Abstract Senescence of bone marrow-derived mesenchymal stem cells (BMSCs) has been widely reported to be closely correlated with aging-related diseases, including osteoporosis (OP). Moreover, the beneficial functions of BMSCs decline with age, limiting their therapeutic efficacy in OP. In the present study, using RNA sequencing (RNA-Seq), we found that leucine-rich repeat containing 17 (LRRc17) expression in BMSCs was highly positively correlated with age. Therefore, we investigated whether LRRc17 knockdown could rejuvenate aged MSCs and increase their therapeutic efficacy in OP. Consistent with the RNA-Seq results, the protein expression of LRRc17 in senescent BMSCs was significantly increased, whereas LRRc17 knockdown inhibited cell apoptosis and reduced the expression of age-related proteins and G2 and S phase quiescence. Furthermore, LRRc17 knockdown shifted BMSCs from adipogenic to osteogenic differentiation, indicating the critical role of LRRc17 in BMSC senescence and differentiation. Additionally, similar to rapamycin (RAPA) treatment, LRRc17 knockdown activated mitophagy via inhibition of the mTOR/PI3K pathway, which consequently reduced mitochondrial dysfunction and inhibited BMSC senescence. However, the effects of LRRc17 knockdown were significantly blocked by the autophagy inhibitor hydroxychloroquine (HCQ), demonstrating that LRRc17 knockdown prevented BMSC senescence by activating mitophagy. In vivo, compared with untransfected aged mouse-derived BMSCs (O-BMSCs), O-BMSCs transfected with sh-LRRc17 showed effective amelioration of ovariectomy (OVX)-induced bone loss. Collectively, these results indicated that LRRc17 knockdown rejuvenated senescent BMSCs and thus enhanced their therapeutic efficacy in OP by activating autophagy. Keywords: BMSCs, LRRc17, Aging, Mitophagy, Osteoporosis Graphical abstract Image 1 [41]Open in a new tab Abbreviations BMSCs bone marrow-derived mesenchymal stem cells O-BMSC Aged mouse-derived BMSC Y-BMSC Young mouse-derived BMSC OP Osteoporosis LRRc17 leucine-rich repeat containing 17 RNA-Seq RNA sequencing RAPA Rapamycin HCQ Hydroxychloroquine OVX Ovariectomy ROS Reactive oxygen species PBS Phosphate-buffered saline α-MEM Alpha minimum essential medium FBS Fetal bovine serum OC Osteoclast OB Osteoblast BMMs Bone marrow-derived macrophages M-CSF Macrophage colony stimulating factor RANKL Receptor activator for nuclear factor-κB ligand TRAP Tartrate resistant acid phosphatase MC3T3-E1 Mouse embryo osteoblast precursor cells ELISA Enzyme linked immunosorbent assay PI Propidium iodide HEK293T Human embryonic kidney 293T JC-1 Tetrechloro-tetraethylbenzimidazol carbocyanine iodide mtROS Mitochondria ROS ATP Adenosine-triphosphate OCR Oxygen consumption rate RT-PCR Real-time polymerase chain reaction DAPI 4’,6-diamidino-2-phenylindole micro-CT Microcomputed tomography BMD Bone mineral density Tb.Th Trabecular bone thickness Tb.N Trabecular bone number BV/TV Bone volume per tissue volume Tb.Sp Trabecular separation Ct.Th Cortical bone thickness BFR Bone formation rate MAR Mineral deposition rate 1. Introduction Mesenchymal stem cells (MSCs) possess proliferative capacity and differentiation potential, and their pluripotency makes them an attractive resource for regenerative cell therapy [[42]1]. MSCs are present in various tissues, including dental pulp, adipose tissue, placenta and bone marrow (BM), among which bone marrow is one of the most common sources used in preclinical and clinical studies due to its high cell number and proliferation activity [[43]2]. Numerous studies have also demonstrated the potential of BM-derived MSCs to treat age-related diseases, including osteoporosis (OP), diabetes mellitus (DM), osteoarthritis (OA), myocardial infarction (MI) and Crohn's disease (CD) [[44]3,[45]4]. Aging is a complex, progressive and inevitable physiological process accompanied by the accumulation of damaged macromolecules and contributes to the development of organ dysfunction. Stem cell senescence results in stem cell exhaustion and leads to tissue failure and pathological and physiological aging [[46]5]. For example, OP is triggered not only by increased bone resorptive activity but also by dysfunction of MSCs that exhibit a distinct shift from osteogenic to adipogenic differentiation and an associated reduction in self-renewal capacity [[47]6]. In addition, the senescence of MSCs may have a profound impact on their therapeutic function. Khan et al. demonstrated that young mouse-derived MSC (Y-BMSC) treatment significantly improved left ventricle systolic and diastolic function in a myocardial infarction mouse model, while the therapeutic effect of MSCs decreased as the age of the donor mice increased [[48]7]. Thus, investigating the mechanism involved in MSC senescence and exploring novel strategies to rejuvenate aged MSCs are essential for their therapeutic applications [[49]8]. The molecular mechanism of MSC senescence is partially related to the p21/p53 and PI3K/AKT pathways [[50]9,[51]10]. In the presence of intracellular or extracellular stimuli, including reactive oxygen species (ROS) and severe inflammation or trauma, the upstream regulator of the p21/p53 pathway is activated, accompanied by diminished telomerase activity and dysfunctional organelle accumulation, especially mitochondria. Moreover, cellular quiescence and changes in differentiation potential are mainly attributed to impaired mitochondria due to decreased autophagy activity [[52]11]. Autophagy is essential for cell metabolism, function and homeostasis and is defined as a lysosomal degradation pathway [[53]12]. Recent evidence has suggested that autophagy deficiency is correlated with impaired osteogenic ability of senescent BMSCs in osteoporosis [[54]13]. In addition, LRRc17, a vital regulator of osteoclast development, is involved in the interactions between osteoclasts and osteoblasts, which are essential to ensure the proper regulation of orthotropic factors in bone metabolism [[55]14]. Both autophagy and LRRc17 are undoubtedly indispensable for bone homeostasis. However, whether there is a correlation between them and the specific mechanism underlying such a correlation have not yet been reported. In this study, we demonstrated that LRRc17 controls BMSC senescence by altering autophagy activity and mitochondrial bioenergetics. These findings indicated that LRRc17 could be a potential therapeutic target for rejuvenating senescent MSCs and treating age-related bone loss. 2. Materials and methods 2.1. Animals All animal experiments were conducted according to the requirements of the Animal Welfare Act. All experimental procedures were performed according to the Ethics Committee of Sichuan University. C57BL/6 mice were divided into three age groups, a young group (<8 weeks), a middle-aged group (5–8 months), and an aged group (18 months), as previously reported [[56]15]. 2.2. Isolation and identification of BMSCs BMSCs were harvested from the bone marrow of C57BL/6 mice as previously described [[57]16,[58]17]. Briefly, bone marrow cells were flushed out and collected from the femur and tibia of mice, plated in T25 flasks, and cultured overnight in a 37 °C incubator with 5% CO[2]. Then, nonadherent cells were removed by rinsing twice with phosphate-buffered saline (PBS). The adherent cells were maintained in alpha minimum essential medium (α-MEM) containing 20% fetal bovine serum (FBS), 2 mM Glutamax, and 1% penicillin and streptomycin (PS). For flow cytometric analysis of surface immunophenotypic markers, BMSCs were incubated with PE-conjugated anti-CD29 (BD Biosciences, USA), SCA-1 (eBiosciences, USA), Alexa Fluor-conjugated anti-CD45 (BD Biosciences, USA), CD11b (BD Biosciences, USA), and FITC-conjugated anti-CD29 (BioLegend, USA) at room temperature for 25 min and then analyzed via flow cytometry (BD Biosciences, USA). 2.3. RNA-sequencing (RNA-seq) Total RNA of Y-BMSCs and O-BMSCs at passage 3 were isolated using TRIzol (Life Technologies, USA). RNA-Seq library construction and RNA high-throughput sequencing were performed by Beijing Genomic Institution (BGI, China) on a BGISEQ-500 high-throughput sequencer. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Set Enrichment Analysis (GSEA), Weighted correlation network analysis (WGCNA) and time course analysis were analyzed according to a previously described method [[59]18]. 2.4. Differentiation potential For osteogenesis and adipogenesis induction of BMSCs, several specific factors were used as previously reported [[60]19]. In brief, for adipogenesis, BMSCs at passage3 (5 × 10^5 cells/cm^2) were cultured α-MEM plus 10% FBS added with 50 μM isobutylmethylxanthine (IBMX), 0.5 μM dexamethasone, 50 μM indomethacin and 10 μg/ml insulin. After 21 d of culture, the cells were fixed with 10% formalin and stained with 0.5% fresh Oil-red O solution (Cyagen, USA) for 30 min. For osteogenesis, BMSCs (4 × 10^5 cells/cm^2) were cultured in α-MEM plus 10% FBS added with 50 μg/ml ascorbate-2, 10 mM β-glycerol phosphate and 0.1 μM dexamethasone. After 14 d of culture, the cells were fixed with 10% formalin, and stained with 0.1% Alizarin red solution (Cyagen, USA) for 30 min. 2.5. Osteoclast (OC) and MC3T3-E1 culture For in vitro osteoclast differentiation, bone marrow cells were harvested from the femora and tibiae of 8-week-old mice. Then, the bone marrow cells (0.5 × 10^6) were differentiated into bone marrow-derived macrophages (BMMs) in α-MEM supplemented with 50 ng/mL recombination macrophage colony stimulating factor (M-CSF), Glutamax, 10% heat-inactivated FBS, and 1% PS in a 12-well plate for 16 h in a 37 °C incubator with 5% CO[2]. The non-adherent cells were then maintained in the presence of 50 ng/mL receptor activator for nuclear factor-κB ligand (RANKL) and 50 ng/mL M-CSF for an additional 5 days. For the coculture assay, osteoclasts were cocultured with BMSCs at a 1:3 ratio for 5 d in a Transwell system. Tartrate resistant acid phosphatase (TRAP) staining was performed using a kit (Solarbio, China) following the manufacturer's protocols. The mouse osteoblast cell line Mouse embryo osteoblast precursor cells (MC3T3-E1) (subclone 4) was purchased from the American Type Culture Collection (ATCC, USA) and cultured in α-MEM containing 10% FBS and 1% PS in a 37 °C incubator with 5% CO[2]. For the coculture assay, osteoblasts were cocultured with BMSCs at a 1:3 ratio for 10 d. Alizarin red staining was conducted using a kit (Cyagen, USA) following the manufacturer's instructions. 2.6. Bone resorption assay To examine osteoclast function, BMMs were seeded on bovine bone slices at a density of 2.5 × 10^4 cells/cm^2 in triplicate. After adhering, cells were stimulated with 50 ng/ml M-CSF and 50 ng/ml RANKL for 7 days. Then, the cells were subsequently washed by mechanical agitation and sonication. Resorption pits were visualized by scanning electron microscope (ZEISS, Germany) and Image J software was used to quantify the area of bone resorption [[61]20]. 2.7. Proliferation assay A proliferation assay was used to determine cell growth kinetics. In brief, 1 × 10^5 BMSCs were cultured in 6-well plates and counted daily for 9 successive days using a hemocytometer. The EdU assay was carried out as previously described [[62]21]. 2.8. CFU analysis BMSCs were rinsed twice with PBS and fixed with 4% formaldehyde for 20 min at room temperature. Then, 0.5% crystal violet was used to stain the BMSCs for 30 min, and the cells were washed with PBS [[63]22]. Colonies composed of 50 or more cells were considered for counting. 2.9. SA-β-gal staining SA-β-gal activity was measured with a staining kit (Beyotime, China) following the manufacturer's protocols. The number of blue SA-β-gal-positive cells was calculated as previously reported [[64]23]. 2.10. Cell death evaluation A cell death detection enzyme linked immunosorbent assay (ELISA) kit (Roche, USA) was applied to measure cell apoptosis. The relative apoptosis ratio was detected at an absorption wavelength of 405 nm and a reference wavelength of 490 nm according to the manufacturer's protocol. 2.11. Cell cycle and apoptosis analysis For the cell cycle assay, BMSCs were harvested and fixed with 70% ethanol overnight at 4 °C. Then, the fixed BMSCs were incubated with 100 μg/mL RNase A (Sigma-Aldrich, USA) and 20 μg/mL propidium iodide (PI) (Sigma-Aldrich, USA) for 30 min. The samples were analyzed by flow cytometry, and the data were analyzed with ModFit LT (version 5.0). For the apoptosis analysis, cells were collected and analyzed with the Annexin V-FITC/PI Apoptosis Detection Kit (BD, USA). 2.12. Telomerase activity BMSCs were lysed to detect telomerase activity. Next, a TeloTAGGG Telomerase PCR ELISA Kit (Roche, USA) was used to perform sequential reaction steps in accordance with the manufacturer’s protocol. The absorbance of the PCR amplification product at 450 nm was determined using a microplate reader within 30 min. 2.13. Lentiviral transfection of BMSCs Construction and transfection of the lentiviral vectors were performed according to previously described methods [[65]24]. In brief, lentiviral sh-LRRc17 or overexpression vectors were cotransfected into human embryonic kidney 293T (HEK293T) cells, and the three packaging plasmids included pHBLV^TM, psPAX2 and pMD2.G. Lentiviral particles were obtained after 48 h of transfection via cell harvest and concentration with ultracentrifugation at 72,000 g for 2 h. Following titer determination, BMSCs were transfected at a multiplicity of infection (MOI) of 50 for 24 h. 2.14. Mitochondrial morphological analyses Mitochondrial morphometric analyses were performed as previously reported [[66]25]. BMSCs were labeled with MitoTracker Green (50 nM, Invitrogen, USA) at 37 °C for 30 min, and representative images were acquired with a laser scanning confocal microscope. Mitochondrial length and complexity were reflected by the aspect ratio (AR, major axis/minor axis) and form factor (FF, 4π × (area/perimeter^2)), respectively, which were calculated with ImageJ software (Wayne; USA). 2.15. Mitochondrial membrane potential (ΔΨ) and mtROS determination Tetrechloro-tetraethylbenzimidazol carbocyanine iodide (JC-1, 10 μg/ml; Beyotime, China) staining was performed at 37 °C for 20 min to determine BMSC mitochondrial membrane potential. For mitochondria ROS (mtROS) detection, BMSCs were stained with 4 μM MitoSOX (Invitrogen, USA) at 37 °C for 20 min, and mtROS quantification was performed using flow cytometry. 2.16. Adenosine-triphosphate (ATP) measurement Intracellular ATP was determined by an ATP measurement kit (Beyotime, China) according to the manufacturer's protocol. The ATP luminescence signal of supernatants mixed with luciferase assay buffer was determined by a luminescence microplate reader and then normalized to the protein concentration. 2.17. Mitochondrial oxygen consumption rate (OCR) measurement The mitochondrial OCR of BMSCs was conducted with a Seahorse XF Cell Mito Stress Test Kit (Agilent, USA) in accordance with the manufacturer's protocol as previously reported [[67]26]. 2.18. Real-time polymerase chain reaction (RT-PCR) Total RNA was isolated using TRIzol (Life Technologies, USA). Then, a high-capacity cDNA reverse transcription kit (Vazyme, China) was used to reverse-transcribe 1 μg of mRNA into cDNA in accordance with the manufacturer’s protocol. RT-PCRs were performed on an ABI7900 PCR system (Applied Biosystems, USA) using SYBR Green MasterMix (Vazyme, China). GAPDH was used as a reference gene. The primers are shown in [68]Supplementary Table 1. 2.19. Immunofluorescence To observe the colocalization of LC3B with mitochondria in BMSCs, BMSCs were incubated with MitoTracker Deep Red (200 nM; Invitrogen, USA) at 37 °C for 10 min, fixed with methanol and then treated with 0.2% Triton X-100 (Sigma-Aldrich, USA) to increase cell membrane penetrability. Cells were incubated with LC3B (1:200 dilution; CST, USA) at 4 °C overnight and then incubated with fluorescently labeled secondary antibodies (1:250 dilution; ABclonal, China) for 1 h at room temperature. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA) for 5 min. Immunofluorescence images were acquired by confocal laser scanning microscopy. 2.20. Western blot Protein expression in BMSCs was determined by immunoblotting as previously described [[69]27]. Anti-LRRc17 (1:1000, Proteintech), anti-p21 (1:1000, Affinity), anti-p16 (1:1000, CST), anti-p53 (1:1000, CST), anti-mTOR (1:500, Affinity), anti-phospho-mTOR (1:500, CST), anti-PI3K (1:1000, CST), anti-phospho-PI3K (1:1000, Affinity), anti-P62 (1:5000, ABclonal), anti-LC3B (1:1000, CST), anti-Beclin1 (1:1000, CST), anti-OPA1 (1:1000, CST), anti-Drp1 (1:1000, CST), anti-JNK (1:1000, Abclonal) abti-phosphor-JNK (1:1000, Abclonal) and anti-Calpain1 (1:1000, Abclonal) antibodies were used to analyze protein expression. The signal was visualized using an Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, USA). Quantification of the gray values of the bands was performed with ImageJ software. 2.21. OVX-induced osteoporosis and BMSC transplantation Six-month-old female C57BL/6 mice were divided into six groups: sham (n = 7), OVX (n = 7), OVX + Y-BMSCs (n = 7), OVX + O-BMSCs (n = 7), OVX + O-BMSCs^LV-GFP (n = 7) and OVX + O-BMSCs^sh-LRRc17 (n = 7). Four month after ovariectomy, ovariectomized mice were anesthetized and immediately received local transplantation of BMSCs (1 × 10^6 cells suspended in 100 μl normal saline) as previously proposed [[70]28]. Briefly, 1 × 10^6 BMSCs in 100 μl of normal saline were delivered with a 22-gauge needle into the marrow cavity through the metaphysis and during needle removal to evenly distribute the cells. One month after transplantation, the mice were euthanized, and samples were collected for subsequent testing. 2.22. Micro-CT and analysis Microcomputed tomography (micro-CT) was applied to determine bone mass. After anesthesia with 1% pentobarbital, the mice were secured on the table in the prone position. Then, femurs were imaged by microcomputed tomography (PerkinElmer, USA) at 55 kV at an 8 μm voxel size. After the image was reconstructed, the area of interest (ROI) of trabecular bone was analyzed within the 0.3-0.8 mm distal metaphysis from the growth plate. Analyze V12.0 software was used to analyze the data and quantify the parameters, including bone mineral density (BMD), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), bone volume per tissue volume (BV/TV), and trabecular separation (Tb.Sp). 2.23. Calcein labeling assay C57/BL6 mice were intraperitoneally injected with 20 mg/kg calcein (Sigma-Aldrich, USA), dissolved in PBS at a concentration of 2 mg/ml with 1 mg/ml NaHCO[3] (Sigma-Aldrich, USA) at 16 d and 2 d before sacrifice. After sacrifice, the left femur was obtained, fixed in 4% formaldehyde, and embedded in methyl methacrylate. A hard tissue slicing machine (SP1600; Leica, Germany) was used to sagittally section specimens into 30-mm sections away from light. Then, cortical endosteum surfaces were evaluated using a fluorescence microscope (STP6000; Leica, Germany) with an excitation wavelength of 488 nm. The bone formation rate (BFR), mineral deposition rate (MAR) and the recommended relevant calculations were used for quantitative analysis using ImageJ 1.47 software [[71]29]. 2.24. Histology and immunohistochemistry The femur was decalcified by 10% EDTA and then embedded in paraffin. Five-micrometer sagittal sections of the metaphysis were prepared, stained with Toluidine blue, TRAP, hematoxylin-eosin (HE) and Masson, and observed by light microscopy (Zeiss, Germany) [[72]30]. For NFATc1 and RUNX2 immunohistochemical staining, after quenching with endogenous peroxidase, achieving antigen retrieval, and blocking nonspecific binding sites, the femur section were incubated with anti-NFATc1 (1:100; Abclonal, China), anti-RUNX2 (1:100; Huabio, China) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibodies (Invitrogen, USA) at room temperature for 30 min. Finally, the sections were exposed to diaminobenzidine peroxidase substrate for 5 min and counterstained with Mayer’s hematoxylin. 2.25. Statistics A two-tailed Student's t-test was applied when two groups were compared. One-way ANOVA with Bonferroni adjustment was performed to determine the differences among multiple groups. Data are shown as the mean ± SD. For all analyses, P values < 0.05 were considered statistically significant. 3. Results 3.1. LRRc17 expressed by BMSCs was positively correlated with age in mice BMSCs were isolated from young, middle-aged and aged C57/BL6 mice and separately cultured to passage three ([73]Figs. S1a and b). Cell death analysis showed that the number of dead cells increased with age in both adherent BMSCs and the culture medium supernatant ([74]Fig. 1a). Furthermore, detection of telomerase activity revealed that telomerase activity gradually decreased, and the reduction was more obvious in female mice ([75]Fig. 1b). In addition, SA-β-gal staining revealed that the ratio of senescent cells in O-BMSCs was notably higher than that in BMSCs isolated from young and middle-aged mice ([76]Fig. 1c). Additionally, the expression of p16, p21 and p53 was also elevated with age ([77]Fig. 1d–g). To reveal the underlying causes of these phenomena, we further analyzed Y-BMSCs and O-BMSCs by RNA sequencing. KEGG pathway analysis showed that the cellular senescence signaling pathway was highly enriched ([78]Fig. 1h). Gene Set Enrichment Analysis (GSEA) showed that pathways were mainly associated with autophagy and p53 signaling ([79]Fig. S2 a, b). Furthermore, weighted gene coexpression network analysis (WGCNA) showed that PTGFR, MX1, IFIT1B, LRRc17, CH3I2, VILL, PENK, GBJ2 and GJB2 might interact with each other ([80]Fig. 1i). Among these genes, LRRc17 (level 1: organismal systems, level 2: development) has been widely reported to be correlated with bone homeostasis regulation, and the log2 (Old/Young) ratio was 2.50. Furthermore, time course analysis showed that LRRc17 was positively correlated with age ([81]Fig. 1j), suggesting that LRRc17 may be a critical factor for regulating senescence. Fig. 1. [82]Fig. 1 [83]Open in a new tab Senescent BMSCs show high expression of LRRc17. BMSCs were separately isolated from young, middle-aged and aged mice and then cultured to passage 3. (a) A photometric enzyme immunoassay was performed to assess the BMSC death ratio via quantitative assessment of cytoplasmic histone-associated DNA fragments (n = 3). (b) Telomerase activity was determined with a commercial TeloTAGGG Telomerase PCR ELISA kit (n = 3). (c) Quantitative analysis of SA-β-gal-positive BMSCs. Five fields from each section were randomly selected to calculate the positive SA-β-gal cell ratio (n = 3). Scale bar, 10 μm. (d–g) BMSCs were lysed and prepared to measure the expression levels of p16, p21 and p53 by Western blotting (n = 3). Aged, middle-aged, and young BMSCs were subjected to RNA-seq, and differentially expressed genes were enriched and subjected to (h) KEGG pathway enrichment analysis, (i) weighted gene coexpression network analysis (WGCNA), (j) and time course analysis (n = 3). All data are shown as the mean ± SD. **P < 0.01, *P < 0.05; NS: not significant (P > 0.05). 3.2. LRRc17 controlled BMSC senescence and differentiation To determine whether LRRc17 is involved in cellular senescence, we knocked down LRRc17 in O-BMSCs via lentiviral transfection and found that LRRc17 silencing significantly decreased the expression levels of p16, p21 and p53 in O-BMSCs compared with control cells, but there were no notable changes in the GFP-transfected cells ([84]Fig. 2a). Furthermore, Y-BMSCs were induced to senescence, characterized by increased expression of p16, p21, and p53, via H[2]O[2] treatment. However, senescence was significantly reversed when LRRc17 was downregulated ([85]Fig. 2b, [86]Figs. S3a–d). In addition, numerous reports have found that in metabolic disorders, senescent cells produce more cytotoxic components, which leads to abnormal cell proliferation and a bias toward apoptosis. Thus, ROS detection, apoptosis and the cell cycle were assessed, and we found that LRRc17 knockdown significantly decelerated ROS accumulation after H[2]O[2] treatment ([87]Fig. S3e). Moreover, apoptosis analysis revealed that the proportion of apoptotic cells declined notably ([88]Fig. S3f), and the number of cells in the quiescent phase (G0/G1) was also much lower in the LRRc17 knockdown group than in the H[2]O[2]-treated group ([89]Fig. S3g). Our data demonstrated that LRRc17 knockdown effectively alleviated BMSC senescence. Fig. 2. [90]Fig. 2 [91]Open in a new tab Silencing LRRc17 alleviates BMSC senescence and alters differentiation potential. O-BMSCs at passage 3 were treated with 100 nM RAPA, followed by transfection of LV^sh-LRRc17 or LV^GFP for 72 h. (a) Quantitative analysis of the protein expression levels of p16, p21, and p53 in 4 different groups (O-BMSCs, RAPA-treated O-BMSCs, LV^sh-LRRc17-transfected O-BMSCs, and LV^GFP-transfected O-BMSCs) by Western blotting (n = 3). Senescence was induced in Y-BMSCs at passage 3 with 400 μM H[2]O[2], followed by treatment with 100 nM RAPA or lentivirus transfection for 72 h. (b) Quantitative analysis of the levels of p16, p21, and p53 in 4 different groups (Y-BMSCs, H[2]O[2]-treated young BMSCs, H[2]O[2]- and RAPA-treated young BMSCs, H[2]O[2]- and LV^sh-^LRRc17-transfected young BMSCs) by Western blotting (n = 3). (c) Quantitative analysis of the protein levels of p16, p21, and p53 in 3 different groups (Y-BMSCs, LV^GFP-transfected Y-BMSCs, and LV^o^v^-^LRRc17-transfected Y-BMSCs) by Western blotting (n = 3). Young, middle-aged, old, young^ov-LRRc17, and old^sh-LRRc17 BMSCs at passage 3 were subjected to osteogenic and adipogenic induction. (d, e) BMSCs (3 × 10^5) were subjected to osteogenic induction for 14 d, and the mineralized nodules were quantified by calculating the ratio of red mineralization area to total area after alizarin red staining (n = 3). Scale bar, 200 μm. (f, g) BMSCs (3 × 10^5) were subjected to adipogenic induction for 21 days. Then, lipid droplets were quantified by calculating the ratio of red lipid droplet area to total area after Oil Red O staining (n = 3). Scale bar, 200 μm. (h, i, j) The expression levels of ALP and RUNX2 were detected by Western blotting after osteogenic induction (n = 3). (k) The expression levels of PPAR-γ and LPL were detected and quantified by Western blotting after adipogenic induction. β-Actin was used as an internal control (n = 3). All data are shown as the mean ± SD. **P < 0.01, *P < 0.05; NS, not significant (P > 0.05). (For interpretation of the references to colour in this figure legend, the