Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Screening of the quinonoid compounds for the treatment of GIOP. (A) Flowchart depicting the screening process of the quinonoid compounds library. The schematic diagram was created by using BioRender.com. (B) Volcano diagram showing the effects of the 153 quinonoid compounds on Runx2 expression in BMSCs. Red and blue dots indicate the specific compounds that up- and down-regulate Runx2 expression in BMSCs, respectively. (C) Heat map showing the effect of the compounds on ALP activity in primary BMSCs. Color from blue to red indicates the ALP activity in primary BMSCs from low to high. (D) Measurement of intracellular ROS level in primary BMSCs treated with three potential compounds by using the fluorescent dye DCFDA. (E) Chemical structure of DUB, the final candidate among the screened drugs. (F) MTT assay for the proliferation of BMSCs treated with different doses of DUB for 2 and 10 days, under osteogenic induction conditions with or without 10 μM Dex. (G) Representative images and quantitative analysis of mineralized nodule formation via Alizarin Red S (ARS) staining in primary BMSCs treated with DUB at a series of concentrations, under osteogenic induction conditions with or without 10 μM Dex. (H) Western blot and quantification for the expression of osteogenesis-related proteins in primary BMSCs under different treatments. (I) Oil Red O staining and quantifications for lipid droplets in primary BMSCs of different groups. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ∗∗∗ p < 0.001 by one-way ANOVA. Scale bars: 200 μm (G), and 50 μm (I).

Article Snippet: For DUB@Lip uptake assay in vitro , primary BMSCs were incubated with DUB@Lip labeled with IR-780 (HY-D1063, MedChemExpress, Shanghai, China) in culture medium.

Techniques: Expressing, Activity Assay, MTT Assay, Staining, Western Blot, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: The effect of DUB on the CD39/CD73/ADO axis. ( A ) The volcano plot showing the differential metabolites in the serum of mice of different groups, as indicated by LC-MS/MS metabolomics analysis. ( B ) The Venn diagram illustrates the overlapping differential metabolites across different groups. ( C ) Heatmap showing the differential metabolites as indicated by Venn analysis. Pseudo-color from blue to red indicated the relative expression level of the metabolites ranges from low to high. ( D ) Relative extracellular ADO level in the serum and bone marrow (BM) in mice of different groups. ( E ) Relative extracellular ATP level in the bone marrow (BM) in mice of different groups. ( F-G ) Western blot and RT-qPCR for CD39 and CD73 expressions in primary BMSCs of different groups. ( H ) Relative extracellular ATP and ADO levels in the conditioned medium (CM) in primary BMSCs under different treatments. ( I ) Western blot validation for the knockdown deficiency of CD39 and CD73 after transfection with si Entpd1 and si Nt 5e, respectively. ( J ) Extracellular ATP and ADO concentrations in the conditioned medium (CM) in primary BMSCs from different groups following transfection with si Entpd1 and si Nt5e . n = 4 mice (A-C) in each group for the high-throughput metabolomics analysis, 8 mice (D-E) in each group for in vivo assays, and 4 independent repeats (F-J) by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA.

Article Snippet: For DUB@Lip uptake assay in vitro , primary BMSCs were incubated with DUB@Lip labeled with IR-780 (HY-D1063, MedChemExpress, Shanghai, China) in culture medium.

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Quantitative RT-PCR, Biomarker Discovery, Knockdown, Transfection, High Throughput Screening Assay, In Vivo, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Roles of the CD39/CD73 axis on DUB's multidirectional protection effects in Dex-treated primary BMSCs. ( A ) ELISA for ROS clearance-related enzyme T-SOD and ROS damage biomarkers 8-OHdG, AOPP, and MDA in primary BMSCs of different groups. ( B ) Western blot and quantification for the expression of ROS clearance-related proteins in primary BMSCs of different groups. ( C ) Representative images and quantitative analysis of immunofluorescence staining for MitoSox (red) in primary BMSCs of different groups, and nuclei were stained with Hoechst (blue). ( D ) Western blot and quantification for the expression of apoptosis-related proteins in primary BMSCs of different groups. ( E ) Cellular apoptosis detection in primary BMSCs of different groups by Annexin V-FITC and PI dual-staining assessment via flow cytometry. The proportion of cells in each quadrant was indicated in the plot. ( F ) Tunel (red) staining and quantification of apoptotic cells in primary BMSCs of different groups, and nuclei were stained with DAPI (blue). ( G ) Representative images and quantitative analysis of Alizarin Red S staining for mineralization in primary BMSCs of different groups under osteogenic conditions. ( H ) Western blot and quantification for the expression of osteogenesis-related proteins in primary BMSCs of different groups. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bars: 50 μm (C), 25 μm (F), and 200 μm (G).

Article Snippet: For DUB@Lip uptake assay in vitro , primary BMSCs were incubated with DUB@Lip labeled with IR-780 (HY-D1063, MedChemExpress, Shanghai, China) in culture medium.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, TUNEL Assay, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: The effect of ADO supplements on primary BMSCs. ( A ) MTT assay for the proliferation of BMSCs treated with different doses of ADO for 2 days and 10 days under osteogenic induction conditions with or without 10 μM Dex. ( B-C ) Representative images and quantitative analysis of mineralized nodule areas by Alizarin Red S staining in primary BMSCs treated with gradient doses of ADO under osteogenic induction with or without 10 μM Dex. ( D ) Western blot and quantification for the expression of osteogenesis-related proteins in primary BMSCs of different groups. ( E ) ELISA for ROS clearance-related enzyme T-SOD and ROS damage biomarkers 8-OHdG, AOPP, and MDA in primary BMSCs of different groups. ( F ) Western blot and quantification for the expression of ROS clearance-related proteins in primary BMSCs of different groups. ( G ) Representative images and quantitative analysis of immunofluorescence staining for MitoSox (red) in primary BMSCs of different groups, and nuclei were stained with Hoechst (blue). ( H ) Western blot and quantification for the expression of apoptosis-related proteins in primary BMSCs of different groups. ( I ) Cellular apoptosis detection in primary BMSCs of different groups by Annexin V-FITC and PI dual-staining assessment via flow cytometry. The proportion of cells in each quadrant was indicated in the plot. ( J ) Tunel (red) staining and quantification of apoptotic cells in primary BMSCs of different groups, and nuclei were stained with DAPI (blue). n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bars: 200 μm (B), 50 μm (G), and 25 μm (J).

Article Snippet: For DUB@Lip uptake assay in vitro , primary BMSCs were incubated with DUB@Lip labeled with IR-780 (HY-D1063, MedChemExpress, Shanghai, China) in culture medium.

Techniques: MTT Assay, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Flow Cytometry, TUNEL Assay, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Roles of A 2b R in ADO-mediated activation of the cAMP/PKA/CREB pathway in primary BMSCs. ( A ) Principal component analysis (PCA) of RNA-seq data from primary BMSCs treated with Dex or Dex + ADO. ( B ) The volcano plot presented the differentially expressed genes (DEGs) as determined by RNA-Seq in primary BMSCs treated with Dex or Dex + ADO. ( C ) Gene Ontology (GO) enrichment analysis in the biological process category for DEGs as determined by RNA-Seq in primary BMSCs treated with Dex, or Dex + ADO. ( D ) The molecular docking of ADO with mus musculus A 1 R, A 2a R, A 2b R, and A 3 R proteins. ADO is displayed in Cyan. The surrounding residues in the binding pocket are shown in green (forming a non-hydrogen bond with ADO) or magenta (forming a hydrogen bond with ADO). The hydrogen bond is labeled as yellow dashed lines. The backbone of the receptor is depicted as gray. ( E ) RT-qPCR analysis of the mRNA levels of Adora1 , Adora2a , Adora2b , and Adora3 in primary BMSCs treated with vehicle, Dex, or Dex + ADO. ( F ) RT-qPCR analysis for the expression of Runx2 in primary BMSCs of different groups. (G) Gene Set Enrichment Analysis (GSEA) plot showing the differentially expressed pathway (cAMP) between the Dex group and the Dex + ADO group as indicated by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. ( H ) Western blot validation for the knockdown deficiency of A 2b R after transfection with si Adora2b . ( I ) ELISA analysis for the relative intracellular cAMP levels in BMSCs of different groups. ( J ) Western blot and quantification for the expression of PKA, p-PKA, CREB, and p-CREB in primary BMSCs. ( K ) Representative images and quantitative analysis of Alizarin Red S staining for mineralization deposit in primary BMSCs of different groups under osteogenic conditions. n = 4 independent repeats by using different biological samples in each group for in vitro experiments. Data were means ± s.e.m. ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bar: 200 μm (K).

Article Snippet: For DUB@Lip uptake assay in vitro , primary BMSCs were incubated with DUB@Lip labeled with IR-780 (HY-D1063, MedChemExpress, Shanghai, China) in culture medium.

Techniques: Activation Assay, RNA Sequencing, Binding Assay, Labeling, Quantitative RT-PCR, Expressing, Western Blot, Biomarker Discovery, Knockdown, Transfection, Enzyme-linked Immunosorbent Assay, Staining, In Vitro

Journal: Bioactive Materials

Article Title: Screening of a quinonoid compounds library identifies decylubiquinone as an antioxidant and anti-apoptotic agent against glucocorticoid-induced osteoporosis via CD39/CD73/adenosine axis

doi: 10.1016/j.bioactmat.2026.03.062

Figure Lengend Snippet: Establishment of DUB-loaded bone-targeted liposomal delivery system. ( A ) Representative images and quantitative analysis of mineralized nodule formation via Alizarin Red S (ARS) staining in primary BMSCs for the effect of DUB@Lip on osteogenesis differentiation under conditions of 10 μM Dex. ( B ) RT-qPCR for the expression of osteogenesis-related genes in primary BMSCs of different groups. ( C-D ) In vitro cellular uptake assay of IR-780-labeled DUB@Lip liposomes in primary BMSCs by confocal microscopy and flow cytometry. ( E-F ) Evaluation of bone-targeting capacity and pharmacokinetic analysis of DUB@Lip and DUB@TLip via ex vivo fluorescence imaging. ( G-H ) Representative reconstructed images and quantification for Tb.BV/TV, Tb.N, Tb.Th, Tb.Sp, and Ct.Th in femora in mice of different groups by Micro-CT. ( I ) H&E staining and quantification for trabecular bone area in distal femora in mice of different groups. ( J ) Masson staining and quantification for collagen deposition fraction (collagen area/trabecular bone area) in distal femora in mice of different groups. n = 4 independent repeats by using different biological samples in each group for in vitro experiments, or 8 mice per group for in vivo assays. Data were means ± s.e.m. ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by one-way ANOVA. Scale bars: 200 μm (A-B), 2 mm (G left & right bottom), 1 mm (G right top), and 100 μm (I-J).

Article Snippet: For DUB@Lip uptake assay in vitro , primary BMSCs were incubated with DUB@Lip labeled with IR-780 (HY-D1063, MedChemExpress, Shanghai, China) in culture medium.

Techniques: Staining, Quantitative RT-PCR, Expressing, In Vitro, Labeling, Liposomes, Confocal Microscopy, Flow Cytometry, Ex Vivo, Fluorescence, Imaging, Micro-CT, In Vivo

Journal: Journal of Nanobiotechnology

Article Title: Supramolecular delivery of senolytics enables targeted anti-senescence therapy and accelerated fracture healing

doi: 10.1186/s12951-026-04138-2

Figure Lengend Snippet: Asp 10 SAC4A enables hypoxia-activated senolysis and promotes osteogenic differentiation in vitro. A ) Heatmap of combination index (CI) values at different D: Q ratios and fraction affected (Fa) levels, showing synergistic effects of Dasatinib and Quercetin (DQ). B ) Cell viability curves of normal BMSCs, senescent BMSCs (Sn-BMSCs), and Sn-BMSCs under hypoxia treated with vehicle, free DQ, Asp 10 SAC4A, or DQ@Asp 10 SAC4A. C ) Representative SA-β-Gal staining images of senescent cells treated with different formulations under normoxic and hypoxic conditions, and quantification of SA-β-Gal-positive area (%) ( n = 4/group). Scale bar: 100 μm. D–F ) Representative Western blot images ( D ) and quantitative analyses ( E , F ) of senescence markers P16 and P21 expression in different treatment groups ( n = 3/group). G ) Representative alkaline phosphatase (ALP, upper panel) and Alizarin Red staining (ARS, lower panel) images demonstrating osteogenic differentiation after indicated treatments under normoxic and hypoxic conditions. H–J ) Representative Western blot images ( H ) and quantitative analyses ( I , J ) showing protein expression levels of osteogenic markers RUNX2 and osteopontin (OPN) ( n = 3/group). (Data are presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001; n = 3–4/group)

Article Snippet: Primary mouse bone marrow mesenchymal stem cells (BMSCs) were purchased from Servicebio (Catalog number STCC6011P).

Techniques: In Vitro, Staining, Western Blot, Expressing

Journal: Regenerative Biomaterials

Article Title: Beyond labeling: differential Ac 4 ManNAz dosing as a tool for functional manipulation of mesenchymal stem cells

doi: 10.1093/rb/rbag044

Figure Lengend Snippet: Analysis of metabolic labeling efficiency and viability of Ac 4 ManNAz-treated BMSCs. ( A ) A schematic overview of the evaluation criteria for MSCs following treatment with Ac 4 ManNAz. ( B ) Visualization of azido groups on MSCs using DBCO-Cy3. Scale bar, 20 μm. ( C ) Relative fluorescence intensity of Cy3 ( n = 3). ( D ) Quantitative analysis of MSCs viability by CCK-8 assay following azido-sugar treatment ( n = 3). ( E ) The cytotoxicity was analyzed by LDH assay ( n = 3). data are expressed as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns represent P > 0.05.

Article Snippet: Primary bone marrow-derived MSCs (BMSCs) were obtained from 3-week-old female Sprague–Dawley (SD) rats (Laboratory Animal Center of Nantong University; Approval ID: S20250516-002) and rats weighing 50–70 g were used in the following experiments.

Techniques: Labeling, Fluorescence, CCK-8 Assay, Lactate Dehydrogenase Assay

Journal: Regenerative Biomaterials

Article Title: Beyond labeling: differential Ac 4 ManNAz dosing as a tool for functional manipulation of mesenchymal stem cells

doi: 10.1093/rb/rbag044

Figure Lengend Snippet: Adaptability of MSCs after treatment with Ac 4 ManNAz. ( A ) Representative images of MSCs after treatment with Ac 4 ManNAz by optical microscopic observation and phalloidin-FITC staining. The red arrow indicates the representative morphology of MSCs. Scale bar, 200 and 20 μm (bottom line). ( B ) A schematic illustration of migration assays. ( C ) Representative images of MSCs with various concentrations of Ac 4 ManNAz treatment by scratch assay. ( D ) Quantification of wound healing ratio by analyze the migration area ( n = 6 from three biologically independent cultures). ( E ) Representative images of Ac 4 ManNAz-treated MSCs migrated through the trans-well membranes by crystal violet staining. Scale bar, 100 μm. ( F ) Quantitative analysis of the number of invasive cells ( n = 4) from three biologically independent cultures. Data are expressed as mean ± SD; * P < 0.05, ** P < 0.01, **** P < 0.0001 and ns represent P > 0.05.

Article Snippet: Primary bone marrow-derived MSCs (BMSCs) were obtained from 3-week-old female Sprague–Dawley (SD) rats (Laboratory Animal Center of Nantong University; Approval ID: S20250516-002) and rats weighing 50–70 g were used in the following experiments.

Techniques: Staining, Migration, Wound Healing Assay

Journal: Regenerative Biomaterials

Article Title: Beyond labeling: differential Ac 4 ManNAz dosing as a tool for functional manipulation of mesenchymal stem cells

doi: 10.1093/rb/rbag044

Figure Lengend Snippet: Analysis of mitochondrial functional index of MSCs following treatment with Ac 4 ManNAz. ( A ) a schematic illustration of mitochondrial functional analysis. ( B ) Representative mitochondrial TEM images of MSC and N 3 -MSC. Scale bar, 5 and 2 μm (magnification). The yellow arrows represent the mitochondrial morphology within the cytoplasm. ( C ) Quantitative analysis of the mitochondrial length ( n = 3). ( D ) TMRE assay was used to detect MMP, and the fluorescence intensity of TMRE was quantitatively analyzed (CCCP represents the positive control). scale bar, 20 μm. ( E ) A representative graph of OCR outputs from Seahorse XFe24 Analyzer of MSCs and N 3 -MSCs (MSCs treated with 10- or 50-μM Ac 4 ManNAz). ( F ) Metabolic indicators were analyzed by quantifying the OCR ( n = 3). Data are expressed as mean ± SD; * P < 0.05, ** P < 0.01 and ns represent P > 0.05.

Article Snippet: Primary bone marrow-derived MSCs (BMSCs) were obtained from 3-week-old female Sprague–Dawley (SD) rats (Laboratory Animal Center of Nantong University; Approval ID: S20250516-002) and rats weighing 50–70 g were used in the following experiments.

Techniques: Functional Assay, Fluorescence, Positive Control

Journal: Regenerative Biomaterials

Article Title: Beyond labeling: differential Ac 4 ManNAz dosing as a tool for functional manipulation of mesenchymal stem cells

doi: 10.1093/rb/rbag044

Figure Lengend Snippet: Immunoregulatory property of Ac 4 ManNAz-treated MSCs on M1 macrophages. ( A ) TGF-β protein secretion in the supernatant from MSC and Ac 4 ManNAz-treated MSCs ( n = 3). ( B ) A schematic illustration of the co-culture assay. ( C ) IF staining of CD86 + macrophages and CD206 + macrophages in LPS-induced macrophages co-cultured with MSC-CM and N 3 -MSC-CM. Scale bar, 20 μm. ( D ) Quantification of CD86 + (M1) macrophages percentage ( n = 4). ( E ) Quantification of CD206 + (M2) macrophage percentage ( n = 4). ( F ) qPCR analysis of marker genes of M1, M2 macrophages, anti-inflammatory, and pro-inflammatory cytokines across experimental groups ( n = 3). Data are expressed as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns represents P > 0.05.

Article Snippet: Primary bone marrow-derived MSCs (BMSCs) were obtained from 3-week-old female Sprague–Dawley (SD) rats (Laboratory Animal Center of Nantong University; Approval ID: S20250516-002) and rats weighing 50–70 g were used in the following experiments.

Techniques: Co-culture Assay, Staining, Cell Culture, Marker

Journal: Regenerative Biomaterials

Article Title: Beyond labeling: differential Ac 4 ManNAz dosing as a tool for functional manipulation of mesenchymal stem cells

doi: 10.1093/rb/rbag044

Figure Lengend Snippet: Transcriptomic analysis of Ac 4 ManNAz-treated MSCs. ( A ) Volcano plot of differentially expressed genes between MSC-10 and MSC-50. ( B ) GO annotations analysis focuses on molecular function, cellular component and biological process between MSC-10 and MSC-50. ( C ) GO enrichment analysis targeting cytokine activity, signaling receptor activator activity and signaling receptor regulator activity. ( D ) KEGG pathway enrichment analysis for cytokine-cytokine receptor interaction signaling pathway. ( E ) Heat map of gene expression related to cytokines and the extracellular space component of interest across experimental groups. ( F and G ) Quantitative qPCR analysis of genes associated with TGF-β signaling pathways ( n = 4). Data are expressed as mean ± SD; *** P < 0.001 and **** P < 0.0001.

Article Snippet: Primary bone marrow-derived MSCs (BMSCs) were obtained from 3-week-old female Sprague–Dawley (SD) rats (Laboratory Animal Center of Nantong University; Approval ID: S20250516-002) and rats weighing 50–70 g were used in the following experiments.

Techniques: Activity Assay, Gene Expression, Protein-Protein interactions

Journal: Regenerative Biomaterials

Article Title: Beyond labeling: differential Ac 4 ManNAz dosing as a tool for functional manipulation of mesenchymal stem cells

doi: 10.1093/rb/rbag044

Figure Lengend Snippet: Analysis of the interaction between SRGN protein and TGF-β in N 3 -MSCs. ( A ) The IF staining of SRGN and TGF-β in the MSC-50 group. Scale bar, 20 μm. ( B ) Plot of pixel intensity along the white line in ( A ). ( C ) Western blot assessment of SRGN and TGF-β homodimer ((H) TGF-β) protein expression. ( D ) The gray value statistical analysis of SRGN proteins ( n = 3). ( E ) The gray value statistical analysis of TGF-β proteins ( n = 3). ( F ) mRNA expression of SRGN in MSCs before and after si-SRGN treatment ( n = 3). ( G ) The rescue assay was performed in SRGN-knocked down MSCs by adding 50-µM Ac 4 ManNAz (Ac). (A)TGF-β represents the mature form of TGF-β protein. ( H ) The protein expression of SRGN was analyzed across the experimental groups ( n = 3). ( I ) Schematic illustration of the mechanism by which the azido monosaccharide metabolic pathway (route 1) and treatment with 50 μM Ac 4 ManNAz modulate the immunological functions of MSCs (route 2). The diagram materials in this article are sourced from CNS knowall.com, provided by Shanghai Sennais (CNS) Biopharmaceutical Technology Co., Ltd. All rights and interpretations belong to the authors. Data are expressed as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns represent P > 0.05.

Article Snippet: Primary bone marrow-derived MSCs (BMSCs) were obtained from 3-week-old female Sprague–Dawley (SD) rats (Laboratory Animal Center of Nantong University; Approval ID: S20250516-002) and rats weighing 50–70 g were used in the following experiments.

Techniques: Staining, Western Blot, Expressing, Rescue Assay