional analytes and can be extended to other complex biological samples. The applicability of the method for the simultaneous determination of selected amino acids was demonstrated, and the method successfully applied to the HaCaT cell lysates.

This study aimed to establish a novel in vitro model by combining jejunal stem cells from commercially available cryopreserved human intestinal mucosal epithelium (CHIM), which can be used without ethical concerns, with 96-well Vitrigel inserts for high-throughput screening. Jejunal stem cells were successfully isolated from CHIM, and stable long-term expansion in spheroid culture was achieved. The differentiated cells exhibited mRNA expression and function of major pharmacokinetic-related genes. Comparable metabolic and transport activities were maintained even with 96-well Vitrigel inserts instead of the conventional 24-well culture inserts. In a 96-well transcellular transport assay, a significant correlation was observed between the in vitro Papp values and the reported FaFg values (the fraction of orally administered drugs reaching the portal vein) in humans for 10 compounds known to be primarily absorbed via passive diffusion, allowing us to construct a fitting curve. Applying this curve, the FaFg values of transporter substrates could also be estimated with reasonably high accuracy. Furthermore, to estimate human Fg values (the fraction of orally administered drugs escaping from intestinal metabolism), we assessed 5 compounds known to be metabolized by intestinal enzymes. Their predicted Fg values closely matched the reported Fg ones, indicating that our cell model enables their accurate prediction. These findings suggest that a combination of CHIM-derived jejunal stem cells and 96-well Vitrigel inserts provides a practical in vitro model for the quantitative prediction of human intestinal absorption of orally administered drugs. SIGNIFICANCE STATEMENT: In this study, we demonstrated that a combination of commercially available cryopreserved human intestinal mucosal epithelium-derived intestinal stem cells, whose use is free from ethical concerns and restricted tissue availability, and 96-well Vitrigel inserts enables a high-throughput assay that quantitatively predicts human intestinal absorption with high accuracy.

Myogenesis involves sequential stages of muscle satellite cell activation, myoblast proliferation, differentiation, and fusion into multinucleated myotubes. Teleost muscle exhibits indeterminate growth and is highly sensitive to environmental temperature, yet the underlying mechanisms by which temperature regulate proliferation and differentiation remain poorly understood. In this study, we established a primary skeletal muscle cell culture from the yellow drum (Nibea albiflora), an economically important marine fish, and integrated morphological observations with comparative transcriptomics analysis to characterize cellular and molecular responses at 28 °C and 20 °C during both proliferation and differentiation stages. Phenotypic analysis revealed that 28 °C significantly enhanced both myoblast proliferation and myogenic differentiation ability compared with 20 °C. Transcriptomic profiling revealed that at 28 °C, differentiation upregulated extracellular matrix(ECM) organization, calcium signaling, and sarcomere assembly, while proliferation enhanced focal adhesion, growth factor signaling, and lipid metabolism. At 20 °C, differentiation was characterized by glutathione metabolism, and ferroptosis, while proliferation involved cytokine-cytokine receptor interaction and negative regulation of signal transduction. Core myogenic regulatory factors (MRFs), particularly myogenin, were strongly upregulated at 28 °C during the differentiation stage, serving as an internal control. Based on these findings, we propose a testable model that elevated temperature coordinates Ca2+-dependent MRF activation with ECM-integrin signaling to drive sarcomere assembly and muscle growth. Key differentially expressed genes (DEGs) regulating myogenesis in N. albiflora were also identified. This study provides a mechanistic framework for temperature adaptation in teleost skeletal muscle and identifies candidate genes for functional validation and marker-assisted selection, as well as a rationale for temperature management strategies to improve aquaculture yield of N. albiflora.

Insulin secretion can be stimulated by immune and neuronal processes prior to a rise in blood glucose, exemplified by the cephalic phase of insulin response in the anticipation of food. Pancreatic α-cells prevent hypoglycemia by releasing glucagon. Here, we identified α-cells as critical mediators of IL-1β- and cholinergic agonist-driven insulin secretion. Cholinergic blockade prevented glucagon-stimulated insulin secretion in mice. Selective ablation of α-cells abolished cephalic phase insulin release. Islets from α-cell-deficient mice also failed to secrete insulin in response to IL-1β or muscarinic receptor activation. However, glucagon, acting on glucagon and GLP-1 receptors, rescued this insulin-stimulatory response. Mechanistically, intracellular Ca2+ mobilization at fasting glucose mediated cholinergic and IL-1β-stimulated insulin release. Short-term high-fat diet impaired glucagon-induced insulin secretion in vivo, while isolated islets showed increased insulin secretion after cholinergic stimulation versus chow-fed controls. These findings reveal α-cell-derived glucagon as a gatekeeper of immune and neuronal control of insulin secretion at fasting glucose.

Gallbladder cancer (GBC) is a rare but aggressive biliary tract malignancy. This study explores the transcriptomic profile of GBC to identify differentially expressed genes (DEGs) and dysregulated pathways involved in its pathogenesis. RNA sequencing was performed on 13 GBC tumors and 6 matched controls. Functional enrichment analysis (FEA) as well as weighted gene co-expression network analysis (WGCNA) were used to identify dysregulated pathways, functionally relevant gene modules and hub genes. Key targets were validated in patient tissues and cell lines. A total of 1319 DEGs were identified (528 upregulated, 791 downregulated). Gene set enrichment analysis revealed activation of E2F targets and G2/M checkpoint, with downregulation of bile acid metabolism and estrogen response pathways. A tumor grade-correlated gene module was identified by WGCNA. FEA of the gene module highlighted pathways related to cell cycle and cell division. Co-expression analysis identified TPX2 as a central hub gene. Inhibitors of aurora kinase, TPX2 dependent enzyme, significantly reduced proliferation, migration, and invasion in GBC cells. Elevated Aurora kinases expression was also observed in GBC. This first transcriptomic analysis of GBC in South-East Asian Indians uncovers key drivers like TPX2 and Aurora kinases in disease progression. The study highlights cell cycle dysregulation and sex-linked signatures, offering insights for biomarker discovery and targeted therapies.

The environmental accumulation of toxic selenite poses a significant threat to public health and ecosystem sustainability. To improve the capability of microorganisms in selenite detoxification and biotransformation, a superior mutant of Lactiplantibacillus plantarum Vse252 was obtained via ARTP mutagenesis integrated with adaptive evolution. The mutant displayed remarkably enhanced selenite tolerance, with a maximum tolerable concentration reaching 300 mM. Furthermore, it converted 60 % of selenite to elemental selenium in 48 h, showing a remarkable improvement in selenium nanoparticles (SeNPs) biosynthesis. Electron microscopic observations showed that mutant maintained intact cell walls under selenite stress and abundantly produced uniformly dispersed SeNPs. Notably, the mutant produced 216.71 mg/L of extracellular polysaccharide (EPS) under selenite induction, a level markedly higher than that of the WT strain. Proteomic and metabolomic analyses revealed a global metabolic reprogramming in mutant, the upregulation of the pentose phosphate pathway and amino/nucleotide sugar metabolism (RfbA/RfbC) supplied NADPH and precursors for cell wall and EPS synthesis, while elevated oxidoreductases (GshR/Gpo) and cofactors (FMN/FAD) drove efficient selenite reduction. Four key mutated genes, including EmrE, MreC, MoaA, and AcpP, were identified via comparative genomic analysis, further confirming that cell wall homeostasis and transmembrane substance transport play critical regulatory roles in shaping the phenotypic characteristics of the mutant strain. This study reveals a tight association between the biosynthesis of extracellular SeNPs and EPS during microbial resistance to inorganic selenium stress, and highlights their pivotal roles in microbial detoxification, providing mechanistic insights and candidate targets for engineering strains toward selenium bioremediation and functional SeNPs production.

Cellular senescence is a stress-induced state characterized by permanent cell-cycle arrest and the development of a distinctive secretory profile that impacts tissue function and contributes to aging and metabolic disease. Senescence-associated β-galactosidase (SA-β-gal) activity is widely used as a marker of senescent cells; however, conventional SA-β-gal assays often rely on subjective visual assessment and provide limited quantitative information. These limitations are particularly evident in primary human cell populations such as the stromal vascular fraction (SVF) derived from adipose tissue, which contains a heterogeneous mixture of preadipocytes, immune cells, and endothelial cells. Here, we present an optimized reflected light confocal microscopy approach for high-resolution, quantitative detection of SA-β-gal activity in human SVF cells. This protocol enables objective single-cell analysis of SA-β-gal activity, allows simultaneous immunocytochemistry for multiplexed detection of additional senescence or lineage markers, and incorporates pH-matched controls. By combining these features, this method provides a sensitive, reproducible, and quantitative approach to studying cellular senescence in heterogeneous primary human cell populations. It offers an improved alternative to conventional SA-β-gal staining.

The nucleus is the characteristic organelle of eukaryotic organisms. Unlike the classic textbook view of static nuclei, nuclear shape is dynamic in live cells. Altered or deformed nuclear shape is a hallmark of cancer in animal cells and environmental stress in plants. Nuclear envelope proteins interact with chromatin to regulate gene expression. Unfortunately, little is known about the impact of abiotic stress on nuclear shape, movement, and chromatin dynamics. To confront this issue, we developed a pipeline using confocal microscopy and particle tracking software to quantify nuclear and chromatin dynamics in Arabidopsis roots under control and abiotic stress condition. This confocal imaging method utilizes a dual fluorescently tagged marker line - nuclear envelope protein and chromatin - to perform live cell imaging of the root in model plant Arabidopsis thaliana under control and salt-stressed conditions. These captured movies are analyzed to quantify nuclear and chromatin dynamics using open-source image processing software Fiji/ImageJ with the help of the TrackMate plugin. To validate this method, we imaged and quantified chromatin movement in control and salt-stressed roots, revealing a decrease in chromatin speed under salt-stressed conditions. This method allows for quantitative live cell imaging of root nuclear shape and chromatin dynamics during plant development and environmental stress, thus enabling analysis of changes in nuclear and chromatin dynamics caused by abiotic stressors.

Nitrite is a harmful substance generated in Litopenaeus vannamei farming systems, largely originating from the inadequate breakdown of surplus feed and shrimp feces. Its accumulation in the water can affect the growth and physiological functions of shrimp, damage the immune system, and even cause mass mortality, thus becoming a key environmental factor restricting the green development of the industry. Under nitrite stress, the eyestalk, as an important neuroendocrine regulatory center in crustaceans, participates in the stress adaptation of the organism and exerts a protective effect by regulating energy metabolism and immune function. However, the molecular regulatory mechanism of the eyestalk in response to nitrite stress remains unclear. In this study, single-cell RNA sequencing (scRNA-seq) technology was used to analyze the heterogeneity of eyestalk cells in L. vannamei under nitrite stress. A total of 18, 394 high-quality cells were obtained, and six major cell subpopulations, including Neurosecretory cell, Motor neuron, Sensory neuron, Interneuron, Neurogliocyte, and Support cell, were identified. Differential expression analysis identified 839 differentially expressed genes, and different cell types showed distinct specific responses to nitrite stress. Functional enrichment analysis indicated that pathways such as glycolysis, oxidative phosphorylation, ribosome function, and endoplasmic reticulum protein processing were significantly activated, while signal transduction and DNA repair-related pathways were inhibited. Further analysis revealed that nitrite stress could induce mitochondrial function changes and trigger oxidative stress, thereby affecting the neuroendocrine system function of the eyestalk. This study provided insights into transcriptomic responses of the eyestalk to nitrite stress at the single-cell level, laying a theoretical foundation for the management of aquaculture environments.

Allergic rhinitis (AR) is a common Th2-mediated inflammatory disease of the nasal mucosa, in which eosinophils serve as pivotal effector cells. The CCR3 receptor, specific for eotaxin, plays a critical role in allergic inflammation. However, the role of CCR3 in the differentiation of bone marrow CD34 ⁺ progenitor cells into eosinophils and its contribution to AR pathogenesis remains incompletely defined. This study aimed to investigate whether bone marrow cell-specific CCR3 deletion is associated with altered CD34 ⁺ progenitor abundance, eosinophil-lineage-related responses, and allergic inflammation in a murine model of allergic rhinitis. An integrative approach was employed. Bioinformatic analyses of transcriptomic data from CCR3-deficient mice, including differential expression screening, WGCNA, functional enrichment, and six machine learning algorithms, were used to identify candidate genes associated with CCR3 deletion. A bone marrow cell-specific CCR3 conditional knockout (CCR3-CKO) mouse model was generated and subjected to an ovalbumin-induced AR protocol. Nasal symptoms, body weight, nasal mucosal histopathology (H&E, PAS), serum cytokine/mediator levels (IL-5, eotaxin, ECP, EPO), and immune cell populations were assessed. Flow cytometry quantified CD34 ⁺ progenitors, CD34 ⁺ CCR3 ⁺ progenitors, and eosinophils in bone marrow, peripheral blood, and nasal lavage fluid. In vitro transwell migration and eosinophil colony-forming assays were performed to evaluate progenitor cell function. Bioinformatic and machine-learning analyses identified CD34 as a candidate hub gene associated with CCR3 deletion. In the AR model, CCR3-CKO mice showed reduced nasal symptom scores, less inflammatory-cell infiltration, and attenuated tissue injury, with a trend toward reduced allergy-associated weight loss. CCR3 deletion was associated with lower CD34 mRNA and protein levels in bone marrow and peripheral blood, as well as reduced proportions of CD34 ⁺ progenitors, CD34 ⁺ CCR3 ⁺ progenitors, and eosinophils across the analyzed compartments. Serum IL-5, ECP, and EPO levels were decreased, whereas eotaxin levels were increased. In vitro, CCR3 deficiency showed a modest reduction in the migratory response of CD34 ⁺ progenitors to eotaxin and partially reduced IL-5/eotaxin-associated eosinophil colony formation. Bone marrow cell-specific CCR3 deletion was associated with reduced eosinophil-lineage-related progenitor abundance, decreased eosinophilic inflammation, and partial improvement of AR-associated phenotypes. These findings suggest that the eotaxin/CCR3 axis may contribute to hematopoietic and eosinophil-lineage regulation in allergic inflammation. However, the direct cell-intrinsic regulation of CD34 expression and the relative contribution of IL-5-dependent pathways require further investigation.

Despite its established role in breast cancer treatment, Doxorubicin treatment remains subject to adaptive resistance mechanisms that extend beyond cancer cell intrinsic alterations ultimately reducing therapy efficacy. Our study in a MMTV-PyMT-driven mouse breast cancer model reveals that prolonged Doxorubicin (Dox) exposure triggers significant reprogramming of the tumour vasculature, substantially altering the angiocrine landscape and shaping treatment outcomes. Notably, tumours that initially respond, but later revert, display an endothelial cell subclustering with activation of proliferative and NF-κB-dependent cytokine pathways. We further identify a novel endothelial subpopulation characterised by higher expression of drug clearance and oxidative metabolism markers, suggesting an active role in mitigating Dox efficacy and angiogenesis promotion. These findings substantiate endothelial plasticity as a critical mediator of therapeutic failure. By uncovering these vascular adaptations, our work provides a new perspective on the underlying mechanisms of Dox resistance and the prolonged efficacy of chemotherapy in breast cancer.

This protocol describes a methodological approach to investigate the expression and functional role of CUB domain-containing protein 1 in nasopharyngeal carcinoma and its potential involvement in epithelial-mesenchymal transition. Clinical tissue samples from patients with nasopharyngeal carcinoma and rhinitis were collected to analyze CDCP1 expression using real-time quantitative PCR and immunohistochemistry. In vitro experiments were performed using CNE2 and HK1 nasopharyngeal carcinoma cell lines. CDCP1 overexpression and knockdown were achieved by transfection with a CDCP1 plasmid or specific siRNA. Cell proliferation was assessed by MTT assay, apoptosis was evaluated by Caspase-3 activity measurement, and the expression of EMT-related markers and phosphorylation levels of ERK1/2 were detected by western blot and quantitative PCR. To validate pathway involvement, rescue experiments were conducted using the ERK1/2-specific inhibitor U0126. This protocol provides a systematic in vitro and ex vivo. framework for elucidating the molecular mechanisms by which CDCP1 may regulate tumor progression in nasopharyngeal carcinoma.

MicroRNAs play wide roles in non-small cell lung cancer (NSCLC). To investigate the clinical value of miR-483-3p in NSCLC and its molecular target, 350 NSCLC patients were recruited in this study. RT-qPCR results showed that tumor histological expression of miR-483-3p progressively decreased as the tumor-node metastasis (TNM) stage increased. The receiver operating characteristic (ROC) curve displayed that histological miR-483-3p level can effectively distinguish NSCLC patients with high TNM stage (III) from those with low stage (I+II) (area under the ROC curve = 0.869). The Kaplan-Meier curve showed that NSCLC patients with low miR-483-3p expression demonstrated a lower 5-year overall survival rate. After adjusting for other confounding factors, multivariate Cox analysis further identified miR-483-3p as an independent protective factor for NSCLC survival. Mechanistically, RNA pull-down assay showed that upregulation of miR-483-3p was co-precipitated with KIF3B mRNA and inhibited its expression, thereby suppressing the malignant phenotypes of NSCLC cells and inducing apoptosis. In conclusion, downregulation of miR-483-3p serves as a poor prognostic marker in NSCLC, potentially affecting cancer progression by negatively regulating KIF3B.

Endoglin (ENG) is expressed in liver sinusoidal endothelial cells (LSECs), where it regulates endothelial activation and inflammation. The 7-ketocholesterol (7-K), a cholesterol oxidation product, accumulates in the liver and induces endothelial dysfunction. ENG has been shown to play an important role in adhesion processes in other endothelial models but its role in LSECs upon 7-K exposure remains unclear. We hypothesized that 7-K treatment of LSECs would confirm the critical role of ENG in endothelial activation, potentially outweighing the contributions of the cell adhesion molecules VCAM-1 and ICAM-1. Human LSECs were exposed to 25 µM 7-K. Protein expression and monocyte adhesion were assessed by flow cytometry, and soluble ENG was measured by ELISA. 7-K reduced ENG expression, while ICAM-1 was markedly upregulated. 7-K increased monocyte adhesion to LSECs, which was abrogated by ICAM-1, but not by ENG neutralization. We showed that 7-K promotes a pro-inflammatory phenotype defined by ICAM-1, rather than ENG. Thus, we propose that individual roles of ENG, ICAM-1, and VCAM-1 must be carefully considered when studying endothelial dysfunction.

The rapid postharvest softening of 'Saiwaihong' apples limits their commercial value. This study evaluated the preservative effects of 1-methylcyclopropene (1-MCP) combined with microporous film (MF), polyethylene film (PF), and silicon window film (SF). During 180 days of storage, fruit quality, physiological metabolism, pectin structure, and related gene expression were analyzed. The 1-MCP + SF treatment was most effective, significantly reducing ethylene production, delaying the respiratory peak by about 60 days, and resulting in the lowest firmness loss (21.50%) and weight loss (6.23%). Cell wall analysis showed that softening was mainly associated with pectin degradation. Moreover, 1-MCP + SF markedly suppressed MdGAL1, MdGAL2, MdPG1, and MdPME1 expression and inhibited β-galactosidase (β-GAL), polygalacturonase (PG), and pectin methylesterase (PME) activities, thereby maintaining cell wall integrity. These findings indicate that 1-MCP combined with SF effectively extends storage life and enhances the commercial potential of 'Saiwaihong' apples.

Prostate cancer (PCa), particularly in its advanced and castration-resistant forms, remains a major threat to men's health, with the tumor microenvironment (TME) playing a crucial role in its progression. Tumor-associated macrophages (TAMs), especially the M2 phenotype, are key components of the TME. Our previous work identified that M2 TAMs in PCa upregulate M-CSF secretion via the MS4A6A-MYC pathway. This study aims to identify the critical downstream effector within PCa cells that mediates the tumor-promoting effects of M-CSF. The impact of M-CSF on PCa cell (PC3 line) viability, invasion, and migration was assessed using CCK-8, Transwell, and wound healing assays. To identify M-CSF-regulated downstream proteins, a comprehensive proteomic analysis (DIA-PASEF) was performed on PC3 cells treated with or without M-CSF. Bioinformatic analyses screened for differentially expressed proteins. Key candidate PCLAF was further validated using Western blot, analysis of TCGA-PRAD data, and immunohistochemistry on a prostate tissue microarray (79 patients). The functional role of PCLAF was confirmed through in vitro experiments and an in vivo xenograft model in nude mice, comparing tumors from PC3 control cells, PC3 cells overexpressing KIAA0101/PCLAF (PC3-KIAA0101+), and PC3 cells with local M-CSF injections. M-CSF stimulation significantly enhanced PC3 cell viability, invasion, and migration in a concentration-dependent manner. Proteomic analysis revealed 95 differentially expressed proteins following M-CSF treatment. Among the top candidates, PCLAF/KIAA0101 was the only protein consistently and significantly upregulated by M-CSF in validation experiments. Analysis of TCGA data confirmed PCLAF's significant overexpression in PCa tumors and its association with poorer disease-free survival. Tissue microarray analysis demonstrated that PCLAF expression was significantly higher in PCa tissues compared to benign tissues and positively correlated with higher Gleason Grade Groups and ISUP risk groups. In the xenograft model, both PC3-KIAA0101+ and PC3 + M-CSF groups exhibited significantly increased tumor growth, volume, and weight compared to the control group. IHC, Western blot, and PCR analyses of the xenograft tumors confirmed that PCLAF expression levels followed the pattern: PC3-KIAA0101+ > PC3 + M-CSF > control, and were positively correlated with tumor growth. This study indicates that M-CSF, secreted by M2 TAMs, promotes prostate cancer progression by upregulating the expression of PCLAF/KIAA0101 in cancer cells. PCLAF is overexpressed in PCa, correlates with tumor malignancy and poor prognosis, and its upregulation is sufficient to enhance tumor growth in vivo. These findings indicate the M-CSF-PCLAF axis as a key mechanism through which TAMs drive PCa invasion and progression, identifying PCLAF as a potential therapeutic target.

Quantitative, spatially resolved analysis of gene expression is essential for assessing cell-type-specific molecular profiles. In the Drosophila visual system, extensive genetic tools open a framework for direct evaluation of both RNA and protein levels in defined neuronal populations. Here, we present a step-by-step protocol that combines expansion-assisted HCR-smFISH (hybridization chain reaction single-molecule fluorescence in situ hybridization) with immunohistochemistry to enable quantitative analysis of cell-type-specific molecular profiles in genetically defined visual system neuronal types. The workflow is optimized for cells labeled with nuclear-localized or membrane-bound markers, allowing measurement of transcript and protein levels in the same neurons. Following tissue expansion, samples are imaged using light-sheet microscopy for rapid volumetric acquisition, with an alternative mounting and imaging workflow demonstrated for standard inverted laser scanning and spinning disc confocal microscopes. We further provide an automated segmentation algorithm that distinguishes nuclear and cytoplasmic transcripts, enabling analyses of transcriptional state and subcellular RNA localization. Practical guidance is provided on experimental parameters and common pitfalls affecting signal quality, tissue integrity, and quantitative performance. Representative applications include validation of cell-type-specific RNA interference by quantifying corresponding changes in RNA and protein levels. By enabling integrated RNA- and protein-level measurements with cell-type specificity, this approach provides a scalable strategy for hypothesis-driven molecular analysis and, in targeted contexts, a practical alternative to single-cell transcriptomic assays. This protocol provides a practical approach for validating cell-type-specific molecular perturbations while preserving the anatomical context of the intact Drosophila brain.