Cell Cycle

Accurate chromosome segregation relies on proper centromere and kinetochore formation and phospho-regulation. We previously demonstrated that a pluripotent state confers a low fidelity of chromosome segregation, however it is unknown how a pluripotent state impacts centromere and kinetochore function. Here, we demonstrate that both centromere and kinetochore structural organization and phosphorylation in mitosis are developmentally regulated. CENP-A, CENP-C, and HEC1 protein abundance is reduced at mitotic centromeres and kinetochores of human pluripotent stem cells (hPSCs) compared to isogenic somatic cells; however, elevating their levels does not improve chromosome segregation fidelity. Rather, we find that reduced phosphorylation of kinetochores is responsible for their low fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs compared to isogenic somatic cells at Cyclin B/Cdk1 and Aurora kinase phospho-sites. Inhibiting PP2A phosphatase activity or differentiation increases HEC1 phosphorylation at hPSC kinetochores decreasing chromosome segregation errors. Thus, mitotic fidelity in non-transformed human cells depends on the developmental regulation of the kinase and phosphatase networks controlling kinetochore phosphorylation. SummaryGalaviz Sarmiento et al show that the developmental regulation of kinetochore phosphorylation governs mitotic fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs during mitosis contributing to their high rate of chromosome segregation errors. While differentiation increases HEC1 phosphorylation improving chromosome segregation fidelity.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Interstrand crosslinks are cytotoxic lesions that inhibit essential processes including replication and transcription. Replication fork reversal occurs in response to interstrand crosslink inducing drug, MMC, but how replication fork reversal promotes repair of interstrand crosslinks is poorly understood. Here, we investigated the role of the RAD54L translocase in interstrand crosslink repair. We found RAD54L is required to promote nascent DNA degradation in FANCD2 and FANCA-depleted cells consistent with a previous study indicating RAD54L promotes replication fork reversal. We further show RAD54L activity is required for formation of radial chromosomes in FANCD2-deficient cells suggesting fork reversal may be required to generate the intermediate undergoing aberrant fusion in FANC-deficient cells. Finally, we demonstrate FANCD2 foci accumulate and DSBs persist in RAD54L-deficient cells indicating RAD54L is required for efficient repair of DSBs. Together, our results indicate RAD54L plays multiple roles in efficient processing and repair of interstrand crosslinks.

BackgroundDynamic chromatin behavior, which is related to chromatin accessibility, plays a critical role in various genome DNA functions such as RNA transcription and DNA replication/repair. Previous studies using highly synchronized cells showed that average local chromatin motion, captured by single-nucleosome imaging and tracking on a second time scale, remained almost constant throughout G1, S, and G2 phases in living human cells, although possible effects of prolonged drug treatments for cell-cycle synchronization could not be excluded. ResultsTo avoid possible effects of prolonged drug treatment, we combined single-nucleosome imaging with Fucci probes to visualize cell-cycle progression through G1, S, and G2. Using HeLa and HCT116 cells expressing H2B-HaloTag and Fucci probes, we found that local nucleosome motion remained similar on average throughout interphase, except for elevated motion in early G1. Transcription inhibition similarly increased nucleosome motion throughout interphase. Local nucleosome motion also increased following replication stress or DNA damage. ConclusionOur findings suggest that near-constant chromatin motion supports housekeeping functions under similar physical conditions during interphase. Our findings also suggest that cells can transiently change chromatin motion to perform ad hoc tasks in response to signals from inside and outside the cell, such as DNA damage.

Cancer-related genomic instability (GI) may cause genetic alterations in spermatozoa, implying health issues not only in cancer survivors, but also in their children [1, 2]. We therefore studied Loss of Y chromosome (LOY), considered as hallmark of GI [3-15], in spermatozoa and blood from survivors of childhood and testicular cancer (CC, TC), and controls (CTRL). We found that LOY was statistically significantly more frequent in spermatozoa from cancer survivors than in controls (Odds Ratio [OR]=2.2 for CC vs. CTRL and OR=2.4 for TC vs. CTRL). Furthermore, LOY was about an order of magnitude more prevalent in spermatozoa than in blood among 18-53-year-old males within all cohorts. Our findings suggest that LOY in spermatozoa might be a clinically useful marker of GI, reduced fertility and disease predisposition in males. Introducing LOY in spermatozoa as a biomarker opens a new research avenue into disease prevention and the causes and consequences of LOY.

The retinoblastoma protein Rb is a cell cycle inhibitor that plays a central role in regulating the G1/S cell cycle transition. Un-/hypo-phosphorylated Rb suppresses E2F transcription activity by binding to E2F/DP dimers and recruiting chromatin remodelers to prevent cells from entering S phase. For cells to progress through the G1/S transition, Rb is inactivated by two mechanisms: the "classic" pathway of Rb hyperphosphorylation by Cyclin-CDK complexes, and a recently identified "degradation" mechanism driven by the E3 ubiquitin ligase UBR5. These two pathways are interconnected, as only the un-/hypo-phosphorylated Rb can be degraded, and the hyper-phosphorylated Rb is stabilized to promote its reaccumulation in preparation for the next cell division cycle. However, the molecular basis for how Rb is stabilized upon phosphorylation remains unclear. In this study, we found that UBR5 preferentially targets chromatin-associated proteins for degradation. Since Rbs chromatin association is modulated by its phosphorylation, we hypothesized that phosphorylation may affect Rb stability by altering its chromatin association. To test this, we constructed a series of un-phosphorylatable Rb variants with graded reductions in chromatin association. Consistent with our hypothesis, we observed a strong correlation between an Rb variants chromatin association and its half-life. Fusing these Rb variants to histone H1 increased chromatin association to similar levels and equalized their protein half-lives. Taken together, these findings show how phosphorylation stabilizes Rb by promoting its dissociation from chromatin. This provides a striking example for how sub-organellar protein localization may be used to regulate stability.

PDCD4 is a tumor suppressor, known to affect protein translation by binding to a component of the eIF4F complex, eIF4A, and reducing its helicase activity, which is necessary for the 48S preinitiation complex formation and scanning of the 5 untranslated region of mRNA. PDCD4 has also been shown to interact with the ribosome and with translation initiation factors eIF4G, eIF4G2, eIF3, and PABP, all of which participate both in initiation and the closed-loop structure that couples initiation and termination. To investigate whether PDCD4 modulates initiation and termination through these interactions, we used a reconstituted mammalian translation system and pre-termination complexes purified from rabbit reticulocyte lysate. We found that PDCD4 suppresses early initiation events prior to eIF4F complex binding to the cap structure on mRNA. Moreover, inhibition of the helicase activity of eIF4A by PDCD4 is lost when the 40S subunit is present. Inhibition of 48S complex formation was also observed in the presence of the truncated eIF4G fragment p50 or the eIF4G2 isoform, both of which interact with eIF4A but lack the eIF4E-binding domain. PDCD4-mediated inhibition of initiation persisted regardless of the presence of PABP. During translation termination, PDCD4 did not affect eIF4A activity, indicating that its regulatory function toward eIF4A is stage-specific and restricted to initiation. Finally, we discovered that PDCD4 additively stimulates peptide release together with eIF3, eIF4G2, and PABP, but competes with eIF4F. Thus, PDCD4 employs a complex molecular mechanism targeting multiple translation factors to regulate different stages of protein synthesis.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Bdelloid rotifers are known to survive desiccation and high doses of ionizing radiation. This extreme resistance is notably due to their capacity to cope with numerous DNA double-strand breaks (DSBs). Genes encoding key components of the non-homologous end joining (NHEJ) DNA repair pathway are strongly upregulated in the bdelloid rotifer Adineta vaga following exposure to ionizing radiation. Considering the notably high doses tolerated by these organisms, their capacity to efficiently restore genome integrity is particularly striking. Although NHEJ is generally regarded as less accurate than homologous recombination (HR), the absence of major genomic rearrangements in the descendants of irradiated rotifers suggests that DNA repair occurs with high fidelity. Terwagne et al. recently reported a delayed repair in germline nuclei, occurring during oocyte development when homologous chromosomes pair, thereby enabling template-based repair through HR. In this study, we established an in situ hybridization approach on A. vaga cryosections to investigate the spatial and temporal expression of key actors involved in NHEJ, HR, and Base excision repair (BER) pathways in somatic and germline tissues. We show that NHEJ (KU80) and BER-related genes (PARPs) as well as A. vaga Ligase E (putatively involved in DNA repair) are expressed early after radiation exposure in the somatic syncytium. In contrast, HR-related genes (Rad51: two paralogs, Rad54), as well as PCNA (involved in DNA replication, NER, BER, HR) are expressed later in maturing oocytes, indicating the activation of a delayed homologous recombination repair pathway in germline nuclei. Nurse cells, which express genes associated with both HR and NHEJ pathways, may rely on both mechanisms for their own DNA repair while also supplying mRNAs to the maturing oocyte. Our results provide new evidence for a differential regulation of DNA DSB repair pathways between soma and germline in bdelloids, with NHEJ predominating in somatic tissues and HR in the germline of A. vaga. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722046v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3b1f3borg.highwire.dtl.DTLVardef@17f5eb5org.highwire.dtl.DTLVardef@122ef14org.highwire.dtl.DTLVardef@7e4413_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure:C_FLOATNO Summary of in situ hybridization results: genes coding for actors of NHEJ are expressed in the somatic nuclei and in the nurse nuclei of Adineta vaga individuals 2.5 hours post X-rays radiation, while genes coding for HR actors and PCNA (involved in multiple pathways including DNA replication and DNA repair: NER, BER, MR, HR) are expressed in the nurse nuclei 2.5 hours post radiation, and later in the maturing oocyte during oogenesis and in the laid eggs. Genes coding for actors highly expressed post-radiation, involved in the BER pathway appear to be only expressed in the somatic syncytium 2.5 hours post radiation, as well as the gene coding for the Ligase E, likely involved in DNA repair. C_FIG

Stress Granules (SGs) are cytoplasmic biomolecular condensates that form in response to a variety of stress conditions, though their function remains unclear. "Canonical" SGs - caused by stressors like sodium arsenite - are dynamic and cytoprotective, allowing cells to evade cell death during periods of stress. Ultraviolet (UV) irradiation is known to elicit a "non-canonical" SG subtype, lacking canonical SG components such as eukaryotic initiation factor 3 and polyadenylated mRNAs. The exact function of UV SGs, and the mechanisms driving their formation, remain unknown. Here we report the findings of a comparative analysis of UVA, UVB and UVC exposures on SG formation in three cell types: osteosarcoma (U2OS), keratinocytes (HaCaT), and mouse embryonic fibroblasts (MEF). We observed that SG formation in response to UV is highly cell type dependent. UVB and UVC induce robust SG formation in U2OS cells. However, only UVC exposure induced modest SG formation in MEFs, and none of the wavelengths caused SGs in HaCaT. While UVC-induced SGs in U2OS cells appear to be cell cycle dependent and specific to G1, UVB induced SG formation regardless of cell cycle stage. We tested the hypothesis that oxidative stress triggered by UV may be driving UV SG formation, and that keratin may buffer this effect, by overexpressing keratin in U2OS. Interestingly, we found that keratin and antioxidant treatment efficiently suppressed arsenite-induced SGs but had no effect on UV SGs. Our work confirms that UV SG formation is cell type specific and is not driven by oxidative stress.

Nucleoli and centromeres play essential roles in cellular proliferation and homeostasis, and are structurally and functionally interconnected. Centromeres frequently cluster around nucleoli, and some centromere assembly factors are known to reside in the nucleoli. To investigate the spatial and temporal relationships between these nuclear domains, we examined their dynamics in living cells. We imaged HeLa cells stably expressing mCherry-NPM1 and GFP-CENP-A using time-lapse microscopy. The results show that a subset of centromeres exhibits dynamic behavior during interphase, migrating over micrometer-scale distances within two hours. On average, 40-50% of centromeres maintain an association with nucleoli throughout interphase, with some cells displaying nucleolar-centromere association and dissociation within hours. Upon entry into mitosis, nucleoli are disassembled, and NPM1 localizes to the periphery of mitotic chromosomes. Nucleolar-centromere interactions are re-established in early G1, coinciding with the assembly of new centromeres. Treatment with actinomycin D, an inhibitor of RNA polymerase I, significantly reduces nucleolar size, nucleolar-centromere interactions, and centromere dynamics. Furthermore, post-mitotic nucleolar reformation is impaired. These findings highlight the dynamic nature of centromeres in interphase nuclei and their interactions with nucleoli. This behavior is partially dependent on rDNA transcription and nucleolar integrity, underscoring the critical roles of nucleoli, centromeres, and their interaction in 4D genome organization.

The cell cycle comprises different phases and is a tightly regulated process at the molecular level. During the cell cycle, two key events occurred: DNA duplication during the S phase and chromosome segregation during mitosis. Accurate cell cycle progression, achieved through faithful chromosome segregation, is essential for maintaining cell fidelity. Long noncoding RNAs are a subclass of noncoding RNA that are longer than 200 bp and form RNA protein complexes (RNPs) to regulate various biological processes. Herein, we demonstrate that lncRNA NORM is involved in regulating the cell cycle by maintaining proper chromosome segregation. NORM exhibited G2 phase-specific expression, and the depletion of NORM resulted in a significant G2/M arrest. NORM-depleted cells failed to progress in mitosis and showed defects in chromosome segregation. We further demonstrated that NORM binds to proteins such as Plk1 and Nsun2. Depletion of NORM hindered the interaction between Plk1 and Bub1, resulting in reduced kinetochore localization of Plk1 during prometaphase. Our results also show that the depletion of NORM affects the binding of Nsun2 protein to CDK1 mRNA and, consequently, the stabilization of CDK1 at the protein level. Altogether, our results demonstrate that NORM regulates chromosome segregation by mediating the interaction between Plk1 and Bub1.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

Recent studies have suggested that S6 kinase 1 (S6K1) contributes to DNA repair (DR). However, the specific pathways and mechanisms involved in this regulation remain unclear. Moreover, it has not been investigated whether S6K2, a functional homologue of S6K1, also contributes to DR. In this study, we investigated the function of both S6K1 and S6K2 (S6K1/2) proteins in DR and demonstrate that both are important for efficient Homologous Recombination-mediated repair (HR). Double knockout of S6K1/2 prevented the formation of BRCA1 and RAD51 foci and increases sensitivity to DNA-damaging agents such as PARP1 inhibitors, cisplatin, and X-ray irradiation. In addition, double knockout of S6K1/2 increased markers of genomic instability, while single knockout had little effect on HR markers and genome stability, which suggests that one kinase can compensate for the loss of the other. Mechanistically, we show that S6K1/2 regulate BRCA1 protein stability, limiting its degradation by the proteasome. Finally, pharmacological inhibition of S6K1/2 sensitised HR-proficient breast cancer cells to Olaparib. Our findings clarify the role of S6K1/2 proteins in HR and suggest that targeting these kinases may be a therapeutic strategy to enhance PARP inhibitor efficacy in HR-proficient tumours.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

The actin polymerization machinery, comprising the ARP2/3 complex and its activators, the WASP family proteins, has been implicated in regulating a broad spectrum of nuclear processes, such as transcriptional regulation and nuclear organization. Here, using clustered protocadherin (cPcdh) and {beta}-globin genes as model systems, we showed that WAVE2, a member of the WASP family, regulates chromatin organization by maintaining heterochromatin dynamics. Specifically, by CRISPR DNA-fragment editing, in conjunction with integrated analyses of ChIP-seq, MeDIP-seq, ATAC-seq, 4C-seq, and RNA-seq, we showed that deposition of H3K9me3, a key heterochromatin mark, is significantly decreased at the cPcdh locus upon WAVE2 deletion, concurrent with aberrant accumulation of CTCF/cohesin complex at promoter regions and spatial reorganization of chromatin architecture around nucleolus. In addition, REST/NRSF exerts a similar heterochromatindependent effect on the cPcdh locus. Finally, genetic and genomic data showed that WAVE2 regulates {beta}-globin gene expression by maintaining heterochromatin status. Together our data suggested that WAVE2 and REST/NRSF regulate clustered gene expression in a heterochromatin-dependent manner.

The cellular response to DNA replication stress (DRS) provoked by anticancer drugs involves activation of the G2/M checkpoint (which promotes transient cell cycle arrest at G2 phase) and DNA repair, followed by induction of apoptosis or senescence. Here, we activated the p53-p21 pathway and ATR using DRS-inducing drugs, and found that that the transition to senescence depends on the duration of the G2 phase. Shortening of G2 duration by G2/M checkpoint inhibitors led not only to a switch in cell fate from senescence to mitotic entry, but also to effective cell death through carry-over of chromosomal aberrations (generated by DRS-inducing drugs) into mitosis and subsequent mitotic progression. Such enhanced cell death was also observed in p53 deficient cells, which do not normally undergo senescence. Thus, we propose that temporal regulation of G2 phase is an approach to enhancing the effects of DRS-inducing drugs in a manner that is independent of p53 status.

PurposeLow-dose radiation-induced adaptive response (LDRIAR) is well documented, but its role in early DNA damage signalling remains unclear. This study aimed to investigate whether adaptive response influences initial DNA double-strand break (DSB) recognition, as reflected by {gamma}H2AX foci formation, and to evaluate its time-dependent expression in human lymphocytes. Materials and MethodsPeripheral blood lymphocytes from three healthy donors were exposed to a priming dose followed by a challenging dose at defined time intervals. DNA damage was assessed using {gamma}H2AX foci analysis, comparing acute and split-dose exposures in both PHA-stimulated (large) and non-stimulated (small) lymphocytes. ResultsA clear time-dependent adaptive response was observed. No significant reduction in {gamma}H2AX foci was detected at 1 h (p > 0.05). At 2 h, a significant decrease was observed ([~]7-8% in large and [~]13% in small lymphocytes; p < 0.01), which increased at 4 h ([~]12% and [~]22%, respectively; p < 0.001). The maximal response occurred at 15 h, with reductions of [~]40- 43% in large and [~]27% in small lymphocytes (p < 0.001). Small lymphocytes exhibited an earlier response, while large lymphocytes showed a greater magnitude at later time points. The temporal trend was consistent across donors, with minor variability at later intervals. ConclusionsThe findings demonstrate that LDRIAR is reflected at the level of DNA damage signalling and follows a defined temporal pattern with cell-type specificity. This suggests that adaptive response may influence early DSB-associated processes, contributing to a better understanding of radiation response mechanisms in radiobiology.

BackgroundAlcohol-induced osteonecrosis of the femoral head (AIONFH) is an orthopedic disorder from chronic alcohol abuse, characterized by disrupted femoral head blood supply, osteocyte death and structural collapse. Current hip-preserving therapy is unsatisfactory, and most patients eventually require total hip arthroplasty. Panax Notoginseng Saponins (PNS), the core active component of Panax notoginseng, exerts pro-angiogenic and anti-osteocyte apoptosis effects, but its specific therapeutic mechanism remains unclear. ObjectiveThis study used network pharmacology, molecular dynamics simulation and animal experiments to identify PNSs active components, core targets and key pathways for AIONFH, verify its in vivo efficacy, and provide a scientific basis for clinical application. MethodsPNS active components, their targets and AIONFH-related targets were screened from databases; intersection targets constructed an interaction network, core targets were screened by three machine learning algorithms, with concurrent GO and KEGG analysis. Molecular docking was performed between core targets and PNS components; Gromacs 2022 conducted 100 ns simulation to evaluate complex stability. AIONFH rat models were grouped with 4-week intragastric intervention; pathology, immunofluorescence and PCR were used for detection. Results and DiscussionNetwork pharmacology identified 127 PNS targets and 18 intersections with 672 AIONFH targets. Six core targets (including FGF2, HSD11B1) were screened; KEGG indicated VEGF pathway as key. Ginsenoside Re bound HSD11B1 with the lowest binding energy (-12.4 kcal/mol), and 100 ns simulation confirmed complex stability. Animal experiments showed PNS improved trabecular structure and regulated osteocyte activity. PNS treats AIONFH via multi-component, multi-target mode, core mechanism being osteocyte apoptosis inhibition. Results and DiscussionNetwork pharmacology screening identified 127 potential targets of PNS, and 18 potential intersection targets were obtained by overlapping with 672 AIONFH-related targets. Six core targets including FGF2 and HSD11B1 were screened out by machine learning, and KEGG analysis indicated that the VEGF pathway and other pathways were the key signaling pathways for PNS action. Molecular docking showed that Ginsenoside Re had the lowest binding energy with HSD11B1 (-12.4 kcal/mol), and 100 ns molecular dynamics simulation confirmed the stable conformation of this complex. Animal experiments demonstrated that PNS could improve trabecular bone structure and regulate osteocyte activity. In summary, PNS exerts a therapeutic effect on AIONFH through a multi-component, multi-target and multi-pathway mode, with the core mechanism of inhibiting osteocyte apoptosis.

Ferroptosis is linked to various diseases, but the role of transferrin (TF) in endometriosis (EM) remains unclear. Expression levels of ferroptosis-related proteins, including transferrin (TF), transferrin receptor (TFRC), and glutathione peroxidase 4 (GPX4), were analyzed by western blotting. Compared to normal endometrial stromal cells, eutopic and ectopic endometrial stromal cells from EM patients exhibited significantly enhanced proliferative and migratory abilities, accompanied by a marked reduction in glutathione (GSH) levels in both eutopic and ectopic tissues. TF and TFRC expression was upregulated in ectopic endometrium relative to normal controls, while GPX4 expression was downregulated. To evaluate the functional role of TF, siRNA-mediated knockdown was performed in endometrial stromal cells, with knockdown efficiency confirmed by western blotting. Functional assays demonstrated that TF knockdown not only suppressed cell proliferation (CCK-8 and clonogenic assays) and migration (wound healing assay) but also significantly increased apoptosis rate (flow cytometry with Annexin V-FITC/PI staining).These findings implicate TF in the pathogenesis and progression of endometriosis, likely through modulating endometrial stromal cell proliferation, migration, and apoptosis.