MsWOXs in different tissues at different levels indicated that the MsWOX genes play various roles in alfalfa. MsWUS, MsWOX3, MsWOX9, and MsWOX13-1 from the three subclades were localized in the nucleus, among which, MsWUS and MsWOX13-1 exhibited strong self-activations in yeast. In addition, various cis-acting elements related to hormone responses, plant growth, and stress responses were identified in the 3.0 kb promoter regions of MsWOXs. Expression detection of separated shoots and roots under hormones including auxin, cytokinin, GA, and ABA, as well as drought and cold stresses, showed that MsWOX genes respond to different hormones and abiotic stress treatments. Furthermore, transcript abundance of MsWOX3, and MsWOX13-2 were significantly increased after rhizobia inoculation. This study presented comprehensive data on MsWOX transcription factors and provided valuable insights into further studies of their roles in developmental processes and abiotic stress responses in alfalfa.The WUSCHEL-related homeobox (WOX) family members are plant-specific transcriptional factors, which function in meristem maintenance, embryogenesis, lateral organ development, as well as abiotic stress tolerance. In this study, 14 MsWOX transcription factors were identified and comprehensively analyzed in the cultivated alfalfa cv. Zhongmu No.1. Overall, 14 putative MsWOX members containing conserved structural regions were clustered into three clades according to phylogenetic analysis. Specific expression patterns of The online version contains supplementary material available at 10.1186/s12870-023-04476-5. Drosophila melanogaster and its orthologs were subsequently found in fungi and plants [KNOTTED-1, functioning in the division of leaf veins [Homeobox (HB) proteins belong to a superfamily of transcriptional factors regulating morphogenesis and development in eukaryotes. The Homeobox domain generally consists of conserved 60–66 amino acids that normally function as a DNA-binding domain, which participates in activating or repressing gene expression . The fird plants , 3. In paf veins . Plant Haf veins .In higher plants, WOX genes encode WUSECHEL-related homeobox domain, a family of plant-specific transcription factors, which have been already identified and characterized in Arabidopsis, rice, maize, soybean, and other plants , 7. WOX WOX gene in Arabidopsis was WUS, which acts as a conserved vital regulator required for shoot apical meristem maintenance and floral organ development [Nicotiana sylvestris [In the model plant Arabidopsis, fifteen WOX members have been identified and described comprehensively. Compared to the ancient and intermediate clades, the WUS clade contained more members and was investigated more extensively. The first identified elopment . Ortholoelopment , and HDLelopment , 14 funcelopment , STF in elopment , and LAMlvestris  play a glvestris , 19. AtWlvestris  and its lvestris , OsWOX3Alvestris , NS1 andlvestris , and NLDlvestris  are requlvestris –27. WOX5lvestris . PFS2/Atlvestris , while rlvestris . WOX8, 9lvestris , 31. WOXlvestris , 33. Thelvestris –37.WOX genes during plant growth and development, although they have some common properties [WOX5 promoter driving WUS to the root meristem or the WUS promoter bringing WOX5 to shoot meristem could complement the wox5 or wus mutant, respectively [MtWOX1/MtSTF promoter, Arabidopsis WUS clade genes could complement the lam1 mutant leaf-attenuated phenotypes in Nicotiana sylvestris [wus mutant by other WUS clade genes in Arabidopsis required accurate expression in the right domain driven by the WUS promoter [Previous reports demonstrated that WOX family members are versatile transcription factors that function in plant growth and development during the whole plant life cycle, from meristem maintenance to embryonic patterning, and from lateral organ formation to abiotic stress tolerance , 38–40. operties . Arabidooperties , 41. Araectively . Driven WOXs participate in some abiotic stress responses. Arabidopsis HOS9-1, sharing similarity with WUS and PRS, functions positively in cold stress independent of the well-known CBF pathway [As sessile organisms, plants are endowed with strong adaptive capacities to adverse environments. Understanding the balance of plant growth and development with abiotic stress tolerance is helpful to improve agricultural productivity. Although WOX transcription factors are well known as development regulators, studies also demonstrated that  pathway . Homolog pathway , 43, 44.MsWOX genes in the cultivated alfalfa based on a genome-wide scan approach referring to genome data of Medicago sativa L. cv. Zhongmu No.1. We predicated their roles by combining analyses of the cis-elements in 3.0 kb promoters and their expression patterns under multiple phytohormonal treatments and abiotic stresses. The study provides a rich resource for further study of MsWOX transcription factors in alfalfa.Genome-wide identification and characterization of WOX family genes have been reported in multiple plant species like Arabidopsis, soybean, wheat, sunflower, and a few others , 45, 46.Medicago sativa L. cv. Zhongmu No.1 was germinated in water for 2–5 days, and the subsequent seedlings were planted in a greenhouse at 24 °C (day) and 20 °C (night) under a 16 h light/8 h dark photoperiod, 60–70% relative humidity and a light intensity of 180 µmol·m− 2 s− 1. Tissues including unfolded leaves, young flowers, mature flowers, nodules, and stems were collected from 12-week-old plants, every sample was harvested from 3 plants at the reproductive stage; and shoots, roots, and shoot apical meristems were harvested from 6 plants at 2-week-old seedlings for each replicate and three biological repeats for RNA extraction.Seeds of Sinorhizobium meliloti 1021 at 0 d,1d,3 d, and 5 d. Shoots and roots of seedlings were split and gathered after the above treatments. 6 plants were collected for each replicate and two biological repeats were performed. All samples were frozen in liquid nitrogen immediately and stored at -80℃ for RNA extraction.For hormone treatments, 2-week-old seedlings were transferred to 1/4 Hoagland containing 10 µM 6-BA, 10 µM 2,4-D, 10 µM GA, and 10 µM ABA for 0 h, 6 h, and 12 h, respectively. For abiotic stress treatments, 2-week-old seedlings were shifted into a 4℃ chamber for cold stress for 0 h, 6 h, 12 h, and 24 h; and 2-week-old seedlings were transferred into 10% (w/v) PEG-6000 solution mimicking drought or inoculated by https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annotation_files/12623960?file=23754059/ZhongmuNo.1.pros.fasta). Arabidopsis WOX protein sequences were retrieved from Plant Transcription Factor Database  and used as queries for a local protein blast against ZhongmuNo.1 protein database. In total, 17 sequences were identified with an E-value cut-off of 0.001. After Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) analysis with the 17 proteins and annotating them with Swiss-prot database (https://www.sib.swiss/swiss-prot), 3 proteins were excluded due to lacking proper WUSCHEL-related homeodomain motif.MsWOX protein database in alfalfa (cv. ZhongmuNo.1), was downloaded from FigShare Brachypodium distachyon, and Medicago truncatula were downloaded from PlantTFDB (http://planttfdb.gao-lab.org/). Afterward, these downloaded sequences and the identified 14 MsWOXs in alfalfa in this study were aligned using the online tool MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft/). Based on the aligned protein sequences, a neighbor-joining tree was created using MEGA11 with 1000 bootstrap replicates. To make the tree more esthetic, R package ggtree was used.WOX amino acid sequences from rice (Japonica), https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annotation_files/12623960?file=23754059/ZhongmuNo.1.gff) and the visualization was done by TBtools. The duplication events and synteny analyses were performed using MCSanX and displayed with Circos and Dual Synteny Plot in TBtools software. The inter-species synteny analyses were done between alfalfa cv. ZhongmuNo.1 and each of the species; Brachypodium distachyon, rice, Arabidopsis, Medicago truncatula, and Glycine max.The chromosomal location information was extracted from the genome annotation file (https://web.expasy.org/protparam/). The length of coding sequences and proteins, the number of introns and exons of the 14 MsWOXs, and the corresponding chromosome number were summarized from the genome annotation file of ZhongmuNo.1 (https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annotation_files/12623960?file=23754059/ZhongmuNo.1.gff). Conserved amino acid motifs of MsWOXs were searched by the online tool MEME Suite 5.1.1 (https://meme-suite.org/meme/), and the motif numbers were set as 10. The phylogenetic tree and conserved motifs of the MsWOXs were visualized by Gene Structure View (Advanced) in TBtools [MsWOXs were submitted to the PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict the putative cis-elements. Simple BioSequence Viewer in TBtools was used to visualize the cis-element distributions on the promoter regions and listed in supplementary Table The gene structure information including the length of coding sequences and proteins, the number of introns and exons of the 14 MsWOXs, and the corresponding chromosome number were summarized from the GFF annotation file downloaded from ZhongmuNo.1 as described in the chromosomal location. The Molecular Weight (MW) and Isoelectric Point (PI) of the 14 MsWOX proteins were calculated by the ProtParam tool . The concentration and quality of RNA were tested by NanoDrop (Thermo). 5 µg of total RNA was used as the template for cDNA synthesis with Uni One-Step gDNA Removal and cDNA Synthesis SuperMix  following the manufacturer’s instructions.G machine with TransStart Green qPCR SuperMix . The qPCR was carried out in a 96-well optical plate using a Quanstudio Real-time PCR system. Each 10 µl reaction contained 3 µl of diluted cDNA template, 5 µl of 2×SuperMix , and 2 µl of forward and reverse primers (1 µM). The thermal cycling for amplification was as follows: 5 min at 95 °C, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s, and then the melting curve from 60 to 95 °C, 15 s with ΔT 1 °C. Each qPCR reaction was performed in 3 or 4 technical replicates for each biological replicate. The relative gene expression levels were calculated using the 2−∆∆Ct method, and MsActin (MsG0380016789.01.T01) was used as an endogenous control for accurate normalization of the qPCR data. Every test was performed with at least two biological repeats. All data were visualized by GraphPad Prism 9.The qPCR was performed using a qTOWER3MsWUS, MsWOX3, MsWOX9-1, and MsWOX13-1 were amplified using designed primers listed in supplementary Tables MsWOXs-GFP were introduced into agrobacteria GV2260 using freeze-thaw transformation and transiently expressed in N. benthamiana leaves mediated by infiltration of GV2260. Subcellular localization was observed after infiltrating 48 h by confocal microscopy .The full-length coding sequences (CDS) without stop codon of MsWUS, MsWOX3, MsWOX9, and MsWOX13-1 were amplified and cloned into pGBKT7-BD vector via a gateway system  as baits. Each bait clone with the empty prey vector pGADT7 was co-transformed into the yeast strain Gold using PEG methods. pGBKT7-p53 co-transformed with pGADT7 was used as a positive control and pGBKT7-lam with pGADT7 as a negative control. All these co-transformed cells were diluted to a gradient concentration of 10− 0, 10− 1, 10− 2, and 10− 3, and then dropped on the SD/-Trp/-Leu/-His + X-a-gal solid medium for incubating at 30 °C for 3 days.The full-length CDS of Error bars in qPCR show the SD of three or four biological or technical repeats, as indicated in the legends. Experimental data were subjected to one-way analysis of variance (ANOVA) and post hoc LSD tests to determine significant differences among mean values at the probability level of 0.05.To isolate MsWOX members in alfalfa, 15 Arabidopsis AtWOX protein sequences were retrieved to perform a protein blast against the alfalfa protein database. In total, 17 proteins were identified in cultivated alfalfa cv. Zhongmu No.1. The conserved domain search and a protein blast with the protein database SwissProt verified the characteristics of these 17 members. Besides the Homeobox or Homeobox Superfamily domain, three of the 17 genes, including MsG0280011271, MsG0280007695, and MsG0380017666, contained a bZIP domain, which suggested that they belong to another family named HD-ZIP Family. Therefore, we excluded them from the WOX family, and 14 MsWOX proteins were finally identified in alfalfa cv. Zhongmu No.1.Medicago truncatula, 13 BdWOXs in Brachypodium distachyon and 14 OsWOX in rice (cv. Japonica), were aligned to construct a phylogenetic tree based on neighbor-joining method with 1000 replicates. These 75 WOX proteins were classified into 3 clades  together with 15 AtWOXs in Arabidopsis, 19 MtWOXs from Medicago truncutula WOX homologs .Gene duplication events drive the evolution of species by increasing the number of functional genes . We condMsWOXs is probably indicated by their spatial expression profiles [MsWOX genes, eight different tissues, i.e., shoots, shoot apices, and roots of 2-week-old seedlings, unfolded leaves, young flowers, mature flowers, nodules, and stems of 12-week-old plants were collected and tested by RT-qPCR. The results showed that different expression patterns of MsWOX genes were displayed in different organs at different stages  were predicted using PSORT and PredictNLS. To gain better insights into the biological roles of MsWOX proteins, we investigated the subcellular localization and self-activation of four MsWOXs selected from the three clades. The full-length coding sequences of MsWUS, MsWOX3, 9 − 1, and 13 − 1 were cloned individually and fused to GFP-containing vectors, which were then infiltrated into MsWOX genes in alfalfa. Numerous cis-acting elements related to the control of different biological processes were observed in all promoters, including plant growth and development conferring meristem and endosperm expression, phytohormone-responsive regulators such as MeJA, salicylic acid, auxin, gibberellin, and ABA, as well as abiotic stress-responsive motifs involving low-temperature, drought, and wound response , and shoots and roots were split and harvested for detecting transcriptional alteration of MsWOXs. Six MsWOXs from three clades  were selected for subsequent analysis by RT-qPCR. Expression of MsWUS, MsWOX3, MsWOX13-1, and MsWOX10/14 were 2,4-D induced slightly in shoots  for further RT-qPCR analysis. Six MsWOXs which were highly accumulated in nodules were analyzed , and abscisic acid (ABA) , and DCaWOX13(a/b) negatively responded to ABA but positively responded to IAA [WOX genes regulate various developmental pathways in close association with phytohormones, but the underlying molecular mechanism of the linkage between WOX and phytohormone responses needs to be further studied.Phytohormones are the main factors for plant growth and development, which appear to have a strong connection to WOX transcription factors , 39, 56.BA) Fig. , and we  pathway . WOX9 ha pathway , 39, andd to IAA , 58, andd to IAA . These fSlWUS and Arabidopsis AtWOX6 are involved in cold stress through CBF-independent pathways [OsWOX11 and OsWOX13 have been reported to be involved in drought resistance [MsWOX promoters (Fig. MsWOXs participated in abiotic stresses too. The strong responses of MsWOX9-1, 13 − 1, and 10/14 to PEG treatment, and MsWOX3, 9 − 1, and 10/14 to cold stress, are consistent with the analysis of cis-regulators in the promoters (Figs. MsWUS and MsWOX11 failed to respond to the PEG and low temperature, which might be due to the lack of corresponding cis-acting elements in their promoters (Figs. Plants can adapt to adverse situations by adjusting their metabolism and altering their morphology. Previous reports mainly focused on WOX regulation of plant development and growth, but few studies investigated the roles of WOXs in diverse stresses. Tomato pathways , 59. Ricsistance , 44. In rs Figs. A and B.MsWOX3, 9 − 1, 9 − 2, 13 − 1, 13 − 2, and 10/14 were highly expressed in nodules (Fig. S.meliloti 1021(Fig. MsWOX13-2 transcript was significantly accumulated continually (Fig. MsWOX3, 9 − 1, and 10/14 were temporarily induced after 1 dpi (Fig. MsWOX9-2, and 13 − 1 both showed no response to rhizobial inoculation (Fig. MtWOX5 and pea PsWOX5 were induced upon nodulation [MsWOXs using GUS staining or GFP fluorescence during nodule development in alfalfa. A comprehensive analysis of the interconnection between phytohormones, nodulation, and specific MsWOX gene expression will advance our understanding of the regulatory steps of nodulation and biological nitrogen fixation.Alfalfa is the most widely cultivated forage crop in the world, and forming a symbiosis with rhizobia for nitrogen-fixing nodules is a vital trait for the legume to improve yields. In this study, transcripts of 1021Fig. . MsWOX13lly Fig. E, while dpi Fig. A, B, F, ion Fig. C, D. Predulation , which iMsWOX genes, and a total of 14 MsWOXs were identified and classified into three subclades in alfalfa cv. Zhongmu No.1. Tissue expression of MsWOXs genes revealed their specific involvement in different organs and developmental programs. Combining the cis-acting element identification of promoters and expression analyses of MsWOXs under different treatments indicated that MsWOXs are involved in multiple biological processes during plant development including modulation and adaptation to adverse environmental conditions. Our results form the basis and provide insight into the diversity and functional significance of MsWOX genes in plant growth and abiotic stress responses in alfalfa.In this study, we performed a genome-wide analysis of Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Radiotherapy is a critical component of cancer care but can cause osteoporosis and pathological insufficiency fractures in surrounding and otherwise healthy bone. Presently, no effective countermeasure exists, and ionizing radiation-induced bone damage continues to be a substantial source of pain and morbidity. The purpose of this study was to investigate a small molecule aminopropyl carbazole named P7C3 as a novel radioprotective strategy. Our studies revealed that P7C3 repressed ionizing radiation (IR)-induced osteoclastic activity, inhibited adipogenesis, and promoted osteoblastogenesis and mineral deposition in vitro. We also demonstrated that rodents exposed to clinically equivalent hypofractionated levels of IR in vivo develop weakened, osteoporotic bone. However, the administration of P7C3 significantly inhibited osteoclastic activity, lipid formation and bone marrow adiposity and mitigated tissue loss such that bone maintained its area, architecture, and mechanical strength. Our findings revealed significant enhancement of cellular macromolecule metabolic processes, myeloid cell differentiation, and the proteins LRP-4, TAGLN, ILK, and Tollip, with downregulation of GDF-3, SH2B1, and CD200. These proteins are key in favoring osteoblast over adipogenic progenitor differentiation, cell matrix interactions, and shape and motility, facilitating inflammatory resolution, and suppressing osteoclastogenesis, potentially via Wnt/β-catenin signaling. A concern was whether P7C3 afforded similar protection to cancer cells. Preliminarily, and remarkably, at the same protective P7C3 dose, a significant reduction in triple-negative breast cancer and osteosarcoma cell metabolic activity was found in vitro. Together, these results indicate that P7C3 is a previously undiscovered key regulator of adipo-osteogenic progenitor lineage commitment and may serve as a novel multifunctional therapeutic strategy, leaving IR an effective clinical tool while diminishing the risk of adverse post-IR complications. Our data uncover a new approach for the prevention of radiation-induced bone damage, and further work is needed to investigate its ability to selectively drive cancer cell death. Due to its high calcium content, bone is estimated to absorb 30%–40% more IR than other tissues, making it a common site for serious ancillary tissue damage.5 Osteoradionecrosis, osteoporosis, pathologic insufficiency fractures, and subsequent fracture nonunions are significant complications associated with radiotherapy in cancer survivors, even with fractionation of treatments.11 As such, the burden of IR-induced damage to healthy bone is a persistent and substantial source of functional impairment, pain, disability, and morbidity.14 The inactive prodrug amifostine has been used as the radioprotector of choice for over the last six decades.17 Despite progress made to improve the effectiveness of this treatment, none of the strategies have resolved the issue of its side effects. Thus, the FDA approved it for limited clinical indications but not for general clinical use.17 Exogenous antioxidants,18 such as melatonin,19 antiresorptive bisphosphonate treatment, and denosumab (a receptor activator of NF-κB ligand (RANKL) inhibitor), are clinically available;20 however, none are capable of completely preventing IR-induced damage, and their long-term efficacy is limited. Thus, no effective preventative countermeasure exists. This issue is important, as the global burden of cancer is expected to increase from 19.3 million new cancer diagnoses and 10 million cancer-related deaths recorded in 202021 to 27.5 million diagnoses and 16.3 million deaths by 2040.22 This increase is largely attributed to the increase in our aging (≥65 years) population, together with increased industrial pollution.24 Thus, a substantial increase in the number of patients to be exposed to radiotherapy is predicted, and the accompanying IR-induced skeletal complications will undoubtedly increase the number and duration of inpatient stays, emergency department visits, and outpatient visits. The exact pathogenesis of IR-induced bone toxicity has not been discovered,26 and new mechanistic insights combined with an effective countermeasure would be of considerable clinical and therapeutic importance. Therefore, novel and improved therapeutic strategies are urgently needed.Cancer remains a leading cause of death worldwide. A combination of surgical resection, radiotherapy, and chemotherapy is commonly used in the treatment of various local and metastasizing cancer cells.27 inability to proliferate and differentiate,29 and apoptosis30 of bone-forming cells. Recent evidence also indicates that oxidative stress caused by excessive reactive oxygen species (ROS),31 macrophage activation and infiltration, and the prolonged release of proinflammatory mediators play an important role in the progression of osteoporosis following IR.32 Molecularly, and during healthy bone turnover, interactions among the transmembrane receptor activator of NF-κB (RANK), the RANK ligand (RANKL), and the secreted decoy receptor osteoprotegerin are critical in regulating the differentiation and maintenance of osteoclastic activity and the subsequent resorption of bone.33 Following IR, the levels of pro-osteoclastic markers  and proadipogenic markers ) increase, while osteogenic mediators ) decrease.34 As a result, excessive bone resorption, combined with impaired bone formation, together with compositional alterations in the adipogenic bone marrow environment, subsequently disrupts cell function and reduces bone quality and strength.Studies have demonstrated increased IR-induced cell cycle arrest,35 senescence, and individual aging.36 NAD exists in an oxidized (NAD+) and reduced (NADH) form. In metabolism, NAD+ acts as an electron-carrying cofactor for oxidation‒reduction reactions, and electron transfer is the main function of NAD.37 As such, NAD+/NADH is an important cellular redox pair, among others, and may regulate ROS generation and scavenging.38 However, this molecule is also used in other cellular processes, adding or removing chemical groups to or from, for example, proteins. Importantly, cellular levels of NAD+ decline significantly during aging,39 and decreased NAD+ contributes to the loss of osteoprogenitors and bone mass in age-related osteoporosis.40 Furthermore, IR therapy causes a significant depletion in NAD+ metabolism and disrupts the processes regulating NAD+ production and consumption.42 Pool 7, Compound 3 (P7C3), is an antiapoptotic aminopropyl carbazole with high oral bioavailability that exerts its activity through activation of the intracellular enzyme nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is an adipokine known to be the rate-limiting enzyme in the NAD+ salvage pathway and directly increases NAD+ levels and NAD+-dependent enzyme activity.44 Mechanistically, P7C3 increases NAD flux in mammalian cells and can restore cell function under conditions of overwhelming energy crisis that would normally lead to cell death.45 Regulation of the NAD+ cascade as a therapeutic in human disease is an area of growing interest.Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH are essential components in various ubiquitous cellular processes, including cell metabolism,50 TAGLN together with the upregulation of integrin-linked kinase (ILK) suggests the increased activation of actin filaments, alterations in cytoskeletal components, and anchorage-dependent cell growth, potentially enhancing cell migration and adhesion, which are important processes that control changes in cell shape, migration, proliferation, survival, and differentiation.51 Furthermore, the immune regulator Toll interacting protein (Tollip) and thioredoxin-binding protein-2 (TBP-2) were significantly upregulated, suggesting that excessive inflammatory resolution52 and reduced osteoclastogenesis via reduced RANKL53 activities may have also contributed to the osteoprotective effect. Finally, an important consideration is whether P7C3 also confers protection to cancer cells during IR. Here, we show that at the same protective P7C3 dose, the metabolic activity of triple-negative breast cancer and osteosarcoma cancer cell lines was significantly reduced in vitro.Here, we describe the role of P7C3 in the prevention of IR-induced bone damage in vitro and in vivo. Most notably, P7C3 inhibited osteoclastic activity and adipogenesis and mitigated tissue loss such that bone maintained its area, architecture, and mechanical strength. The mechanistic novelty revealed is based on P7C3 significantly increasing myeloid cell differentiation and the proteins low-density lipoprotein receptor-related protein 4 (LRP-4) and transgelin (TAGLN), with downregulation of growth differentiation factor-3 (GDF-3), SH2B adapter protein 1 (SH2B1), and OX-2 membrane glycoprotein, also named cluster of differentiation 200 (CD200). Together, this pattern of protein expression may favor osteoblastic at the expense of adipogenic bone mesenchymal stem cells (BMSC) differentiation.54 Our previous studies demonstrated that exposure to 7 Gy IR significantly increased MNGC formation and activated osteoclastic activity in RAW264.7 macrophages.55 In this study, we assessed the ability of P7C3 to inhibit the formation of MNGCs and levels of tartrate-resistant acid phosphate+ (TRAP+) staining, which indicates activated osteoclastic activity in macrophages and following IR. Macrophages were cultured in the absence of osteoclastic differentiation factors, and our results revealed that a dose of 10 μmol·L−1 P7C3 significantly decreased cell size and resulted in fewer multinucleated cells than those of the IR and DMSO control groups  and the osteoclastic differentiation of macrophages. Radiation-induced MNGC formation typically indicates the presence of chronic inflammation, a condition known to promote osteoclastogenesis and increase osteoclastic activity, leading to elevated bone resorption.ups Fig. . A signioup Fig. . Our res IR Fig. .Fig. 1Ir−1 and 10 μmol·L−1) was nontoxic to cells, with no apparent change in the number of live/dead cells when compared with that of the untreated control cells after 3 days of culture  at this time point. However, following 6 days of culture, 10 μmol·L−1 P7C3 treatment resulted in a significant increase in cell growth (1.04 ± 0.1) compared to that of the IR control cells . hBMSC morphology was qualitatively evaluated, and as shown in Fig. −1 of P7C3 treatment, the level and intensity of actin filaments appeared to increase compared to those of the IR control group. The effect of P7C3 on the metabolic activity of RAW264.7 cells showed a similar pattern. A significant increase in macrophage activity was found following supplementation with 10 μmol·L−1 P7C3 (P < 0.001) compared with that of the control cells on Day 3 of culture  than both the IR- and DMSO-treated cells. Quantitative analysis revealed that 10 μmol·L−1 P7C3-treated hBMSCs resulted in a significant >1.5-fold  increase in new bone mineral at 14 days and a > 3-fold  increase at 28 days post-IR compared with those of the control group (0.36 ± 0.05)  Fig. , despite56 Senescent cells cause damage to surrounding healthy cells via a bystander effect and through the release of various mediators known as the senescence-associated secretory phenotype (SASP). We next assessed whether P7C3 treatment protected cells against IR-induced cell senescence using SA-β-Gal staining. As shown in Fig. −1 P7C3 treatment showed markedly decreased β-gal-positive staining compared with the IR only or DMSO-treated cells. Additionally, 1 μmol·L−1 P7C3-treated hBMSCs reduced β-gal-positive staining following exposure to IR, indicating decreased IR-induced cell senescence.The persistence of unrepaired DNA damage leads to senescent cell cycle arrest.−1 P7C3-treated hBMSC group displayed significantly decreased (P < 0.001) staining , PPARG, lipoprotein lipase (LPL), leptin (LEP), fatty acid binding protein 4 (FABP4), CCAAT/enhancer-binding protein alpha (CEBPA), perilipin-1 (PLIN1), and fatty acid synthase (FASN) were investigated on Day 12 following IR exposure. qRT‒PCR results demonstrated a significant reduction following P7C3 treatment in LEP (P < 0.05), ADPOQ, PPARG, LPL, CEBPA, PLIN1 , FABP4 and FASN (both P < 0.000 1) expression compared with that of the control group . In rats, 7 Gy/day for 5 days (35 Gy) is the human equivalent to a 70 Gy dose.60 Therefore, the exposure of rats to 8 Gy on Days 1, 3 and 5 represents a hypofractionated model delivering a human equivalent of 48 Gy . This, for example, represents the hypofractionated dose delivered to lung13 or breast cancer patients,57 where nontraumatic rib fracture can commonly occur following IR,62 and hypofractionated IR increases this risk.63 Although the risk of late-onset fragility fracture is heightened in patients,65 in rodent models, and similar to other published studies,67 our previous research showed significant levels of cell dysfunction and bone damage as early as 7 days following IR32.Having established the favorable impact of P7C3 on cells in vitro, we next analyzed its impact in vivo. We utilized a rat hind limb model and applied a hind limb localized accumulative IR dose of 24 Gy (three treatments of 8 Gy/dose) Fig. . For com32 while 20 mg·kg−1 P7C3-treated rats showed reduced bone loss compared to the IR-treated rats. Skeletal changes were further assessed histologically and by H&E staining. As shown in Fig. −1 P7C3-treated and IR-exposed rats exhibited marked improvement, with results demonstrating a bony structure similar to that of the non-IR exposed, healthy control group. Additionally, qualitative histological analysis showed more and larger, darkly stained cuboidal osteoblasts on the bone surface following 20 mg·kg−1 P7C3 treatment. This finding suggests the presence of activated osteoblasts and was noted to be in contrast to the quiescent bone surfaces observed in the control and IR-treated groups. Quantification of %bone area/total area (BA/TA%) .To validate IR-induced bone loss and whether P7C3-treated rats were able to maintain their bone structure, we qualitatively examined histomorphological bone parameters using nano-CT analysis. As shown in Fig. A%) Fig.  demonstr+ and therefore active osteoclasts was significantly higher in the IR-induced rats than in the control animals (P < 0.000 1). Furthermore, the number of active osteoclasts was significantly reduced following P7C3 treatment compared with that of the IR-exposed control group , ultimate load , fracture stress , and ultimate stress  compared to those of the IR only group (58.20 ± 8.33 N (fracture load); 66.25 ± 13.77 N (ultimate load); 51.65 ± 13.06 MPa (fracture stress), and 57.71 ± 11.15 MPa (ultimate stress)). Additionally, no significant differences were found between the non-IR control animals and the IR + P7C3 animals. These results show that IR negatively impacts bone strength, making it more susceptible to fracture. Remarkably, treatment with P7C3 had a significant and positive impact by maintaining a healthy level of bone strength despite exposure to harmful levels of IR.Next, we evaluated whether the P7C3-treated rats were able to maintain their cortical bone structure and strength. The 3-point bending test fixture is presented in Fig. To better understand the systemic effect of P7C3 in irradiated animals, we quantified cytokine and chemokine expression in serum using a cytokine array that examined 500 rat proteins simultaneously. Hierarchical clustering of the 500 biomarkers is shown in Fig. KEGG pathway analysis is presented in Fig. + staining. Quantification of positively stained adipocytes demonstrated a significant and >20-fold increase in adipocyte number in the IR group compared with the control group . The results also revealed an IR-induced increase in adipocyte area  and diameter (P < 0.000 1) compared with those of the IR-exposed animals. Together, these results indicate a reduction in lipid accumulation and bone marrow adiposity (as defined by a reduction in the proportion of the bone marrow cavity volume occupied by adipocytes) following P7C3 treatment.Our in vitro data showed that P7C3 inhibited adipogenesis, and the cytokine array results revealed that P7C3 upregulated LRP-4, TAGLN, ILK, and Tollip and downregulated GDF-3, SH2B1, and CD200, uncovering many factors able to regulate preferential myeloid progenitor BMSC differentiation toward osteoblastogenesis at the expense of adipogenesis. Notably, some of these factors facilitate inflammatory resolution (Tollip) and suppress RANKL expression and osteoclastogenesis (TBP-2), thereby supporting our in vivo RANKL and TRAP staining results presented in Fig. + cells compared with those of the non-IR control animals . However, P7C3 treatment resulted in a significant reduction in perilipin+ cell number . Importantly, P7C3 treatment significantly reduced lipid staining ~20-fold (P < 0.000 1) compared to that in the animals in the IR group ; however, analyses showed significantly reduced SA-β-gal+ activity in the P7C3-treated rats compared to the IR-treated rats . A significant reduction in SASP TNF-α+, p21+, and p16+ cells were found in the P7C3-treated animals compared to the control IR-treated animals  of P7C3 ± IR in vitro.Our results have shown that P7C3 appears nontoxic to host cells and radioprotects against IR-induced cell damage, tissue loss, and bone fragility. However, it is critical to determine whether a protective effect is also afforded to cancer cells. To this end, we quantified changes in the metabolic activity of MDA-MB-231, PC3, and OS cells in response to increasing concentrations (10 μmol·LP < 0.01), a reduction in growth of 34.7%. On Day 3, cell metabolic activity was reduced by 43.8% when IR was applied. Remarkably, our results revealed that in the absence of IR and on Day 1 of culture, P7C3 at concentrations of 20 μmol·L−1 and 40 μmol·L−1 significantly reduced MDA-MB-231-GFP cell activity by 40.0% and 80.0%, respectively (P < 0.000 1 in both groups). By Day 3 of culture, cell activity was also significantly reduced (37.5%) in the 10 μmol·L−1 P7C3 group (P < 0.001) compared with the control cell group. At this time point, cell activity was further reduced in the 20 μmol·L−1 and 40 μmol·L−1 groups by 86.3% and 91.3%, respectively. The inhibitory activity of P7C3 on MDA-MB-231-GFP cells was maintained following exposure to IR; however, no radiosensitizing effect was measured. Live cell imaging revealed a marked decrease in MDA-MB-231-GFP cell activity following all doses of P7C3 ± IR, especially at the higher concentrations of 20 μmol·L−1 and 40 μmol·L−1  was measured and compared between groups .As shown in Fig. L−1 Fig. . The resups Fig. . The resP < 0.01), signifying an IR-induced decrease in activity of 40%. On Day 3, and following IR exposure, activity had further decreased to 57.1% (P < 0.000 1) compared with that of the control non-IR cells. Following supplementation with P7C3 in the absence of IR, a trend of decreased PC3 activity was observed with a significant reduction measured in the 40 μmol·L−1 group (P < 0.000 1) on Day 1 and in the 20 μmol·L−1 and 40 μmol·L−1 groups on Day 3 (both P < 0.000 1). Similar to that of the MDA-MB-231-GFP group, the inhibitory activity of P7C3 was maintained in the presence of IR; however, no synergistic effect was measured. Live cell imaging revealed a dose-dependent inhibitory response to P7C3 ± IR . Furthermore, our results showed that when OS cells were treated with P7C3 at concentrations of 10 μmol·L−1, 20 μmol·L−1, and 40 μmol·L−1 in the absence of IR, a significant decrease in cell activity was found on Day 1  and Day 3 . No synergistic effect was found when P7C3 was combined with IR. Live cell imaging revealed a dose-dependent inhibitory response to P7C3 ± IR  through which P7C3 was able to decrease senescence and increase osteogenic commitment despite DNA damage remains unclear. This phenomenon may be explained in part by the p53 protein. Bai and colleagues79 showed that IR-induced DNA damage suppressed cell viability and osteogenic differentiation potential and facilitated senescence via p53/p21 senescence-related gene expression. Our microarray results revealed the substantial upregulation of p53 in the P7C3-treated animals compared to the control IR animals. p53 is coupled to various post-translational modifications , critical steps in mediating the cellular response to IR-induced DNA damage. Furthermore, p53 activates de novo synthesis of proapoptotic molecules, and in this study, its upregulation may have reduced the formation of cellular senescence and instead favored apoptosis or cell survival where damaged DNA can be subsequently repaired.83 However, this is cautiously speculated, as the differential activation of cell cycle arrest or cell death by p53 is not completely understood, and p53 protein levels have been shown to be proportional to the extent of DNA damage in a cell.84 It is also important to highlight that our results reflect the short-term (7 days) post-IR, and cells that survive immediate genotoxic IR stress display genomic instability up to 36 days post IR.88 Thus, any dysfunction may not be observed until longer after IR exposure.89 As such, further longer-term studies are warranted. Finally, although supplementary data showed that P7C3 offered no protection against IR-induced DNA damage, it is important to acknowledge that further investigation at earlier 4–8-h time points post-exposure, and when most DNA damage occurs, is needed.Cellular DNA is highly vulnerable to IR via direct damage and indirect ROS-mediated damage.91 and various pathological conditions.96 Bone marrow adipocytes are derived from the same progenitor cells  as osteoblasts. A switch in lineage commitment toward adipogenic differentiation at the expense of osteogenic differentiation following IR-induced injury65 or via transdifferentiation of osteoblasts into adipocytes66 has been identified. Interestingly, IR exposure directly and preferentially reduced the osteogenic potential of BMSCs compared to more resistant adipogenic differentiation.98 However, the underlying mechanisms involved are yet to be clarified. Our in vitro studies show that supplementation of cells with P7C3 significantly reduced IR-induced master adipogenic transcription factor PPARγ expression, along with other key adipogenic transcription factors, and with significantly reduced levels of lipid droplet formation. Notably, a >2-fold increase in bone mineral deposition was found in the P7C3-treated cells compared to the control IR-exposed cells. Our in vivo findings further support preferential IR-induced adipocyte formation and the antiadipogenic activity exerted following P7C3 treatment. Seven days following IR exposure, an ~20-fold increase in bone marrow adiposity and lipid formation was measured in the control group. Notably, pretreatment and daily i.p. administration of P7C3 significantly reduced the aberrant changes in the number and size of bone marrow adipocytes and lipid formation, despite exposure to harmful levels of IR. This finding indicates the potential of P7C3 to play a critical role in the suppression of adipogenesis alongside preferentially enhancing osteoblastogenesis. Although bone was able to maintain its structure and strength, new bone formation in vivo was not measured in this study and warrants further important investigation in future studies.An increase in bone marrow adiposity has been observed in most bone loss conditions, including aging99 Five hundred serum proteins were screened following in vivo exposure to IR ± P7C3. The results revealed that exogenous administration of P7C3 reduced the expression of proteins related to adipogenesis and upregulated those associated with promoting osteogenesis. For example, the protein LRP-4 was strongly upregulated in the P7C3-treated animals post-IR. LRP-4 is highly expressed by osteoblasts and early osteocytes and is considered to play an important role in bone metabolism and homeostasis, potentially through interaction with SOST/sclerostin.100 PPARγ is essential for the production of sclerostin,101 a protein that mediates endocrine communication between fat and bone tissue, and its expression was significantly inhibited by P7C3. Sclerostin increases adipogenesis while decreasing osteogenesis via Wnt/β-catenin signaling activation.103 Interestingly, Bullock et al.104 reported the prevention of disuse osteoporosis in an LRP-4 knock-in murine model. The authors concluded that LRP-4 may serve as a high yield target for the development of compounds that protect skeletal integrity. Paradoxically, Chang et al.100 demonstrated that LRP-4 deficiency resulted in increased sclerostin serum levels and a progressive gain in cancellous and cortical bone as measured in a LRP-4 knockout murine model. A study by Kim et al.46 demonstrated the differential impact of LRP-4 and sclerostin expression in adipocytes vs. osteoblasts and concluded that the impact on adipocyte physiology was distinct from the effect on osteoblast function. With the support of our findings, these studies highlight a key and multifaceted role of LRP-4 in bone regulation and an emerging area that warrants further research.Notably, the Wnt/β-catenin signaling pathway is a well-studied endogenous regulator of mesenchymal cell fate. Decreased Wnt/β-catenin signaling is a key step in the commitment of progenitor cells to the adipogenic lineage, adipocyte maturation, and de novo lipogenesis, while upregulation promotes osteoblastogenesis.105 This is important, as during lineage commitment, BMSCs undergo a significant modification in morphology and actin cytoskeletal organization, which participates in cell fate determination.106 Furthermore, actin levels can directly influence osteogenesis vs. adipogenesis via Yes-associated protein and the regulation of Runx2,107 as well as during proliferation.108 Elsafadi et al.47 reported that TAGLN overexpression enhanced the osteogenic and adipogenic differentiation of hBMSCs, together with their migration in vitro. Increased bone formation was also reported in vivo. TAGLN deficiency resulted in impaired osteogenic and adipogenic differentiation and reduced cell motility, which was determined to be due to downregulation of the actin cytoskeletal and focal adhesion pathways. Furthermore, and in vivo, BMSCs are not in isolation but physically interact with components in the microenvironment, including mechanical stimuli that influence osteogenic vs. adipogenic differentiation.109 To this end, mechanical promoters  of cytoskeleton contractility are considered osteogenic, while those disrupting contractility promote adipogenesis.110 Our findings suggest that TAGLN plays a key role in the osteoprotective response observed, and this may occur through the optimal activation of actin filaments, alterations in cytoskeletal components, and potentially enhanced cell migration and adhesion. This finding is further supported by the strong upregulation of ILK, a protein also involved in cell-matrix interactions, cell adhesion, and anchorage-dependent cell growth.111 Important processes that control cell shape change, migration, proliferation, survival, and differentiation via interaction with the actin cytoskeleton.51Similarly, the protein TAGLN was upregulated. Transgelin is a transforming growth factor beta-inducible gene and is considered a novel regulator of BMSC commitment to the osteogenic and adipogenic lineage. Transgelin regulates differentiation via the distribution of actin filaments and alterations in cytoskeletal organization.112 Low levels of GDF-3 expression may have further facilitated osteogenic commitment vs. adipogenesis. For example, Shen et al. 48 reported that GDF-3 deficiency protected mice against diet-induced obesity. Other supporting studies have demonstrated that overexpression of GDF-3 via PPARγ suppressed lipolysis, facilitated adipose accumulation,113 and increased adipose-tissue mass and weight gain in mice.114 Finally, P7C3 treatment was also associated with substantially reduced levels of SH2B1 and CD200, two proteins associated with increasing the level of tissue adiposity.50Our results also reveal substantial downregulation of GDF-3. GDF-3 is highly expressed in adipose tissue macrophages and adipocytes and has been reported to play a major role in regulating adipose tissue homeostasis and energy balance.115 which play a pivotal role in the promotion of osteoclastogenesis, osteoclastic activity, and subsequent bone resorption.116 Our in vitro results showed that P7C3 prevented macrophage MNGC formation, a hallmark indicator of chronic inflammation. Notably, in vivo P7C3 facilitated the strong upregulation of Tollip, a multifunctional immune regulator that prevents an excessive proinflammatory response.52 Under strong and acute inflammatory conditions, Tollip serves as a negative inhibitor of the NF-κB signaling pathway and facilitates the resolution of inflammation, promoting autophagy and clearance of cellular debris as well as excessive lipids by enabling vacuole transport.118 Thus, Tollip may also contribute a critical osteoprotective response against IR-induced damage. Our in vivo findings also demonstrated a significant reduction in the number of RANKL+ cells and active TRAP+ osteoclasts in the P7C3-treated animals post-IR. Notably, TBP-2 was also strongly upregulated following P7C3 administration. For example, Aitken et al. 53 demonstrated that TBP-2 overexpression inhibited osteoclastogenesis, potentially via RANKL signaling events. Thus, TBP-2 may also play an important role in regulating bone-resorbing osteoclasts following IR insult. Notably, TBP-2 is also a key regulator of lipid and glucose metabolism; however, the mechanism remains elusive.119A further consequence of IR exposure is cell dysfunction due to increased levels of inflammatory cytokines ,+ levels, the NAD+/NADH ratio, and SIRT1 activity.122 Notably, NAMPT underexpression resulted in a reduced level of osteogenesis.123 Furthermore, higher levels of NAD were reported to inhibit osteoclastogenesis via altered RANKL activity, while lower levels stimulate osteoclastogenesis.123 Li et al.124 recently reported that osteogenic committed BMSCs exhibited elevated intracellular NAD+ levels in vitro, while a decline in intracellular NAD+ levels was found in adipogenic committed BMSCs. The authors concluded that NAD+-mediated processes were indispensable for osteogenic commitment and in bone repair. NAD+, NAMPT, and SIRT1 levels were not measured in this study and therefore warrant further investigation. However, these studies support the potential and important role of P7C3-driven NAD formation in osteogenesis and in contributing to the osteoprotective response observed.Mechanistically, P7C3 increases intracellular NAD flux through overexpression of NAMPT. NAMPT has been shown to promote osteogenesis via increases intracellular NAD125 demonstrated that P7C3, at similar doses, without damaging normal cells, suppressed the malignant growth of glioma cells in vitro and in vivo by regulating aerobic glycolysis. Cancer cells prefer glycolysis to supply advancing energy requirements under normal oxygen conditions. Therefore, this and other studies129 suggest that glycolysis is feasible for targeted therapy, and as such, P7C3 may be an important and undiscovered agent in suppressing cancer progression.Radiotherapy is mainly based on the principle that normal tissue cells exhibit a greater DNA repair capacity than carcinoma cells following IR damage. In light of the protective activity of P7C3s against host cells, it is difficult to consider that the drug may not also provide a protective response against cancer cells. However, our preliminary findings show that P7C3 had a cytotoxic effect on MDA-MB-231, PC3, and OS cancer cell lines. Chen et al. 130 It is therefore difficult to simulate these confounding clinical factors using our rodent model. A further limitation is that exposure of both hind limbs to IR does not mimic cancer radiotherapy where high-dose focal radiation is applied to a local region, thereby potentially altering the subsequent mechanistic cellular response to IR.131 Finally, due to anatomical factors, the level of IR to tissue can vary by 23%–32%.132 In this respect, and as a dosimeter was not used, confirmation that reproducible and accurate IR doses were delivered to bone was not provided in this study.The degree of radiotherapy-induced bone damage to patients varies due to a number of factors, including sex, age, dose per fraction, total dose, chemotherapy, any comorbidities, and the cancer itself.Controlling the adipo-osteogenic lineage commitment of BMSCs in favor of osteogenesis is considered a promising approach for bone regeneration and repair. The exogenous administration of P7C3 in vivo resulted in bone being able to maintain its area, architecture, and mechanical strength despite exposure to IR. While the underlying mechanisms remain unclear, they are potentially primarily driven via the upregulation and downregulation of a series of proteins that together preferentially favor osteogenesis at the expense of adipogenesis, facilitate inflammatory resolution, and suppress RANKL and osteoclastogenesis Fig. . Finally® PCS-500-012™), murine-derived macrophages , human osteosarcoma cells , metastatic prostate cancer cells , and triple-negative breast cancer cells  were investigated in this study. hBMSCs, hOS cells, and MDA-MB-231/GF cells were maintained in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum  and 1% (v/v) penicillin/streptomycin in a humidified incubator containing 5% CO2 at 37 °C. RAW 264.7 cells were maintained in DMEM with 10% heat-inactivated FBS and 1% (v/v) penicillin/streptomycin in a humidified incubator containing 5% CO2 at 37 °C. PC3 cells were maintained in ATCC-formulated F-12K medium  with 10% FBS and 1% (v/v) penicillin/streptomycin in a humidified incubator containing 5% CO2 at 37 °C.Human bone marrow-derived mesenchymal stromal cells , cells were subjected to 7 Gy irradiation with a 160 kV tube voltage, 4 mA tube current, and a distance of 30 cm between the source and the surface . After IR, the cell media was replenished with either 1 μmol·L−1 or 10 μmol·L−1 P7C3. Cells in the solvent control group received 1 μmol·L−1 or 10 μmol·L−1 DMSO.P7C3  was purchased from MCE . For the in vitro analyses, P7C3 was dissolved in dimethyl sulfoxide  at a stock concentration of 10 mmol·L2 at 37 °C for 1 or 3 days. Cells were fixed with 4% paraformaldehyde for 20 min. Cell morphological characteristics were studied using Alexa Fluor™ 488 Phalloidin and DAPI double staining. Images were captured using confocal laser scanning microscopy , and the cell area (µm2) was quantified using ZEN 3.1 software . Additionally, tartrate-resistant alkaline phosphatase (TRAP) staining was used to determine the formation of activated osteoclastic cells from macrophages. Images were captured using an inverted phase microscope .Following IR-induced damage, RAW264.7 macrophages were cultured in DMEM containing 10% FBS with or without P7C3 in a humidified incubator containing 5% COThe toxicity of P7C3 on hBMSCs was determined using a live/dead assay according to the protocol developed by ibidi, USA. After staining, images of live (green) and dead (red) cells were taken using an inverted phase microscope .133 Absorbance was measured using a microplate reader  at 570 nm. Cell number and morphological changes were further evaluated using confocal laser scanning microscopy. Briefly, cells were fixed with 4% paraformaldehyde for 20 min. The actin filaments and nuclei were stained using Alexa Fluor™ 594 Phalloidin  or Alexa Fluor™ 488 Phalloidin  and 4′,6-diamidino-2-phenylindole dihydrochloride , respectively. Images were captured using confocal laser scanning microscopy , and the cell area (µm2) was quantified using ZEN 3.1 software .An MTT  assay  was further performed as per our previous study.®  was used to assess IR-induced DNA damage to hBMSCs. Briefly, the cells were detached gently using a cell scraper, pelleted and then resuspended in ice-cold PBS (Ca++ and Mg++ free). A 50 µL cell suspension was mixed with 500 µL of molten LMAgarose at 37 °C. Fifty microliters of the mixture was then pipetted immediately onto a CometSlide™. After a 10 min solidification period at 4 °C, the slides were immersed in a prechilled lysis buffer overnight at 4 °C. After cell lysis, slides were immersed in a freshly prepared alkaline unwinding solution for 20 min, followed by 30 min of electrophoresis at 21 volts. The slides were washed twice in deionized H2O, followed by washing in 70% ethanol. One hundred microliters of diluted SYBR™ Green I Nucleic Acid Gel Stain was used to stain the slides in the dark for 10 min. The slides were visualized using confocal laser scanning microscopy .A Comet Assay−1 β-glycerophosphate, 100 μmol·L−1 L-ascorbic acid 2-phosphate, 10 nmol·L−1 dexamethasone). The medium was changed every 3 days and replenished with either 10 μmol·L−1 P7C3 or solvent control.After P7C3 pretreatment (24 h) and 7 Gy of IR, hBMSCs were cultured in DMEM containing 10% FBS with osteogenic components  Kit  was used to assess ALP expression at 14 days. Briefly, the cells were stained with ALP staining solution for 30 min. Images were captured using an inverted phase microscope .A Sirius Red/Fast Green Collagen Staining Kit  was used to detect total collagen levels at 14 days. Images were captured using an inverted phase microscope .Alizarin red S staining  was performed to evaluate mineral deposition at 14 and 28 days. Briefly, cells were fixed with 4% paraformaldehyde and stained with 2% pH 4.1 alizarin red S solution for 20 min at room temperature. Images were captured using an inverted phase microscope . Cetylpyridinium chloride  was used to dissolve the mineral deposition, and the optical density (OD) values were determined at 562 nm .55Cellular senescence in hBMSCs was detected using a senescence-associated β-gal  staining kit  after 28 days of culture, as per our previous study.After P7C3 pretreatment (24 h) and 7 Gy of IR, hBMSCs were cultured in a MesenCult™ Adipogenic Differentiation Kit . The medium was changed every 3 days and replenished with either P7C3 or solvent control. Gene expression of adipogenic-related marker genes in hBMSCs was examined via qRT‒PCR using the methods described above.At 14 and 21 days, cells were fixed with 4% paraformaldehyde and stained with LipidSpot™ Lipid Droplet Stains  according to the manufacturer’s protocol. Images of stained sections were captured using an inverted phase microscope .ADPOQ, PPARG, LPL, LEP, FABP4, CEBPA, PLIN1, and FASN) was examined via qRT‒PCR at 12 days. Briefly, total RNA was extracted using a PureLink™ RNA Mini Kit , and genomic DNA contamination was removed from samples using DNase . RNA concentration was measured using a NanoDrop 8000 spectrophotometer (NanoDrop Technologies). Five hundred nanograms of total RNA was used as a template for reverse transcription, and cDNA synthesis was performed using SuperScript™ III First-Strand Synthesis SuperMix . qRT‒PCR was performed using Fast SYBR™ Green Master Mix  on an ABI Prism 7500 Thermal Cycler . The primers used in this study were KiCqStart™ Primers and were purchased from Millipore Sigma. The fold change in relative mRNA expression was calculated using the comparative Ct (2−ΔΔCT) method.Gene expression of adipogenic-related marker genes in hBMSCs (n = 6): control, X-ray, and X-ray + 20 mg·kg−1 P7C3. The main body of the rat was shielded for protection using a lead blanket , and the hind limbs (only) were subjected to a local fractionated X-ray dose of 8 Gy on Days 1, 3, and 5 of the study . X-ray exposure was applied at a tube voltage of 160 kV, a 4 mA tube current, and at a distance of 30 cm between the source and the surface of the animal . This biological irradiator produces a polyenergetic beam similar to the irradiators used clinically. Animal positioning, lead shielding during exposure , dose rate, distance from the source, and radiation intensity were kept consistent. P7C3 (20 mg·kg−1) was administered as a pretreatment prior to IR and then daily via intraperitoneal (i.p.) injection until the end of the study (7 days).Eighteen male SAS Sprague Dawley rats, 8–9 weeks of age (~200 g), were investigated in vivo. Animals were acclimatized for 1 week before commencing the experiment. All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Central Florida  and were performed in accordance with the American Veterinary Medical Associated guidelines. Rats were randomly divided into three groups  staining kit was used to detect the bony structure . Images of stained sections were captured using an inverted phase microscope .Briefly, harvested bone was fixed in 10% neutral buffered formalin, dehydrated in graded ethanol, and embedded in paraffin wax. Longitudinal sections were prepared through the distal femoral condyle. Briefly, 5 μmol·L−1) were rehydrated and incubated with TRAP staining solution containing 0.2 mol·L−1 sodium acetate buffer , 50 mmol·L−1 L-(+) tartaric acid , 0.5 mg·mL−1 naphthol AS-MX phosphate , and 1.1 mg·mL−1 Fast Red TR salt  for 2 h at 37 °C. Nuclei were then counterstained with hematoxylin  for 5 min before mounting with VectaMount™ AQ Mounting Medium . Slides were visualized using an inverted phase microscope .TRAP staining was used to detect osteoclastic activity using the standard naphthol AS-BI phosphate postcoupling method. Paraffin sections . The DICOM files were imported, and a label map was created. A threshold was used to automate the segmentation process, and a smooth crushing tool was used to manually clean the segments.Tibiae were prepared for high-resolution X-ray computed tomography (CT-scanning) using a GE V|TOME|X M 240 Nano CT scanner . Samples were scanned using a 180 kv X-ray tube with a diamond-tungsten target under the following settings: 75 kV, 150 mA, a 0.5 second detector time, averaging of three images per rotation and a voxel resolution of 12.4 μmol·L® 43, MTS, Eden Prairie, MN, USA), a vertical force was applied to the tibial mid-shaft using a 3 mm diameter leading roller and a 5 kN load cell. Each tibia was loaded until failure at a displacement rate of 0.02 mm·s−1. As the cross-sectional area of the tibia was nonuniform and similar to other studies,136 we assumed that the cross-sectional area was circular and obtained the mechanical properties from the load‒displacement curve and using the following formulae:σ is the stress (Pa), F is the applied load (N), L = 0.008 is the span distance between the supports (m), 4) calculated as follows:per bone and from each experimental group were selected, and both inner and outer diameters were calculated from each cross-section. The mean value of the inner-to-outer diameter from each cross-section was calculated to determine the inner radius. The ultimate stress (σu) and fracture stress (σf) were calculated. The mechanical strength parameters were adjusted for body size .137Immediately after dissection, the retrieved tibiae were plastic wrapped and stored at −20 °C. Samples were thawed at room temperature, and 3-point bending analyses were carried out within 96 h of tissue retrieval. Each tibia was positioned horizontally on support bars positioned 8 mm apart. With the tibial anterior bow facing downward and using a universal testing machine (Criterionhttps://www.abcam.com/ps/pdf/protocols/ihc_p.pdf). Briefly, sections (5 μmol·L−1) were deparaffinized in xylene and hydrated in descending alcohol and deionized H2O before immersion in an antigen retrieval buffer. Antigen retrieval was performed by boiling slides in 10 mmol·L−1 sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was inactivated in a hydrogen peroxide blocking reagent  for 10 min. After PBS washes, sections were blocked using a protein block  at room temperature for 30 min. The following antibodies were used as primary antibodies overnight incubation: RANKL  and perilipin . Goat anti-mouse IgG secondary antibody [HRP Polymer]  or goat anti-rabbit mouse IgG secondary antibody [HRP Polymer]  was used as secondary antibodies. Samples were stained with the SignalStain® DAB Substrate Kit  and counterstained with hematoxylin for 3 min. All stained sections were dehydrated in ascending alcohol baths, cleared in xylene, and coverslipped using Fisher Chemical™ Permount™ Mounting Medium . Images were captured using an inverted phase microscope .Immunohistochemistry (IHC) staining was performed according to the Abcam protocol , and sera were collected for cytokine analysis . The signal intensities of 500 protein targets were plotted as a heatmap in which the different colors represent biomarker expression levels. The targets were then subjected to hierarchical clustering based on Euclidean distance. Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted as overrepresentation evaluations of the differentially expressed biomarkers involved in the various KEGG pathway/GO terms. All of the targets measured formed the background. All analyses were conducted in the R programming language V3.6.3 (R Core Team 2017). Pathway/GO overrepresentation was implemented using the R package clusterProfiler.Five-micron-thick sections were prepared from paraffin-embedded samples, followed by deparaffinizing and rehydrating. Then, the tissue sections were stained with Sudan black B solution  for 3 h. After rinsing with 70% isopropyl alcohol and distilled water, nuclear fast red  was used for counterstaining. Images were captured using an inverted phase microscope .−1) were used for Oil red O staining . Briefly, slides were rinsed with PBS and 60% isopropanol and stained with Oil Red O solution for 15 min. Hematoxylin was used for counterstaining. Images were captured using an inverted phase microscope .Frozen tissue sections (20 μmol·L−1) were prepared for the detection of β-gal activity using the methods described above. Nuclear fast red  was used for counterstaining. Images were captured using an inverted phase microscope . TNF-α IHC staining was performed according to the methods described above. TNF-α  was used as the primary antibody, and goat anti-rabbit mouse IgG secondary antibody [HRP Polymer]  was used as the secondary antibody. Anti-p21/CIP1/CDKN1A antibody (NB1001941) was purchased from Novus Biologicals, USA. Anti-CDKN2A/p16INK4a antibody (ab54210) was purchased from Abcam, USA.Frozen sections (20 μmol·L−1) or solvent control for 1 or 3 days. Cells were subjected to irradiation as described above. After X-ray exposure, the cell medium was replenished with P7C3. A cell proliferation assay and morphological analysis were performed using the MTT assay or phalloidin and DAPI staining as described above. A 3D MDA-MD-231 spheroid culture was performed according to the Thermo Fisher protocol (https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/3-d-cell-culture-protocol/mda-mb-231-cell-line-spheroid-generation.html). Spheroids were imaged using confocal microscopy, and spheroid size (µm2) was quantified using ZEN 3.1 software  after 14 days of culture.To investigate the effect of P7C3 and IR on the cancer  cell response, we applied (1.) P7C3 treatment only or (2.) IR + P7C3. Cells were treated with P7C3 . Statistical analysis was carried out using GraphPad Prism , and groups were compared using the nonparametric Mann‒Whitney test. Supplementary Figures


Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L−1 2,4-dichlorophenoxyacetic acid , respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L−1 2,4-D. At higher concentrations of 2,4-D , the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L−1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L−1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of  Daphne belongs to the Thymelaeaceae family and contains over 90 species that are distributed throughout Asia, Europe, and North Africa [Daphne genkwa Sieb.et Zucc. Is a well-known medicinal shrub plant in China and Korea [Daphne is quite diverse and it produces several classes of secondary metabolites including coumarins, flavonoids, lignans, terpenes and other alkaloids [D. genkwa have been used for diuretic, antitussive, anticancer, and abortifacient purposes [D. genkwa exhibit anti-inflammatory, analgesic and immunomodulatory activities [Daphne can be considered an important biological resource both for the treatment of various diseases and as possible leads in the discovery of new medications [The genus h Africa . In partnd Korea . The phylkaloids . Especiapurposes ,4,5. Flativities ,6. Thus,ications .Daphne species are very popular because of their delightful, fragrant and attractive flowers [Daphne is typically from seed or vegetative cutting [Daphne. Seed propagation often produces non-uniform materials because of genetic segregation. Moreover, Daphne has been categorized as a ‘‘difficult-to-root’’ group, both with conventional propagation via cuttings and following micropropagation [Daphne’s cultivation is susceptibility to fungal root pathogens [ flowers . The com cutting . Howeverpagation . Furtherathogens  and viruathogens . These lathogens .Daphne species. An in vitro propagation system of several Daphne species was established from nodal explants by shoot multiplication using cytokinins such as BA, 2iP and TDZ [Daphne species were propagated using media enriched with natural ingredients including coconut water and pineapple pulp for rooting. Nowakowska et al. [D. mezereum cv. Alba. They insisted that the type and concentration of plant growth regulators had an essential effect on the regeneration and growth of the shoots of D. mezereum cv. Alba in the in vitro culture. Recently, Nowakowska and Pacholczak [Daphne mezereum. Although studies related to in vitro shoot multiplication from nodal cultures of Daphne species have been reported, no study has yet reported successful plant regeneration through the somatic embryogenesis of Daphne species.Micropropagation may be a good alternative method to produce ornamental and medicinal shrubs . However and TDZ ,13. Wisz and TDZ  also repa et al.  achievedcholczak  reportedD. genkwa. To achieve this goal, the effect of several auxin  concentrations and types of culture media on the formation of embryogenic structures and the development of somatic embryos from leaf explants were examined. In addition, we investigated the effects of cytokinin and light requirements on the process of plant regeneration through shoot development from embryogenic calluses or embryonic structures.Therefore, this study aimed to establish an efficient plant regeneration system through the somatic embryogenesis of Daphne genkwa was established in this study , they proliferated well  were also able to regenerate whole plants via somatic embryogenesis from the leaf explants of D. genkwa . The leaf explants did not form any embryogenic structures at 5 mg·L−1 2,4-D, even with the prolonged incubation period. Similar to the MS medium, leaf explants were able to form embryogenic structures when they were cultured on WPM medium supplemented with 0.1 to 1 mg·L−1 2,4-D in the dark   suppleme−1 2,4-D . After 8the dark . The freD. genkwa was examined  on embryogenic structure formation from leaf explants was affected in the presence of light see . In the ents see . Even inD. genkwa on culture medium containing 2,4-D, IBA, and NAA, and white embryogenic structures (WESs) were formed on culture medium containing IBA and NAA. When the YES was subcultured twice in MS1D medium, embryogenic calluses (EC) were formed  were simultaneously formed from the leaf explants of e formed A. To invtermined B,C. The d  were formed on culture medium containing IBA and NAA. When the YES was subcultured twice more in MS1D medium, embryogenic calluses (EC) were formed  classified maize calluses into three types, namely, I-, II-, and III-type calluses, based on the callus characteristics. Among these types, only the II-type callus, known as the embryonic callus, has cell totipotency and the ability to regenerate into whole plants [Miscanthus x giganteus including the shoot-forming callus, embryogenic-like callus, friable callus and yellow root-forming callus was also reported [D. genkwa could be maintained for long-term culture periods (up to 1 year) by periodic subculture.Interestingly, yellow embryogenic structures (YES) were simultaneously formed from the leaf explants of e formed A. The fre formed . These re plants . The difreported . The losreported ,46,47. Ireported . Thus, tDaphne genkwa Sieb. et Zucc. were purchased from a botanical garden in Kyung-gi province in Republic of Korea. A voucher specimen (accession number KRIB 0093099) was preserved at the herbarium of the Korea Research Institute of Bioscience and Biotechnology.Mature plants of D. genkwa were used for the experiments. The leaves were collected and surface-sterilized in 70% alcohol for 1 min, and then soaked in a sodium hypochlorite solution containing 0.8% active chloride for 15 min with occasional agitation, and then washed five times with sterilized distilled water. Remaining moisture on the leaves was removed with sterile filter papers  in a laminar hood.Fully expanded young leaves of 2 in area) in a laminar hood. The leaf segments were placed onto callus-inducing medium. In this study, we used two types of culture medium for embryogenic structure induction. One was MS [−1 thiamine·HCl, 0.5 mg·L−1 pyridoxine·HCl, 0.5 mg·L−1 nicotinic acid, 2 mg·L−1 glycine, 100 mg·L−1 myo-inositol, 30 g·L−1 sucrose and 4 g·L−1 Gelrite. The WPM medium consisted of full-strength WPM inorganic salts, 1 mg·L−1 thiamine·HCl, 0.5 mg·L−1 pyridoxine·HCl, 0.5 mg·L−1 nicotinic acid, 2 mg·L−1 glycine, 100 mg·L−1 myo-inositol, 30 g·L−1 sucrose and 4 g·L−1 Gelrite. The pH of all media was adjusted to 5.8 with 1 N NaOH before autoclaving.After surface sterilization, leaf explants were cut into small segments . Each treatment consisted of 10 explants in a plastic Petri dish (90 mm × 15 mm) and was repeated three times. After 8 weeks of culture, the frequency of embryogenic structure formation from leaf explants was determined. The embryogenic structures formed on leaf explants were transferred onto MS or WPM medium containing 1 mg·L−1 2,4-D and maintained at 25 °C in the dark. The subculture was conducted at 4-week intervals. Similar to 2,4-D, the effect of IBA and NAA treatments on embryogenic structure formation from leaf explants was examined in the same manner. Leaf segments were transferred onto MS and WPM medium supplemented with 0, 0.1, 0.5, 1, 5, and 10 mg·L−1 IBA or NAA, respectively. All IBA and NAA treatments were prepared and cultured in the same way as the 2,4-D treatment. As mentioned above, the frequency of embryogenic structure formation from leaf explants was examined after 8 weeks of incubation. Subculture was carried out in the same manner as mentioned above.To examine the effect of 2,4-D on embryogenic structure formation, leaf segments were placed onto MS and WPM medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·LD. genkwa when they were incubated on medium containing auxins. The embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L−1 2,4-D. To investigate whether the embryogenic calluses and embryogenic structures from leaf explants have different potentials for plant regeneration, the EC, YES and WES were transferred onto MS medium supplemented with or without 0.1 mg·L−1 BA, respectively. Each treatment consisted of 12 explants in a plastic Petri dish (90 mm × 15 mm) and was repeated three times. After 4 weeks of incubation in the light, the frequency of somatic embryo development and the total number of somatic embryos from these three types of structures were determined. After a further 4 weeks of incubation in the light with subculture on the same medium, the frequency of shoot differentiation and the total number of elongated shoots from these three types of structures were determined.In preliminary studies, we found that the yellow embryogenic structures (YESs) and white embryogenic structures (WESs) were formed from leaf explants of −1 IBA. The cultures were kept at 25 °C in the light . Each treatment consisted of 10 explants in a plastic Petri dish (90 mm × 15 mm) and was repeated three times. After carefully removing the agar, rooted plantlets were washed with running water and transferred to potting soil. After sealing with wrap to maintain humidity, the plants were grown under light culture conditions for 10 days. When new leaves emerged from the plant, the wrap was removed and the plants were grown in a greenhouse.For whole plant regeneration, regenerated shoots from somatic embryos were transferred onto 1/2 MS medium containing 0.1 mg·Lt-test. At least three biological replicates were performed for each analysis. Quantitative data are expressed as mean ± standard deviation (SD). Student’s t-test was conducted in Excel.Statistical analysis between different groups was evaluated with D. genkwa was established in this study. These systems can be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa. In addition, we are going to investigate the possibility of the mass production of somatic embryos through cell suspension cultures for the production of useful secondary metabolites.In conclusion, an efficient plant regeneration system from leaf-derived embryogenic structure cultures of


The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this investigation, we employed cryoelectron microscopy to elucidate the three-dimensional structure of the uroplakin complex embedded within the porcine AUM at a resolution of 3.5 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane. The urothelium, which lines the urinary tract, performs a dual function: it serves as a permeability barrier, effectively preventing the leakage of urine components into surrounding tissue while undergoing morphological changes to adapt to the distention and contraction that occurs during the micturition cycle . The apiEscherichia coli’s attachment to the urothelium , and the resulting co-expression plasmid was introduced to E coli. BL21 (DE3) (The cDNA of 21 (DE3) . The bac2 and subdivided into 10 frames. The angular range of the tilt series was from − 60° to 60° with 3.0° increments using the dose-symmetric scheme , blotted from both sides for 3 seconds at 4 °C under 99% humidity and plunge frozen in liquid ethane using Vitrobot Mark IV . Images were recorded using a Titan Krios G4 microscope at the University of Tokyo (Thermo Fisher Scientific) at 300 keV, a Gatan Quantum-LS Energy Filter  with a slit width of 20 eV, and a Gatan K3 BioQuantum direct electron detector in the electron counting mode. The nominal magnification was set to 64,000 × with a physical pixel size of 1.35 Å/pixel. Movies were acquired using the SerialEM software , and thec scheme  or the c3 dimensions were extracted from 8 × binned tomograms and were averaged using the PEET software ; in-source CID voltage, 0 V (off) and 35 V. MS-DIAL 4.9 software . After centrifugation , the supernatant was further diluted (1:10) by 2-propanol, and 3 μL was analyzed by LC-MS. The LC-MS system consisted of an Acquity UPLC system , connected to an Exactive mass spectrometer equipped with a HESI-II ion source . An Acquity UPLC BEH C8 column  was used for reversed-phase chromatography with binary gradient elution with mobile phase A (10 mM ammonium formate in acetonitrile/water/formic acid (60/40/0.1)) and B (10 mM ammonium formate in 2-propanol/acetonitrile/water/formic acid (90/9.5/0.5/0.1)) at a flowrate of 100 μL/min. Following time program was used [time (%B)]: 0 min (30%) – 12 min (55%) – 18 min (70%) – 20 min (80%) – 22 min (99%) – 27 min (99%) – 27.1 min (30%) – 28.5 min (30%). MS was operated in following conditions to perform data-independent, all-ions fragmentation (AIF) MS/MS acquisition: ionization polarity, positive and negative; scan range, 100–2,000 m/z; mass resolution, 25,000; maximum injection time, 500 ms; AGC target, balanced (1×10software . was use