Experimental cell research最新文献

Individuals with type 2 diabetes mellitus frequently display heightened levels of palmitic acid (PA) in their serum, which may lead to β-cell damage. The involvement of ferroptosis, a form of oxidative cell death in lipotoxic β-cell injury remains uncertain. Here, we have shown that PA induces intracellular lipid peroxidation, increases intracellular Fe²⁺ content and decreases intracellular glutathione peroxidase 4 (GPX4) expression. Furthermore, PA causes distinct changes in pancreatic islets and INS-1 cells, such as mitochondrial atrophy and increased membrane density. Furthermore, the presence of the ferroptosis inhibitor has a significant mitigating effect on PA-induced β-cell damage. Mechanistically, PA increased ceramide content and c-Jun N-terminal kinase (JNK) phosphorylation. The ceramide synthase inhibitor effectively attenuated PA-induced β-cell damage and GPX4/Fe²⁺ abnormalities, while inhibiting JNK phosphorylation. Additionally, the JNK inhibitor SP600125 improved PA-induced cell damage. In conclusion, by promoting ceramide synthesis, PA inhibited GPX4 expression and increased intracellular Fe²⁺ to induce β-cell ferroptosis. Moreover, JNK may be a downstream mechanism of ceramide-triggered lipotoxic ferroptosis in β-cells.

Liver fibrosis is a significant health concern globally due to its association with severe liver conditions like cirrhosis and liver cancer. Histone lactylation has been implicated in the progression of hepatic fibrosis, but its specific role in liver fibrosis, particularly regarding H3K18 lactylation, remained unclear. To investigate this, we established in vivo and in vitro models of liver fibrosis using carbon tetrachloride (CCl4) injection in rats and stimulation of hepatic stellate cells (HSCs) with TGF-β1, respectively. We found that histone lactylation, particularly H3K18 lactylation, was upregulated in both CCl4-induced rats and TGF-β1-activated HSCs, indicating its potential involvement in liver fibrosis. Further experiments revealed that lactate dehydrogenase A (LDHA) knockdown inhibited H3K18 lactylation and had a beneficial effect on liver fibrosis by suppressing HSC proliferation, migration, and extracellular matrix (ECM) deposition. This suggests that H3K18 lactylation promotes liver fibrosis progression. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that H3K18 lactylation facilitated the transcription of SOX9, a transcription factor associated with fibrosis. Importantly, overexpression of SOX9 counteracted the effects of LDHA silencing on activated HSCs, indicating that SOX9 is downstream of H3K18 lactylation in promoting liver fibrosis. In summary, this study uncovers a novel mechanism by which H3K18 lactylation contributes to liver fibrosis by activating SOX9 transcription. This finding opens avenues for exploring new therapeutic strategies for hepatic fibrosis targeting histone lactylation pathways.

Glaucoma is characterized by pathological elevation of intraocular pressure (IOP) due to dysfunctional trabecular meshwork (TM), which is the primary cause of irreversible vision loss. There are currently no effective treatment strategies for glaucoma. Mitochondrial function plays a crucial role in regulating IOP within the TM. In this study, primary TM cells treated with dexamethasone were used to simulate glaucomatous changes, showing abnormal cellular cytoskeleton, increased expression of extracellular matrix, and disrupted mitochondrial fusion and fission dynamics. Furthermore, glaucomatous TM cell line GTM3 exhibited impaired mitochondrial membrane potential and phagocytic function, accompanied by decreased oxidative respiratory levels as compared to normal TM cells iHTM. Mechanistically, lower NAD + levels in GTM3, possibly associated with increased expression of key enzymes CD38 and PARP1 related to NAD + consumption, were observed. Supplementation of NAD + restored mitochondrial function and cellular viability in GTM3 cells. Therefore, we propose that the aberrant mitochondrial function in glaucomatous TM cells may be attributed to increased NAD + consumption dependent on CD38 and PARP1, and NAD + supplementation could effectively ameliorate mitochondrial function and improve TM function, providing a novel alternative approach for glaucoma treatment.

Mouse HORMAD1 is a phospho-protein involved in multiple functions during meiotic prophase I. To obtain insight into the significance of its phosphorylation, we generated phospho-specific antibodies against two serine residues, Ser307 and Ser378, representing each of two serine clusters in mouse HORMAD1. The Ser307 phosphorylation is detectable from early leptotene substage in both wild-type and Spo11^−/− spermatocytes, indicating that Ser307 is a primary and SPO11-independent phosphorylation site. In contrast, the Ser378 phosphorylation is negligible at earlier substages in wild-type and Spo11^−/− spermatocytes. After mid-zygotene substage, the Ser378 phosphorylation is abundant on unsynapsed chromosome axes in wild-type spermatocytes and is detected only in a part of unsynapsed chromosome axes in Spo11^−/− spermatocytes. We also generated a non-phosphorylated Ser307-specific antibody and found that Ser307 is phosphorylated on sex chromosome axes but is almost entirely unphosphorylated on desynapsed chromosome axes in diplotene spermatocytes. These results demonstrated a substage-specific phosphorylation status of mouse HORMAD1, which might be associated with multiple substage-specific functions.

Colorectal cancer (CRC) poses a significant challenge in oncology due to its increasing global incidence and treatment complexities. This study delved into the role of the dual-specificity protein kinase CLK3 in CRC progression and its potential as a therapeutic target. By analyzing clinical data and experimental models comprehensively, we found that CLK3 expression was markedly elevated in CRC tissues compared to normal colon tissue. High CLK3 levels were associated with advanced clinical stages and poor prognosis in CRC patients, suggesting its utility as a prognostic biomarker. Functional assays demonstrated that CLK3 overexpression boosted CRC cell proliferation and ATP production, whereas genetic CLK3 knockdown hindered cell proliferation in vitro and curbed tumor growth in vivo. Mechanistically, we uncovered that CLK3 positively influenced the IL-6/STAT3 signaling pathway by stabilizing JAK2 protein levels. These findings propose targeting CLK3 signaling as a promising therapeutic approach for CRC. Further investigation into CLK3's molecular mechanisms and clinical implications is necessary to fully harness its potential in managing CRC.

Bladder cancer(BC) is one of the most prevalent cancers in the urinary tract, with high recurrence and fatality rates. Research indicates that go-ichi-ni-san complex subunit 1 (GINS1) crucially influences cancer progression by regulating DNA replication through cell cycle modulation. Thus, suppressing the active proliferation of cells in tumor tissues may require silencing GINS1. However, the consequences of GINS1 in bladder cancer aren't to be determined. In this paper, we examine the role and mechanism of GINS1 in the development of bladder cancer. GINS1 expression levels and prognostic relevance in bladder cancer were validated using Western blotting, immunohistochemistry, and Kaplan-Meier survival analysis. The influence of GINS1 on bladder cancer was investigated using a variety of approaches, including cell transfection, cell counts, transwell migrations, colony formation, and flow cytometry. Immunohistochemistry studies demonstrate that GINS1 expression is increased in bladder cancer tissues. GINS1 silencing resulted in an arrest of the cell cycle at the phase of G0/G1, which inhibited BC cell growth both in vitro and in vivo. GINS1 knockdown also hindered the AKT/mTOR pathway. Furthermore, increased GINS1 expression affects the cell cycle and stimulates the AKT/mTOR pathway, allowing BC to develop more quickly. Consequently, GINS1 occurs as a latent therapeutic target, particularly for individuals with BC.

Prostate cancer (PCa) is the most prevalent malignant tumor of the genitourinary system, and metastatic disease has a significant impact on the prognosis of PCa patients. As a result, knowing the processes of PCa development can help patients achieve better outcomes. Here, we investigated the expression and function of ORC6 in PCa. Our findings indicated that ORC6 was elevated in advanced PCa tissues. Patients with PCa who exhibited high levels of ORC6 had a poor prognosis. Following that, we investigated the function of ORC6 in PCa progression using a variety of functional experiments both in vivo and in vitro, and discovered that ORC6 knockdown inhibited PCa cell proliferation, growth, and migration. Furthermore, RNA-seq was employed to examine the molecular mechanism of PCa progression. The results revealed that ORC6 might promote the expression of PLK1, a serine/threonine kinase in PCa cells. We also discovered that ORC6 as a novel miR-361-5p substrate using database analysis, and miR-361-5p was found to lower ORC6 expression. Additionally, RNA immunoprecipitation (RIP) and luciferase reporter tests revealed that the transcription factor E2F1 could regulate ORC6 expression in PCa cells. PLK1 overexpression or miR-361-5p inhibitor treatment effectively removed the inhibitory effects caused by ORC6 silencing. Notably, our data showed that therapeutically targeting the miR-361-5p/ORC6/PLK1 axis may be a viable therapy option for PCa.

Firefly luciferase (Fluc) from Photinus pyralis is one of the most widely used reporter proteins in biomedical research. Despite its widespread use, Fluc's protein phase transition behaviors and phase separation characteristics have not received much attention. Current research uncovers Fluc's intrinsic property to phase separate in mammalian cells upon a simple cell culture temperature change. Specifically, Fluc spontaneously produced needle-shaped crystal-like inclusion bodies upon temperature shift to the hypothermic temperatures ranging from 25 °C to 31 °C. The crystal-like inclusion bodies were not associated with or surrounded by membranous organelles and were likely built from the cytosolic pool of Fluc. Furthermore, the crystal-like inclusion formation was suppressed when cells were cultured in the presence of D-luciferin and its synthetic analog, as well as the benzothiazole family of so-called stabilizing inhibitors. These two classes of compounds inhibited intracellular Fluc crystallization by different modes of action as they had contrasting effects on steady-state luciferase protein accumulation levels. This study suggests that, under substrate insufficient conditions, the excess Fluc phase separates into a crystal-like state that can modulate intracellular soluble enzyme availability and protein turnover rate.

CCAAT enhancer binding protein delta (CEBPD) is a transcription factor and plays an important role in apoptosis and oxidative stress, which are the main pathogenesis of ischemic stroke. However, whether CEBPD regulates ischemic stroke through targeting apoptosis and oxidative stress is unclear. Therefore, to answer this question, rat middle cerebral artery occlusion (MCAO) reperfusion model and oxygen-glucose deprivation/reoxygenation (OGD/R) primary cortical neuron were established to mimic ischemic reperfusion injury. We found that CEBPD was upregulated and accompanied with increased neurological deficit scores and infarct size, and decreased neuron in MCAO rats. The siRNA targeted CEBPD inhibited CEBPD expression in rats, and meanwhile lentivirus system was used to blocked CEBPD expression in primary neuron. CEBPD degeneration decreased neurological deficit scores, infarct size and brain water content of MCAO rats. Knockdown of CEBPD enhanced cell viability and reduced apoptosis as well as oxidative stress in vivo and in vitro. CEBPD silencing promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the expression of heme oxygenase 1 (HO-1). Newly, CEBPD facilitated the transcription of cullin 3 (CUL3), which intensified ischemic stroke through Nrf2/HO-1 pathway that was proposed by our team in the past. In conclusion, targeting CEBPD-CUL3-Nrf2/HO-1 axis may be contributed to cerebral ischemia therapy.

Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of β-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.