Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: The impaired ANG1 signaling and angiogenesis in diabetic wound healing. ( A ) A comparison of the wound healing process between normal mice and diabetic mice. ( B ) Quantitative analysis showing the dynamic changes in wound areas during the healing process in normal and diabetic mice. n = 6. ( C ) Representative immunostaining images of CD34, a marker of vascular endothelial cells, in normal and diabetic wounds at 14 or 28 days post-wound creation. W, wound central areas and E, wound edges. ( D ) Quantitative analysis of CD34 + vessel-like structures in the wound edge area of diabetic wounds compared to normal wounds. n = 6. ( E ) The results of q-PCR revealing the differential expression levels of various molecules regulating angiogenesis on days 7, 14, 21, and 28 during normal and diabetic wound healing. Notably, the expression levels of ANG1 were significantly lower throughout the diabetic wound healing process compared to normal healing. All data are presented as mean ± SD. Scale bar: 100 μm in C and 20 μm in the magnified figure in C. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Comparison, Immunostaining, Marker, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: Construction and characterization of engineered MSCs stably expressing ANG1 (MSC ANG1 ). ( A ) Schematic illustration of the lentiviral plasmids carrying GFP, or both ANG1 and GFP. ( B ) Representative immunostaining images of GFP showing the presence of GFP in MSCs infected with GFP (MSC GFP ) and ANG1 (MSC ANG1 ) lentivirus. ( C ) Quantitative analysis of GFP intensity in MSC GFP and MSC ANG1 . n = 6. ( D ) Statistical analysis of GFP intensity in the diabetic wounds engrafted with MSC GFP and MSC ANG1 for 7 days. ( E ) Representative images of GFP immunostaining showing the survival of MSC GFP or MSC ANG1 engrafted onto the diabetic wounds for 7 days. The areas in the white boxes were enlarged and shown in the right panel. ( F ) WB showed the expression levels of ANG1 in MSC ANG1 and MSC GFP , as well as the levels in CM of MSC ANG1 and MSC GFP . Full-length blots are presented in Fig. ( G ) WB showed the expression levels of ANG1 in CM of MSC ANG1 cultured for 12 h, 24 h, 48 h, and 72 h, respectively. Full-length blots are presented in Fig. . Scale bar: 50 μm in B, 100 μm in left panel of D and 20 μm in right panel of D. All data are presented as mean ± SD

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Stable Transfection, Expressing, Immunostaining, Infection, Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 promoted the survival, tubulogenesis and Akt activation in HUVECs. ( A ) CCK8 assay showing the total number of HUVECs treated with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 48 h. n = 6. ( B ) Flow cytometry of PI and Annexin-V in HUVECs treated with Ctrl, MSC GFP -CM or MSC ANG1 -CM for 48 h. ( C ) Statistical analysis of apoptosis (PI + /Annexin-V + ) in HUVECs treated with Ctrl, MSC GFP -CM or MSC ANG1 -CM for 48 h. n = 3. ( D ) Representative images of Calcein-AM and PI staining, illustrating HUVECs treated with Ctrl, MSC GFP -CM, or MSC ANG1 -CM for 48 h. ( E and F ) Statistical analysis of the mean fluorescence intensity (MFI) for Calcein-AM ( E ) and PI ( F ). n = 6. ( G ) Representative images showing the tube formation of HUVECs treated with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 4 h. ( H ) Quantitative analysis of the length of tubes in HUVECs. n = 9. ( I ) Representative WB images of p-Tie2, Tie2, p-Akt and Akt expression levels in HUVECs after treatment with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 48 h. β-Actin was the internal control. Full-length blots are presented in Fig. S3. ( J ) Semi-quantitative analysis of the phosphorylation of Tie2 (p-Tie2/Tie2) and Akt (p-Akt/Akt) in HUVECs. n = 9. Scale bar: 100 μm in C. All data are presented as mean ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Activation Assay, CCK-8 Assay, Flow Cytometry, Staining, Fluorescence, Expressing, Control

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 promoted VE-Cadherin expression, vascular integrity, and inhibited Src activation. ( A ) Schematic diagram illustrating the evaluation of endothelial barrier integrity. ( B ) Statistical analysis of the fluorescence intensity of FITC-Dextran that permeated through the monolayer of HUVECs treated with Ctrl, MSC GFP -CM, or MSC ANG1 -CM for 48 h. n = 3. ( C ) Representative immunostaining images of VE-Cadherin in HUVECs following treatment with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 48 h. ( D ) Quantitative analysis of VE-Cadherin fluorescence intensity in HUVECs post-treatment. n = 9. ( E ) Representative WB images depicting the expression levels of p-Src, Src, and VE-Cadherin in HUVECs after treatment with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 48 h. β-Actin served as the internal control. Full-length blots are presented in Fig. S3. ( F ) Semi-quantitative analysis of Src phosphorylation levels (p-Src/Src) and VE-Cadherin expression in HUVECs. n = 9. Scale bar: 50 μm in C. All data are presented as mean ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Expressing, Activation Assay, Fluorescence, Immunostaining, Control

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 promoted angiogenesis and activated Tie2/Akt pathway in diabetic wound healing. ( A ) Representative images of double staining for CD34 and αSMA in the diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. W, the center area of wounds, and E, the transitional area of wounds. The area in the dashed box was enlarged and shown in the insets. Arrows indicated the vessel-like structure positive for both CD34 and αSMA. ( B ) Quantitative analysis of the vessel-like structures positive for CD34, or CD34 and αSMA. n = 6. ( C ) Representative WB image revealing the expression levels of CD34 in diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. β-Actin was set as the internal control. Full-length blots are presented in Fig. S3. ( D ) Semi-quantitative analysis of CD34 expression in diabetic wounds. n = 9. ( E ) Representative WB images of p-Tie2, Tie2, p-Akt and Akt expression levels in diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. β-Actin was set as the internal control. Full-length blots are presented in Fig. S3. ( F ) Semi-quantitative analysis of the phosphorylation of Tie2 (p-Tie2/Tie2) and Akt (p-Akt/Akt) in the diabetic wound healing. n = 9. Scale bar: 100 μm in A, 20 μm in the insert panel of A. All data are presented as mean ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Double Staining, Control, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 promoted the VE-Cadherin expression and inhibited Src activation in diabetic wound healing. ( A ) Representative images of double staining for CD34 and VE-Cadherin in the diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. W, the center area of wounds, and E, the transitional area of wounds. The area in the dashed box was enlarged and shown in the insets. Arrows indicated the vessel-like structures positive for both CD34 and VE-Cadherin. ( B ) Quantitative analysis of vessel-like structure positive for both CD34 and VE-Cadherin in diabetic wounds. n = 6. ( C ) Representative WB images showing the expression levels of p-Src, Src VE-Cadherin in diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. β-Actin served as the internal reference. Full-length blots are presented in Fig. S3. ( D ) Semi-quantitative analysis of Src activation (p-Src/Src) and VE-Cadherin expression levels in the diabetic wounds. n = 9. Scale bar: 100 μm in A, 20 μm in the insert panel of A. All data are presented as mean ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Expressing, Activation Assay, Double Staining, Control

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 treatment promoted diabetic wound healing process. ( A ) Representative images displaying the dynamic changes in skin wounds of diabetic mice treated with control, MSC GFP , and MSC ANG1 . ( B ) MSC ANG1 treatment resulted in a substantial reduction in wound area in diabetic mice models at various time points compared to control and MSC GFP treatment. The percentage of wound healing area was calculated as 100% × (initial wound area - wound area at different time points) / initial wound area. n = 6. ( C ) Hematoxylin and Eosin (H&E) staining of diabetic wounds transplanted with control, MSC GFP , and MSC ANG1 for 14 and 28 days, respectively. ( D ) Representative immunostaining images of K14 in diabetic wounds transplanted with control, MSC GFP , and MSC ANG1 for 14 and 28 days, respectively. The epithelial gap (EG) signifies the area of the wound that remained uncovered by the K14-positive epidermis. ( F ) Representative images of double immunostaining for K14 and K17 in diabetic wounds transplanted with control, MSC GFP , and MSC ANG1 for 14 and 28 days, respectively. The area within the dashed box was magnified and presented in the right panel. Arrows indicate hair follicles positive for both K14 and K17. ( F ) Statistical analysis indicating that MSC ANG1 -treated diabetic wounds depicted a notably reduced EG compared to the control or MSC GFP -treated wounds. n = 6. ( G ) Statistical analysis illustrating that MSC ANG1 and MSC GFP -treated wounds exhibited significantly increased dermal thickness (THK) of the regenerated tissues compared to the control. THK was defined as the dermal thickness in the central area of the regenerated tissues. n = 6. ( H ) Statistical analysis demonstrating that MSC ANG1 -treated diabetic wounds displayed significantly more hair follicles positive for both K14 and K17 compared to the control and MSC GFP -treated wounds. n = 6. Scale bar: 1 mm in C and D, 100 μm in the left three panel of E and 20 μm in the right panel of E. All data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Control, Staining, Immunostaining, Double Immunostaining

Journal: bioRxiv

Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

doi: 10.1101/2020.10.15.340604

Figure Lengend Snippet: ( A ) Generation of pseudotyped lentiviral vectors. Second generation LVs pseudotyped with S protein were generated by transfection of HEK-293T cells with a packaging plasmid encoding HIV-1 gag/pol, a transfer vector plasmid with a lacZ reporter gene and one of two envelope plasmids encoding codon-optimized SARS-CoV-2 S with or without (SΔ19) the 19 C-terminal amino acids. The C-terminal endoplasmic reticulum retention signal (purple) and the receptor binding domain (RBD, orange) are indicated. ( B ) Incorporation of S protein into LVs determined by Western blotting. S-LV and SΔ19-LV particles (V) and lysates of their producer cells (C) were stained for the presence of S protein (top) and p24 as particle loading control. Top blot was exposed for 30 s, bottom blot for 5 s. ( C ) Gene transfer activities on the indicated cell lines. The indicated dilutions of 5 μl vector stock of SΔ19-LV or VSV-LV were added to the cells. Cell lysates were prepared three days after vector addition and lacZ reporter activity was quantified as a luminescence readout. Symbols represent means of technical triplicates. Grey shaded area indicates 95% CI of signals from untransduced cells (blanks). ( D ) Effect of ACE2-overexpression on reporter transfer. 293T cells transfected with ACE2 expression plasmid or mock plasmid were incubated with 0.2 μL of SΔ19-LV or VSV-LV. Cell lysates were prepared three days after vector addition and reporter activity was quantified as a luminescence readout. Bars represent geometric means of technical triplicates ±95% CIs.

Article Snippet: The expression plasmid for N-terminally myc-tagged hACE2 (pCMV3-SP-Myc-ACE2) was purchased from Sino Biological (HG10108-NM).

Techniques: Generated, Transfection, Plasmid Preparation, Binding Assay, Western Blot, Staining, Activity Assay, Over Expression, Expressing, Incubation

Journal: bioRxiv

Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

doi: 10.1101/2020.10.15.340604

Figure Lengend Snippet: ( A ) Principle of quantifying cell-cell fusion. Effector cells expressing S protein and the α-fragment contain active β-galactosidase when they fuse with target cells expressing ACE2 and the ω-fragment. ( B-C ) Effector cells transfected with different amounts of S-protein expression plasmid (7500-7.5 ng/T75 flask) were assessed for S protein expression by flow cytometry ( B ) and then applied in the fusion assay ( C ). ( B ) Mean fluorescence intensity (MFI, orange bars) and the percentage of S-positive cells (yellow bars) were determined in flow cytometry. Bars represent means ± 95% CIs, n=3. P-values are from one-way ANOVA. ( C ) Activities of β-galactosidase in cocultures of effector and target cells in absence of ACE2 overexpression. Effector cells were transfected with the indicated amounts of S protein encoding plasmid, detached with trypsin and cultivated for the indicated time periods with target cells which were transfected with the ω-fragment encoding plasmid only. Bars represent means ± 95% CIs, n=3. ( D ) Influence of proteolytic processing and ACE2 overexpression on cell fusion activity. Left panel: Effector cells were detached without trypsin using 5 mM EDTA in PBS. Right panel: Target cells were co-transfected with the ω-fragment and ACE2 encoding plasmids. Bars represent means ± 95% CIs, n=3.

Article Snippet: The expression plasmid for N-terminally myc-tagged hACE2 (pCMV3-SP-Myc-ACE2) was purchased from Sino Biological (HG10108-NM).

Techniques: Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Single Vesicle Fusion Assay, Fluorescence, Over Expression, Activity Assay

Journal: bioRxiv

Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

doi: 10.1101/2020.10.15.340604

Figure Lengend Snippet: The neutralizing activities of an S-protein specific antibody and sera from two convalescent Covid-19 patients were determined against S-protein mediated particle entry ( A ), cell-cell fusion ( B ) and FFWO ( C-D ). Bars represent means, n=3. P-values are from two-way ( A, C ) or one-way ( B ) ANOVA. ( A ) 0.2 μL of SΔ19-LV or VSV-LV were incubated for 30 min with the indicated antibodies or sera before being added to HEK-293T cells transfected with ACE2 expression plasmid. Cell lysates were prepared 3 days after vector addition and reporter activity was quantified as luminescence readout. ( B ) Effector cells coexpressing S protein (7.5 ng S plasmid per T75) and the α-fragment were incubated for 30 min with antibodies or sera before being added to target cells cotransfected with ω-fragment and ACE2 expression plasmids Bars represent means, n=3. ( C ) 5×10 8 SΔ19-VLPs or bald VLPs were incubated for 30 min with antibodies or sera before being added to the cocultures of α/ACE2 and ω/ACE2 cells. Luminescence was quantified after overnight incubation. Select multiplicity-adjusted p-values are reported. ( D ) Neutralizing capacity of the indicated dilutions of antibodies and sera on FFWO. Dilution factors apply to pure serum 80 μg/mL for the antibodies. ( E ) Semi-quantitative Western blots comparing S protein dose applied to the three fusion assays as SΔ19-VLPs, SΔ19-LV particles and effector cells. 2.5×10 5 effector cells transfected with 750 - 7.5 ng/T75 of S protein encoding plasmid were compared with three-fold dilutions of the particles starting with 2.5×10 9 VLPs or 3 μL of LVs. Samples corresponding to the material used in the neutralization experiments are marked with arrowheads.

Article Snippet: The expression plasmid for N-terminally myc-tagged hACE2 (pCMV3-SP-Myc-ACE2) was purchased from Sino Biological (HG10108-NM).

Techniques: Incubation, Transfection, Expressing, Plasmid Preparation, Activity Assay, Western Blot, Neutralization

Journal: bioRxiv

Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

doi: 10.1101/2020.10.15.340604

Figure Lengend Snippet: ( A ) Principle of the assay. HIV-1 derived SΔ19-VLPs were added as effector triggering fusion from without (FFWO) of cocultures of ACE2 overexpressing HEK-293T cells transfected with plasmids encoding the α-fragment or the ω-fragment of β-galactosidase. Cocultures were lysed and galactosidase activity of the lysates determined in luminescence reactions. ( B ) The indicated numbers of SΔ19-VLPs or bald VLP, produced without the S protein, were added to the coculture. Activities were quantified after overnight incubation. Bars represent means ± 95% CIs, n=4. ( C-D ) Effect of proteolytic processing on the induction of FFWO. 5×10 8 SΔ19-VLPs, bald VLPs or no VLPs were incubated in 2 mg/mL trypsin for the indicated time periods before addition to cocultures (10 5 cells). Luminescence was quantified after overnight cultivation. ( C ). Bars represent means of technical triplicates ± 95% CIs. ( D ) Processing of the S protein after trypsin treatment of the VLPs for indicated times by Western blotting for S and p24 proteins.

Article Snippet: The expression plasmid for N-terminally myc-tagged hACE2 (pCMV3-SP-Myc-ACE2) was purchased from Sino Biological (HG10108-NM).

Techniques: Derivative Assay, Transfection, Activity Assay, Produced, Incubation, Western Blot

Journal: Journal of Virology

Article Title: Porcine Deltacoronavirus Engages the Transmissible Gastroenteritis Virus Functional Receptor Porcine Aminopeptidase N for Infectious Cellular Entry

doi: 10.1128/JVI.00318-18

Figure Lengend Snippet: Flow cytometry analysis of cell surface expression of pAPN. (A) Soluble pAPN-mFc (10 μg/ml) preincubation was able to block TGEV-S1-hFc and PDCoV-S1-hFc binding to permissive LLC-PK1 and ST cells (gray-filled histogram), whereas preincubation with soluble mFc (10 μg/ml) did not block TGEV-S1-hFc or PDCoV-S1-hFc binding (black-filled histogram). Preincubation with pAPN-mFc followed by hFc binding was used as the control (dashed line). Cell surface binding was detected by an FITC-conjugated anti-human IgG Fc. (B) TGEV-S1-hFc and PDCoV-S1-hFc bound to BHK-21 or Vero cells stably expressing pAPN (BHK-pAPN and Vero-pAPN, respectively; red histograms) but not to BHK-21 or Vero (green histograms) or to BHK-21 or Vero cells expressing ACE2 cells (blue histograms). Soluble pAPN-mFc (10 μg/ml) preincubation was able to block TGEV-S1-hFc and PDCoV-S1-hFc binding to BHK-pAPN and Vero-pAPN cells (yellow histogram). Cell surface binding was detected as described in the legend of Fig. 1A and ​andBB.

Article Snippet: The expression plasmid harboring the full-length cDNA of the SARS-CoV cellular receptor ACE2, pCMV3-Flag-ACE2 (HG10108-NF), was purchased from Sino Biological, Inc. (Beijing, China).

Techniques: Flow Cytometry, Expressing, Blocking Assay, Binding Assay, Stable Transfection