Fig 1: GFP+/GFP− co-cultures confirm distinct roles for Jag1 and Dll1.(A) Schematic of GFP+/GFP− co-culture experiment. GFP+ cells were generated using a GFP adenovirus and co-cultured at a 1:50 ratio with GFP− cells under differentiation conditions (TGFβ1±). GFP+ cells were collected after 72 h of culture by flow sorting for downstream RNA isolation and qRT-PCR analysis. (B) qRT-PCR analysis of Alb, Opn, and Sox9 mRNA transcripts in GFP+ cells from co-cultures of every GFP+/GFP− combination of control-, shDll1-, or shJag1-infected BMEL cells. Results were normalized to expression in cultures grown under basal conditions in parallel with co-cultures. For each gene, Student’s t-tests were performed against ControlGFP+ (ControlGFP−) for every combination of GFP− and GFP+ cells. P < 0.05 indicated separately for TGFβ1− (*) and TGFβ1+(^). Numeric callouts show y-axis values (not P-values). Data presented as mean ± s.e.m. with n ≥ 3. See also Supplemental Fig. S4.
Fig 2: Jag1 and TGFβ1 coordinate cholangiocytic fate specification.(A) qRT-PCR analysis of Dll1, Dll4, Jag1, and Jag2 mRNA transcripts in BMEL cells under basal (growth) and differentiation (TGFβ1±) conditions. Student’s t-tests were performed against basal for TGFβ1±. (B) Representative immunoblot against JAG1 in BMEL cells under differentiation conditions (TGFβ1±). Cells were further treated with an equivalent volume of vehicle (DMSO), GSI X (5 μM, GSI), or SB-431542 (10 μM, SB). Molecular weight markers shown in kDa (left) and β-actin control at 45 kDa (bottom). (C) Quantification of JAG1 immunoblots described in (B). Student’s t-tests were performed against DMSO for TGFβ1±. (D) qRT-PCR analysis of Alb and Opn mRNA transcripts in BMEL cells infected with lentiviral shRNA constructs against a non-target sequence (control), Dll1 (shDll1), and Jag1 (shJag1). For shDll1 and shJag1, Student’s t-tests were performed against the same treatment condition (TGFβ1±) in control cells. Numeric callouts show y-axis values (not P-values). Data presented as mean ± s.e.m. with n ≥ 3. P-values indicated for P < 0.05 (*). See also Supplemental Figs S2 and S3.
Fig 3: Peripheral biliary differentiation is dependent on both Notch signaling and substrate stiffness.(A) Immunolabeling for OPN of BMEL cells presented with DLL4 on 30 kPa and 4 kPa substrates. Cells were treated with vehicle control (DMSO) or an inhibitor of Notch signaling (γ-secretase inhibitor X, GSI, 5 µM). (B) Quantification of OPN+ cell counts on 30 kPa and 4 kPa substrates after treatment with DMSO or GSI. (C) Immunolabeling for SOX9 and HNF4A of BMEL cells on 30 kPa and 4 kPa substrates. (D) Quantification of SOX9 and HNF4A intensity on 30 kPa and 4 kPa substrates. (E) RNA in situ hybridization for Jag1, Dll1, and Notch2 on 30 kPa and 4 kPa substrates. Cells were exogenously presented with IgG or DLL4. (A, C, E) Scale bars indicate 150 µm. (B, D) Mean ± 95% CI.10.7554/eLife.38536.013Figure 2—source data 1.Summary table for OPN data in Figure 2B and Figure 2—figure supplement 1.10.7554/eLife.38536.014Figure 2—source data 2.Summary table for SOX9 and HNF4A data in Figure 2D.
Fig 4: MiR‐199b targets the Notch ligand Jagged1. Pre‐199b suppressed the expression of Notch ligand Jagged1 (JAG1) (A) and subsequently the Notch 1 (B) in mRNA and protein levels (C, and quantification in D) during endothelial cell (EC) differentiation from induced pluripotent stem (iPS) cells (data are means ± SEM [n = 3], *, p < .05). LNA‐199b induced the expression of both JAG1 and Notch (A–D). (E): iPS cells were forced to differentiate toward ECs for 4 days, and cotransfections of Pre‐199b or LNA‐199b with the luciferase plasmid of the 3′UTR‐JAG1 were performed. Forty‐eight hours later, the cells have subjected to luciferase analysis demonstrating that the 3′UTR of the JAG1 is a direct target of miR‐199b (data are means ± SEM [n = 3], *, p < .05). The data presented were representative or average of three independent experiments. Abbreviations: LNA, locked nucleic acid; 3′UTR, three prime untranslated region.
Fig 5: MiR‐199b modulates STAT3‐VEGF activation through targeting JAG1. Mouse induced pluripotent stem (iPS) cells were seeded on 5 µg/ml Jag‐1‐coated plates and cultured in differentiation medium (DM) supplemented with vascular endothelial growth factor (VEGF) for 6 days. Then the cells have been harvested and subjected to further analysis and the expression of endothelial cell (EC) markers were detected by real‐time PCR. (A): The differentiated cells in Jag1‐mediated substrate significantly reduced the expression of EC marker CD144, the expression of VEGF and VEGF receptor (FLK‐1), when compared with the control experiments (cells seeded on collagen IV‐coated dishes) (data are means ± SEM [n = 3], *, p < .05) at day 6 during EC differentiation. Control: iPS cells seeded on collagen IV‐coated dishes, Jag1 Substrate: the recombinant rat Jagged 1 FC Chimera (Product Code: 599‐JG100). (B): iPS cells were forced to differentiate toward ECs on collagen IV‐coated dishes in DM media supplemented with VEGF and the JAG1 was knocked down by shRNA. Then, the inhibitory effect of the LNA‐199b inhibitor in EC markers and VEGF and VEGFR (FLK‐1) was ablated in both mRNA (B) and protein levels (C), and quantification in (D). Knockdown of JAG1 by shRNA during EC differentiation showed a robust induction of STAT3 and VEGF in the presence of the miR‐199b inhibitor in protein level as displayed by Western blots (C, D) (data are means ± SEM [n = 3], *, p < .05). The data presented were representative or average of three independent experiments. Abbreviations: LNA, locked nucleic acid; shJAG1, short hairpin RNA JAG1; shNT, short hairpin RNA nontargeting.
Supplier Page from R&D Systems, a Bio-Techne Brand for Jagged 1 Fc Chimera Protein
Available conjugates: Sizes Available: 100 ug