Cytotechnology最新文献

Colorectal cancer is a major cause of cancer-related mortality, and its clinical management is still limited by recurrence, metastasis, and therapy resistance. Although ferroptosis is increasingly recognized as a therapeutically relevant vulnerability in colorectal cancer, the upstream regulators that link malignant signaling to ferroptosis-related homeostasis remain poorly defined. TRIM21 is a multifunctional E3 ubiquitin ligase with emerging roles in cancer biology, but its involvement in USP4/TGF-β-associated regulation and ferroptosis-related phenotypes in colorectal cancer is not well understood. Public transcriptomic datasets were analyzed to evaluate the clinical significance of TRIM21 expression across different clinicopathological categories. Gain- and loss-of-function approaches were applied in HCT116 cells to evaluate proliferation, migration, and invasion using CCK-8, wound-healing, and Transwell assays. We examined the relationship between TRIM21 and USP4 by co-immunoprecipitation and a rescue design involving TRIM21 silencing with USP4 re-expression. Ferroptosis-associated markers (SLC7A11 and GPX4) were evaluated using immunoblotting, and ferroptosis-related biochemical indices (Fe²⁺, SOD activity, and MDA content) were quantified. Ferrostatin-1 was used to pharmacologically investigate ferroptosis under TRIM21/USP4 perturbations. TRIM21 was clinically associated with advanced clinicopathological features and was elevated in colorectal cancer cell models. Functional studies revealed that TRIM21 promotes proliferative and invasive/migratory phenotypes, accompanied by coordinated changes in USP4 and TGFB1 transcript levels. USP4 re-expression in TRIM21-silenced cells partially restored malignant traits and reshaped ferroptosis-associated molecular and biochemical readouts. Pharmacological inhibition of ferroptosis modulated TRIM21/USP4-linked phenotypes and corresponding ferroptosis-related indices. These findings identify TRIM21 as a clinically relevant regulator that links USP4/TGF-β-associated signaling to ferroptosis-related homeostasis, thereby promoting malignant behaviors in colorectal cancer and providing a targetable vulnerability for therapeutic development.

Non-small cell lung cancer (NSCLC) is a major cause of cancer-related deaths worldwide. One protein involved in RNA processing and splicing, SNRPA1, has been suggested to play a role in the pathogenesis of NSCLC. Therefore, investigating the regulatory mechanisms involving small nuclear ribonucleoprotein polypeptide A' (SNRPA1) in NSCLC could provide valuable insights into the disease progression. The study involved the analysis of SNRPA1, methyltransferase 3, n6-adenosine-methyltransferase complex catalytic subunit (METTL3), insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) and twist family bHLH transcription factor 1 (TWIST1) expressions in lung tissues and normal lung tissues using data obtained from the TCGA, CPTAC, and/or ENCORI databases. The prognostic value of SNRPA1 in lung tissues was assessed through the Kaplan-Meier Plotter database and TCGA database. mRNA expression was quantified via qRT-PCR, while protein expression was evaluated using western blotting assay or IHC assay. Cell viability, proliferation, migration, and invasion were analyzed through various in vitro assays. The interaction between SNRPA1 and METTL3 or IGF2BP2 was studied using RIP assay, dual-luciferase reporter assay, and actinomycin D assay, while the association of SNRPA1 with TWIST was determined through Co-IP assay and CHX assay. The effects of METTL3 silencing and SNRPA1 overexpression on malignant growth of NSCLC cells were confirmed using a xenograft mouse model assay and a lung metastasis model. SNRPA1 expression was significantly upregulated in NSCLC tissues and cells. Depletion of SNRPA1 led to the inhibition of NSCLC cell proliferation, migration, and invasion. Additionally, METTL3 and IGF2BP2 were found to stabilize SNRPA1 mRNA expression through m6A methylation modification. SNRPA1 overexpression attenuated the effects induced by METTL3 knockdown on NSCLC cells in vitro. Furthermore, SNRPA1 was observed to interact with TWIST1 in NSCLC cells, and TWIST1 overexpression attenuated SNRPA1 knockdown-induced effects on the key malignant phenotypes. In vivo experiments showed that SNRPA1 overexpression rescued the effects of METTL3 depletion on the malignant growth of NSCLC cells. The findings of this study highlight the crucial role of the METTL3/IGF2BP2-SNRPA1-TWIST1 axis in promoting NSCLC development through m6A methylation modification. Targeting this pathway may offer novel therapeutic strategies for the treatment of NSCLC.

Graphical abstract: The METTL3/IGF2BP2-mediated m6A methylation modification of SNRPA1 binds to TWIST1 to promote NSCLC cell proliferation, migration, and invasion, ultimately leading to NSCLC progression.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-026-00924-w.

Photothermal therapy has emerged as a promising minimally invasive approach for biomedical applications, where nanoparticle-mediated heat generation plays a critical role. The present study aims to investigate the photothermal efficiency of green-synthesized bimetallic silver-palladium nanoparticles (AgPd NPs) and gold-palladium nanoparticles (AuPd NPs) prepared using Aloe barbadensis Linn. leaf gel as a natural reducing and stabilizing agent. The formation and physicochemical properties of the synthesized nanoparticles were characterized by UV-visible spectroscopy, transmission electron microscopy (TEM), and inductively coupled plasma-atomic emission spectroscopy (ICP-AES), confirming their optical properties, morphology, and elemental composition. Both AgPd NPs and AuPd NPs exhibited efficient photothermal conversion under irradiation, resulting in a significant temperature increase. Cytocompatibility was evaluated using an MTT assay on L6 rat myofibroblast cells at concentrations ranging from 20 to 100 µg/mL for 48 h. AgPd NPs showed concentration-dependent cytotoxicity, with pronounced toxicity at higher concentrations, whereas AuPd NPs maintained comparatively higher cell viability across the tested range. These findings indicate that Aloe vera-mediated AuPd NPs combine effective photothermal performance with superior biocompatibility, highlighting their potential as environmentally sustainable and safe nanomaterials for photothermal therapeutic applications.

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Macrophage polarization represents a potential therapeutic target for ameliorating osteoarthritis (OA). Milk fat globule EGF and factor V/VIII domain containing (MFG-E8) can modulate macrophage polarization; however, its underlying mechanism in OA remains elusive. This study aims to investigate MFG-E8's effects on macrophage polarization in OA. Nine healthy volunteers and twenty-nine patients with knee OA were recruited to collect knee synovial fluid for detecting interleukin (IL)-1β, tumor necrosis factor (TNF)-α, transforming growth factor-β1 (TGF-β1), and MFG-E8. Lipopolysaccharide (LPS) was used to induce M1 polarization in RAW264.7 cells, with interventions of recombinant MFG-E8 (500 ng/mL) and the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) agonist nigericin (10 µmol/L). Enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence, quantitative real-time polymerase chain reaction, and western blotting were used to analyze M1 macrophage biomarkers and the NLRP3 signaling pathway. IL-1β and TNF-α levels were elevated in OA patients, and positively correlated with Kellgren Lawrance grading, whereas TGF-β1 and MFG-E8 showed the opposite trend. M1/M2 macrophages were increased in OA patients and inversely correlated with MFG-E8 levels. In LPS-stimulated macrophages, mRNA and protein levels of IL-6, IL-1β, TNF-α, NLRP3, and IL-18 were upregulated, along with increased expressions of inducible Nitric Oxide Synthase, cleaved-caspase 1, and N-terminal domain of Gasdermin-D; these were reduced by MFG-E8 addition. Nigericin reversed the effect of rmMFG-E8 on LPS-stimulated macrophages. This study proves that MFG-E8 inhibits NLRP3 to suppress M1 macrophage polarization, laying a foundation for the application of MFG-E8 in OA treatment.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-026-00936-6.

To explore the intervention mechanism of the new compounds Frehmaglutin F (Fre F) and Frehmaglutin H (Fre H) in Rehmannia glutinosa Libosch. (RG) on pulmonary hypertension (PH). An in vitro model of PH was established by using hypoxia-induced aberrant proliferation of Pulmonary Arterial Smooth Muscle Cells (PASMCs). Firstly, the activity and safety of Fre F and Fre H compounds were evaluated. Subsequently, the PH gene expression profile chip dataset GSE244830 and GSE113439 were downloaded from the gene expression omnibus (GEO) database to analyze the differential genes. Last, The cell migration and proliferation ability, the level of oxidative stress, mitochondrial damage, indicators related to the SIRT3/HIF-1α/PKM2 pathway, and the level of glycolysis were examined by adding the SIRT3 inhibitor (3-TYP) or silencing the SIRT3 gene to explore the the mechanism of action of Fre F and Fre H in improving PH. The SIRT3 gene was significantly reduced in both patients and mice with PH, and further molecular docking showed that the binding energies of Fre F and Fre H to the SIRT3 protein receptor were - 12.4 kJ/mol and - 11.2 kJ/mol. This result indicated that Fre F and Fre H had better binding activity to SIRT3 protein. The results of in vitro experiments showed that Freh F and Fre H significantly inhibited the proliferative and migratory abilities of PASMCs, decreased the intracellular levels of ROS, regulated mitochondrial damage, modulated the expression of SIRT3, HIF-1α, PKM2, and inhibited the glycolytic ability of PASMCs, but the above effects were completely or partially reversed upon the addition of 3-TYP and the silencing of SIRT3. Fre F and Fre H may exert anti-PH effects by reducing glycolysis levels via SIRT3/HIF-1α/PKM2 signal pathway.

The ATP-binding cassette, subfamily G, member 2 (ABCG2) transporter, or breast cancer resistance protein (BCRP), plays a crucial role in the absorption and distribution of various compounds, including anti-seizure medications (ASMs). ABCG2 genetic polymorphisms have been shown to affect the pharmacokinetics of several drugs, including anti-cancer drugs and statins. Clinical studies suggested an association between the ABCG2 variations and ASMs pharmacokinetics. Thus, the aim of the present study was to evaluate the effect of the coding ABCG2 variants, 742T > G and 886G > C on the ABCG2-mediated transport of ASMs, carbamazepine and N-desmethyl clobazam by employing cell-based bidirectional transport experiments. Human cerebral microvascular endothelial cells (hCMEC/D3) were transfected with ABCG2, carrying either the wild-type allele (742T, 886G) or its mutated counterparts (742G or 886 C). The cells overexpressing ABCG2 variants exhibited significant reduction in mRNA and protein expression levels compared to those with wild-type protein (742T, 886G). This suggests that ABCG2 variants lead to a downregulation in ABCG2 expression. ABCG2 wild-type protein (742T, 886G) actively transported DCLB, with an efflux ratio > 2 that significantly decreased upon addition of ABCG2-specific inhibitor Ko143, confirming its role in DCLB transport. In contrast, CBZ showed an efflux ratio < 1.5 with no notable reduction upon ABCG2 inhibition. ABCG2 742T > G or 886G > C variants markedly reduced the efflux ratio of DCLB to 1, compared to wild-type protein. In contrast, these variants did not affect CBZ transport. These findings indicate that the ABCG2 genetic polymorphisms studied may account for variability in individual ASM responses, supporting more personalized therapy for epilepsy patients.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-026-00909-9.

Background: Diabetic neuropathic pain (DNP) is a common chronic complication of diabetes mellitus (DM). Schwann cells (SCs) dysfunction plays an important role in the pathogenesis of DNP. Early growth response protein 1 (EGR1) is related to apoptosis and immune regulation and plays important roles in neuropathic pain. However, its exact role in DNP-induced SCs injury is still unclear. Methods: A DNP mouse model was established using a high-fat diet combined with STZ, and a high glucose (HG)-induced RSC96 cell model was used for the experimental studies. The metabolic status of the mice was assessed by fasting blood glucose (FBG) levels. Pathological damage to the sciatic nerve was detected by HE staining. Pain sensitivity was comprehensively evaluated by the von Frey test and hot plate test. Cell viability was determined by a CCK-8 assay, and apoptosis was detected by TUNEL staining. Western blotting, immunohistochemistry, immunofluorescence, and ELISA were used to detect the expression of related proteins. Results: EGR1 was highly expressed in DNP mice and HG-induced RSC96 cells. Knockdown of EGR1 alleviated the symptoms of DNP mice, including a reduction in FBG levels, an increase in the tactile response threshold, a reduction in thermal response delay, and alleviation of sciatic nerve injury. In addition, EGR1 knockdown inhibited the apoptosis of SCs and inflammatory responses. In terms of molecular mechanisms, EGR1 competitively binds to MDM2 to inhibit MDM2-mediated P53 ubiquitination and increase P53 expression, thereby promoting HG-induced SC injury, whereas inhibition of MDM2 reverses the protective effect of EGR1 knockdown on SCs. Conclusion: EGR1 inhibits MDM2-mediated P53 ubiquitination and degradation by competitively binding to MDM2, resulting in the upregulation of P53 expression, thereby promoting HG-induced SC function injury and inflammatory responses and aggravating DNP.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-026-00935-7.

Tumor-associated macrophages (TAMs) polarized to the M2 phenotype play a pivotal role in fostering an immunosuppressive tumor microenvironment (TME) that drives colorectal cancer (CRC) progression. Ubiquitin-specific peptidase 16 (USP16) is a deubiquitinating enzyme, yet its function in CRC-associated macrophage polarization remains undefined. The present study demonstrated that USP16 was a critical promoter of M2 macrophage polarization in CRC. We found that USP16 silencing in macrophages skewed polarization towards the antitumor M1 phenotype, enhanced phagocytic capacity, and increased lysosomal enzyme activity. Conversely, USP16 overexpression promoted M2 polarization and suppressed M1 characteristics. Mechanistically, USP16 directly interacted with and deubiquitinated the transcription factor E2F1, thereby stabilizing it. Stabilized E2F1 transcriptionally activated Notch1, a key regulator of the Notch signaling pathway. This USP16/E2F1/Notch1 axis was essential for driving M2 polarization, as silencing either E2F1 or Notch1 abrogated the effects of USP16 overexpression. Furthermore, conditioned medium from USP16-overexpressing macrophages significantly enhanced the proliferation and invasion of CRC cells, effects that were dependent on this molecular axis. In conclusion, our findings identify a novel USP16/E2F1/Notch1 signaling circuit that orchestrates protumorigenic M2 macrophage polarization in CRC, highlighting USP16 as a potential therapeutic target for reprogramming the immunosuppressive TME and improving CRC treatment.

Vascular endothelial dysfunction plays a central role in the progression of gestational hypertension (GH). A portion of GH cases progress to preeclampsia, suggesting shared angiogenic imbalances and placental dysfunction as underlying mechanisms. Laminin subunit alpha-4 (LAMA4) downregulation has been linked to preeclampsia pathogenesis. This study explored the precise role of LAMA4 in HUVEC dysfunction induced by hypoxia/reoxygenation (H/R) insults. HUVECs were treated with H/R conditions to mimic the in vitro model of GH. LAMA4 mRNA expression was analyzed using quantitative PCR. Protein expression was detected by immunoblotting. The effects on HUVEC dysfunction under H/R were evaluated by measuring cell viability, proliferation, apoptosis, tube formation, and ROS and MDA levels. The relationship between SP1 and LAMA4 was confirmed by chromatin immunoprecipitation (ChIP) and luciferase assays. The interaction between NEDD4 and LAMA4 was confirmed by protein stability analysis and immunoprecipitation (IP) assay. LAMA4 expression was decreased in GH serum and H/R-treated HUVECs. Rescue of LAMA4 expression recovered HUVEC growth and tube formation capacities and diminished cell apoptosis and oxidative stress under H/R insults. Mechanistically, SP1 upregulated LAMA4 by transcriptionally activating its promoter. Furthermore, NEDD4 downregulated LAMA4 expression through ubiquitination. Silencing LAMA4 abrogated the attenuated effects of SP1 upregulation or NEDD4 depletion on HUVEC dysfunction induced by H/R. Our study demonstrates that LAMA4, which is transcriptionally upregulated by SP1 and post-translationally downregulated by NEDD4, mediates H/R-induced dysfunction in HUVECs by modulating cell growth, tube formation, and oxidative stress.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00861-0.

Neuropathic pain (NP) is a common and disabling condition characterized by microglial polarization-evoked neuroinflammation. Pedunculoside has been implicated in several inflammation-related diseases and exerts the neuroprotective effects However, its role in NP remains unclear. In this study, pedunculoside dose-dependently suppressed LPS-induced activation of BV2 microglial cells by reducing expression of the microglial marker IBA-1, but without obvious cytotoxicity. Immunofluorescence assay further confirmed that pedunculoside decreased % of CD32⁺ M1 microglia and increased % of CD206⁺ M2 microglia in LPS-stimulated microglia, accompanied by reduced expression of M1 microglial marker CD32 and iNOS and increased expression of M2-like microglial marker CD206 and Arg-1, indicating that pedunculoside could reverse LPS-induced microglial polarization from M1 phenotype towards M2 phenotype. Moreover, pedunculoside also attenuated inflammatory response in LPS-treated microglia by lowering pro-inflammatory cytokine levels (IL-1β, TNF-α, and IL-4) and increasing anti-inflammatory IL-10 levels. Mechanistically, the activation of the TLR4-NF-κB pathway in LPS-treated microglia was suppressed by pedunculoside. Furthermore, reactivating this signaling by TLR4 overexpression abrogated pedunculoside-mediated effects on microglial polarization towards M2 and inflammation. In vivo, administration of pedunculoside alleviated pain sensitivity and modulated microglial polarization from M1 to M2 in chronic constrictive injury (CCI)-induced NP mice. Additionally, pedunculoside also alleviated neuroinflammation and suppressed activation of the TLR4-NF-κB pathway in NP mice. Collectively, these findings indicate that pedunculoside may ameliorate the progression of NP by affecting microglial polarization from M1 towards M2 phenotype through inhibition of the TLR4-NF-κB pathway, supporting its potential as a promising therapeutic agent for NP.