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: 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 3: Arrayed Notch ligands drive cholangiocytic fate specification.(A) Immunolabeling of arrayed Fc-recombinant JAG1 and DLL1. Scale bar is 50 μm. (B) Immunofluorescence micrograph showing BMEL cells in TGFβ1−. Arrowhead shows spatial specificity of cholangiocytic (OPN+) differentiation at the edge of the island surrounding an OPN—core. Scale bar is 150 μm. (C) ALB quantification of shRNA-infected BMEL cells in TGFβ1− on five ECM proteins. (D) OPN quantification of shRNA-infected BMEL cells in TGFβ1− on five ECM proteins. Break in y-axis applies only to bar for JAG1/laminin/shDll1 condition. Data presented as mean ± s.e.m. with n ≥ 3. Hypothesis testing in (C,D) was performed as follows: For control cells, Student’s t-tests were performed against IgG for each arrayed Notch ligand within each ECM condition with P-values indicated for P < 0.05 (*). For shDll1 and shJag1 cells, Student’s t-tests were performed against the corresponding arrayed Notch ligand for control cells, again within each ECM condition with P-values indicated for P < 0.05 (^). See also Supplemental Figs S8 and S9.
Fig 4: Schematic summary of approach and findings.Cellular microarrays enable controlled studies of the combined effects of microenvironmental signals, including TGFβ, Notch, and ECM. Analysis of BMEL cell differentiation within cellular microarrays and complementary co-culture formats is further suggestive of the following roles for distinct Notch ligands: TGFβ1 and cell-extrinsic Notch ligands (JAG1 and DLL1) cooperate to induce cholangiocytic fate; cell-intrinsic DLL1 plays a role in the suppression of hepatocytic fate in response to TGFβ1; and cell-intrinsic DLL1 inhibits the generation of double-positive (ALB+/OPN+) cells during differentiation.
Fig 5: 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.
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