jagged1  (R&D Systems)

Journal: Stem Cells

Article Title: Notch Signaling Regulates the Chondrogenic Potential of Both Articular Chondrocytes and Their Progenitors During Expansion

doi: 10.1093/stmcls/sxad031

Figure Lengend Snippet: Properties and expanding conditions for murine artSPCs. ( A , B ) The pattern of CD73 expression in the articular cartilage of 6-day-old mice. The square in B is magnified in ( C ). ( D-F ) Flow cytometric analysis of CD73 high cells separated by MACS confirmed their expression of both Sox9 and CD73. ( G-I ) Formation of colonies by CD73 high cells obtained from Prg4CreERT:R26-Confetti mice and visualized by hematoxylin staining (G) or by direct fluorescence of ( H ) RFP or ( I ) GFP. H and I exemplify clonal colonies made by a single original cell harboring one Confetti allele after recombination. ( J ) Adipogenic (stained with Oil red O) and (K) osteogenic (stained with alizarin red) differentiation of CD73 high cells isolated by MACS and cultured in the corresponding medium for 21 days. ( L-N ) CD73 high cells were MACS-isolated, and their expansion rate was assessed in various conditions. The cumulative number of cells (CNN) after 3 passages in cultures containing (L) DAPT or Jagged1; (M) XAV or LiCl; or (N) FGF2 or fibronectin were compared to those of corresponding control cells. ( O ) The formation of chondrogenic pellets of different sizes from the same number of cells after 21 days of culture in the presence of different factors. ( P ) The areas of the pellets shown in (O) were determined by ImageJ and normalized to the corresponding controls. * P < .05 is a comparison to the corresponding control group (DMSO in the case of DAPT, XAV, and LiCl; Fc for Jagged1; and PBS for Fibronectin and FGF2). ANOVA with the post hoc Dunnett’s test was used to access differences between more than 2 groups (ie, DMSO, DAPT, XAV, and LiCl; PBS, Fibronectin, and FGF2) and Student’s unpaired t -test to compare Jagged1 with the Fc control. In all cases cells were pooled together from 5 to 6 mice, 3 independent experiments were performed, and the values shown are the means and SDs of the 3 values obtained. Representative images and gating strategy are shown in A-K and O. EC, epiphyseal cartilage; M, meniscus in A-C; FSC-A= forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area.

Article Snippet: In brief, 20 ug/mL Fc-specific human IgG (Sigma-Aldrich) diluted in PBS was incubated in the culture dishes at room temperature for 2 h. Thereafter, 10 ug/mL of either recombinant rat Jagged1 fused to human Fc (R&D Systems) or human Fc control protein (R&D Systems) dissolved in PBS was added to the dishes pre-coated with Fc-specific human IgG and incubated at 4 °C overnight.

Techniques: Expressing, Staining, Fluorescence, Isolation, Cell Culture, Control, Comparison

Journal: BMC Pulmonary Medicine

Article Title: Notch1 activation of Jagged1 contributes to differentiation of mesenchymal stem cells into endothelial cells under cigarette smoke extract exposure

doi: 10.1186/s12890-022-01913-3

Figure Lengend Snippet: Jagged1 expression is regulated by Notch signaling in mesenchymal stem cells (MSCs). A , B Efficiency of Notch-1 knockdown in MSCs by lentiviral vectors harboring shRNA. MSCs were infected with lentiviral vectors GV248-Notch-1-shRNA1, 2, and 3 targeting Notch-1 or a scrambled shRNA vector (Mock) as detailed in Materials and Methods. Notch-1 protein expression was determined by western blotting assays. Notch-1 protein expression was normalized to β-actin levels. * P < 0.05 compared with non-infected MSCs. C – F The Notch signaling pathway was inhibited by incubation with CSE; N1ICD overexpression increased expression levels of Hey1, HeyL, Hes3, and Hes5, which were decreased after transfection with GV248-Notch-1-shRNA2. G , H CSE treatment downregulated Hey1 and Jagged1 expression levels; N1ICD overexpression rescued the decrease in MSCs, whereas the expression levels of Hey1 and Jagged1 were markedly decreased in GV-248-Notch1-shRNA2 transfected MSCs. * P < 0.05 vs. control group, # P < 0.05 vs. CSE group; CSE, cigarette smoke extract

Article Snippet: Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (D-MEM/F-12, Gibco, USA), bovine serum albumin (BSA), and trypsin/EDTA (Gibco-BRL); EGM-2 Bullet Kit (mixture of ascorbic acid, hydrocortisone, EGF, IGF-I, heparin, VEGF, and FGF2,; Lonza); insulin-transferrin-selenium sodium pyruvate (ITS; Invitrogen); Jagged1 Fc Chimera (599-JG; R&D Systems); rat immunoglobulin G (IgG) (I4131; Sigma Aldrich); PE-mouse anti-Rat CD31 (BD PharmingenTM); anti-Hif-1α antibody (Proteintech); R-PE-conjugated Donkey Anti-Goat IgG(H + L) (Proteintech); anti-Jagged-1 antibody (Affinity); anti-N1ICD antibody (CST); anti-Hey1 antibody (Abcam); anti-VEGF receptor2 antibody (Abcam); anti-Von Willebrand factor (vWF) antibody (Abcam); goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen); TRIzol (Takara, Biotechnology, Dalian, China); 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes); reverse Q-PCR kit, and RT-PCR kit, protein lysis buffer kit (Beyotime Biotechnology); anti-actin antibody (Sigma); horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG (Beyotime Biotechnology); Matrigel (Corning); Dual-GLO Luciferase Assay System (Promega); Fugen6 (Roche).

Techniques: Expressing, Knockdown, shRNA, Infection, Plasmid Preparation, Western Blot, Incubation, Over Expression, Transfection, Control

Journal: BMC Pulmonary Medicine

Article Title: Notch1 activation of Jagged1 contributes to differentiation of mesenchymal stem cells into endothelial cells under cigarette smoke extract exposure

doi: 10.1186/s12890-022-01913-3

Figure Lengend Snippet: Notch signaling via RBP-Jκ promotes Jagged-1 expression. Jagged-1 enhanced the transcriptional activation of RBP- Jκ, which was endogenous Notch signaling-dependent. A 2%CSE treatment inhibited Notch1 reporter activation. Incubation with Jagged1 Fc significantly enhanced Notch1 reporter activation by greater than 8.1-fold relative to that in vehicle-treated mesenchymal stem cells (MSCs), whereas this improvement was suppressed by Notch-shRNA; Jagged1/Notch signaling also upregulated Jagged1 protein expression in MSCs and this response was blocked by Notch1-shRNA2 accordingly B, C * P < 0.05 vs. control group, # P < 0.05 vs. CSE group; CSE, cigarette smoke extract. D MSCs, Lenti-V-MSCs, and N1ICD over-expressing MSCs were co-cultured with MSCs expressing a Notch reporter gene 24 h after transfection. E After 24 h of co-culture, Notch reporter expression was significantly increased in N1-ICD-overexpressing MSCs in the lower compartment relative to that in control co-cultures, whereas a significant decrease was observed when the lower compartment cells were pre-transfected with RBP-Jκ shRNA. F , G Jagged1 protein expression in lower compartment cells was determined by western blotting, and these cells exhibited similar expression trends with Notch reporter expression. * P < 0.05

Article Snippet: Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (D-MEM/F-12, Gibco, USA), bovine serum albumin (BSA), and trypsin/EDTA (Gibco-BRL); EGM-2 Bullet Kit (mixture of ascorbic acid, hydrocortisone, EGF, IGF-I, heparin, VEGF, and FGF2,; Lonza); insulin-transferrin-selenium sodium pyruvate (ITS; Invitrogen); Jagged1 Fc Chimera (599-JG; R&D Systems); rat immunoglobulin G (IgG) (I4131; Sigma Aldrich); PE-mouse anti-Rat CD31 (BD PharmingenTM); anti-Hif-1α antibody (Proteintech); R-PE-conjugated Donkey Anti-Goat IgG(H + L) (Proteintech); anti-Jagged-1 antibody (Affinity); anti-N1ICD antibody (CST); anti-Hey1 antibody (Abcam); anti-VEGF receptor2 antibody (Abcam); anti-Von Willebrand factor (vWF) antibody (Abcam); goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen); TRIzol (Takara, Biotechnology, Dalian, China); 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes); reverse Q-PCR kit, and RT-PCR kit, protein lysis buffer kit (Beyotime Biotechnology); anti-actin antibody (Sigma); horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG (Beyotime Biotechnology); Matrigel (Corning); Dual-GLO Luciferase Assay System (Promega); Fugen6 (Roche).

Techniques: Expressing, Activation Assay, Incubation, shRNA, Control, Cell Culture, Transfection, Co-Culture Assay, Western Blot

Journal: BMC Pulmonary Medicine

Article Title: Notch1 activation of Jagged1 contributes to differentiation of mesenchymal stem cells into endothelial cells under cigarette smoke extract exposure

doi: 10.1186/s12890-022-01913-3

Figure Lengend Snippet: Jagged1 promotes endothelial cell (ECs) differentiation, which is dependent on endogenous Notch1 signaling. A , B Jagged1-Fc-activated Notch1 signaling in mesenchymal stem cells (MSCs), as shown by the upregulated protein expression of N1ICD and Hey1; this response was blocked by Notch1-shRNA2. C – H Jagged1 enhanced the expression levels of CD31, eNOS, VE-cadherin and VEGFR2; however, a significant reversal of this effect was observed when induced cells were transfected with Notch1-shRNA2 lentivirus. * P < 0.05

Article Snippet: Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (D-MEM/F-12, Gibco, USA), bovine serum albumin (BSA), and trypsin/EDTA (Gibco-BRL); EGM-2 Bullet Kit (mixture of ascorbic acid, hydrocortisone, EGF, IGF-I, heparin, VEGF, and FGF2,; Lonza); insulin-transferrin-selenium sodium pyruvate (ITS; Invitrogen); Jagged1 Fc Chimera (599-JG; R&D Systems); rat immunoglobulin G (IgG) (I4131; Sigma Aldrich); PE-mouse anti-Rat CD31 (BD PharmingenTM); anti-Hif-1α antibody (Proteintech); R-PE-conjugated Donkey Anti-Goat IgG(H + L) (Proteintech); anti-Jagged-1 antibody (Affinity); anti-N1ICD antibody (CST); anti-Hey1 antibody (Abcam); anti-VEGF receptor2 antibody (Abcam); anti-Von Willebrand factor (vWF) antibody (Abcam); goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen); TRIzol (Takara, Biotechnology, Dalian, China); 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes); reverse Q-PCR kit, and RT-PCR kit, protein lysis buffer kit (Beyotime Biotechnology); anti-actin antibody (Sigma); horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG (Beyotime Biotechnology); Matrigel (Corning); Dual-GLO Luciferase Assay System (Promega); Fugen6 (Roche).

Techniques: Expressing, Transfection

Journal: BMC Pulmonary Medicine

Article Title: Notch1 activation of Jagged1 contributes to differentiation of mesenchymal stem cells into endothelial cells under cigarette smoke extract exposure

doi: 10.1186/s12890-022-01913-3

Figure Lengend Snippet: Notch1 signaling promotes vessel assembly. A, B Hif-1α protein expression levels were increased in N1ICD-MSCs (mesenchymal stem cells), but were decreased in the 2%CSE group. A statistically significant difference in vessel assembly capacity, as evidenced by the number of tubule-like structures, was also found between cultures of N1ICD MSCs and the CSE-treatment group ( C , D ). MSCs infected with Notch shRNA2 displayed significantly reduced protein levels of Hif-1α, as well as vessel assembly capacity, compared to those in the CSE treatment group, * P < 0.05 vs. control group, # P < 0.05 vs. CSE group; CSE, cigarette smoke extract. E – H Treatment with Jagged1 Fc improved the expression of HIF-1α, as well as vessel assembly capacity, whereas Notch1 shRNA2 blocked the promotion of vessel assembly induced by Jagged1, as shown by a significant reversal in the number of tubule-like structures and the protein expression of Hif-1α. * P < 0.05

Article Snippet: Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (D-MEM/F-12, Gibco, USA), bovine serum albumin (BSA), and trypsin/EDTA (Gibco-BRL); EGM-2 Bullet Kit (mixture of ascorbic acid, hydrocortisone, EGF, IGF-I, heparin, VEGF, and FGF2,; Lonza); insulin-transferrin-selenium sodium pyruvate (ITS; Invitrogen); Jagged1 Fc Chimera (599-JG; R&D Systems); rat immunoglobulin G (IgG) (I4131; Sigma Aldrich); PE-mouse anti-Rat CD31 (BD PharmingenTM); anti-Hif-1α antibody (Proteintech); R-PE-conjugated Donkey Anti-Goat IgG(H + L) (Proteintech); anti-Jagged-1 antibody (Affinity); anti-N1ICD antibody (CST); anti-Hey1 antibody (Abcam); anti-VEGF receptor2 antibody (Abcam); anti-Von Willebrand factor (vWF) antibody (Abcam); goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen); TRIzol (Takara, Biotechnology, Dalian, China); 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes); reverse Q-PCR kit, and RT-PCR kit, protein lysis buffer kit (Beyotime Biotechnology); anti-actin antibody (Sigma); horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG (Beyotime Biotechnology); Matrigel (Corning); Dual-GLO Luciferase Assay System (Promega); Fugen6 (Roche).

Techniques: Expressing, Infection, Control