Fig 1: Effects of soluble DLK1 on glioma cell stem cell characteristics. (A, B) Representative images and quantification of primary and secondary sphere forming assays in U3082MG, U3084MG, U3065MG, and PIGPC cells grown at 0 or 50 ng/ml recombinant DLK1. (C) qPCR data for relative mRNA expression of OCT4, NANOG, and SOX2 in U3082MG, U3084MG, U3065MG, and PIGPC cells grown at 0 or 50 ng/mL recombinant DLK1 for 72 hours. Data are expressed as fold change of untreated control. (D) 8xCSL-luciferase experiments showing Notch activity in cell lines untreated or treated with 50 ng/mL recombinant DLK1 for 24 and 72 hours. Results are expressed as fold change of respective controls. Statistical analysis: independent experimental replicates are as follow, (B) n = 5 for U3082MG, U3084MG, and U3065MG primary spheres and n = 3 PIGPC cells, (C) n = 8 except PIGPC where n = 3, (D) n = 4. Statistical significance was determined by t test, in C and D Welch's correction for unequal variances was applied. In the whole figure significance is represented as *P < 0.05, **P < 0.01, and ***P < 0.001 vs untreated controls. DLK1, delta-like noncanonical Notch ligand 1; PIGPC, primary murine glioma cell.
Fig 2: Effects of soluble DLK1 on glioma growth in vivo. (A) Representative images and densitometric analysis of western blots showing HIS-tagged DLK-S expression in DF1 cells transfected with empty or DLK-S RCAS vectors. (B) Kaplan-Meier survival plot of PDGFB-induced tumors with (DLK-S, red line) or without (EMPTY, black line) DLK1 overexpression. (C, D) Representative images and quantification of immunofluorescent stainings showing Ki67 positive cells expressed as percentage of tumor cells identified by OLIG2 staining. Scalebars represents 50 µm. (E) Kaplan-Meier survival plot of DLK1-HIGH (red line) and DLK1-LOW (black line) patients in TCGA GBMLGG data set. Statistical analysis: independent experimental replicates are as follow, (A) n = 4, (B) n = 21 for PDGFB and n = 27 for DLK-S, (C) n = 6, E n = 333, events = 82 for DLK1-low and n = 334, events = 157 for DLK1-high. Statistical significance was determined by t test with Welch's correction (A), Kaplan-Meier survival plot with log-rank Mantel-Cox test (B, C) and Mann-Whitney (D). In the whole figure significance is represented as *P < 0.05, **P < 0.01 vs control. DLK1, delta-like noncanonical Notch ligand 1; DLK-S, soluble part of DLK1; PDGFB, platelet-derived growth factor B; RCAS, replication-competent avian sarcoma-leukosis virus long-terminal repeat (LTR) with splice acceptor; TCGA, The Cancer Genome Atlas. (Color version is available online.)
Fig 3: Effects of soluble DLK1 on glioma cells. (A) Proliferation curves of U3082MG, U3084MG, U3065MG, and PIGPC cells grown at increasing concentrations of recombinant DLK1 for 72 hours. Data are expressed as fold change of untreated control. (B, C) Representative images and quantification of colony forming ability of U3082MG, U3084MG, U3065MG, and PIGPC cells grown at increasing concentrations of recombinant DLK1. Data are expressed as fold change of untreated control. Statistical analysis: independent experimental replicates are as follow, (A) n = 6, except U3065MG where n = 4, (C) n = 4, Statistical significance was determined by 1-way ANOVA, followed by Bonferroni post hoc test. In the whole figure significance is represented as *P < 0.05 and **P < 0.01 vs untreated controls. DLK1, delta-like noncanonical Notch ligand 1; PIGPC, primary murine glioma cell.
Fig 4: DLK1 expression in murine glioma. (A-C) Representative images of immunofluorescent stainings showing tumor-associated astrocytes and DLK1 localization in bulk tumor (A) and perinecrotic (N) and perivascular (V) areas (B, D, respectively) of shp53-induced murine gliomas. Scalebars represent 25 µm. (D, E) Representative images of immunofluorescent stainings showing tumor-associated astrocytes and N-terminal, secreted, DLK1 localization in bulk tumor (D) and perinecrotic areas (N) (E) of shp53-induced murine gliomas. (F-G) Colocalization analysis of immunofluorescent staining showing DLK1 and GFAP expression in bulk tumor vs perinecrotic (F) and perivascular (G) niches. (H) Colocalization analysis of immunofluorescent staining showing secreted DLK1 and GFAP expression in bulk tumor vs perinecrotic niche. Scalebars represent 25 µm. Statistical analysis: (A-H) n = 3. Statistical significance was determined with t test with Welch's correction for unequal variances applied to Pearson's coefficients (F-H). In the whole figure significance is represented as *P < 0.05 and **P < 0.01 vs bulk tumor. DLK1, delta-like noncanonical Notch ligand 1; GFAP, glial fibrillary acidic protein.
Fig 5: Effects of soluble DLK1 on HIF-2alpha activity under hypoxia. (A) Representative images and densitometric analysis of western blots showing HIF-1alpha (HIF-1a) and HIF-2alpha (HIF-2a) expression in U3082MG cells after treatment with 50 ng/mL recombinant DLK1 or hypoxia exposure as indicated in the figure. (B) Representative images and densitometric analysis of western blots showing HIF-1alpha (HIF-1a) and HIF-2alpha (HIF-2a) expression in U3084MG cells after treatment with 50 ng/mL recombinant DLK1 or hypoxia exposure as indicated in the figure. (C) HRE-luciferase time course experiments showing hypoxia response in cell lines untreated or treated with 50 ng/mL recombinant DLK1 and grown in 1% O2 for up to 72 hours. Results are expressed as fold changes of respective normoxic controls. (D) Representative images and colocalization analysis of immunofluorescent staining showing DLK1 and HIF2-alpha (HIF2) expression in perivascular and hypoxic niches. Scale bars represent 50 µm. Statistical analysis: independent experimental replicates are as follow, (A, B) n = 3, (C) n = 5 for U3082MG and n = 4 for the other cell lines, (D) n = 3. Statistical significance was determined by 1-way ANOVA (A, B) followed by Bonferroni post hoc test, 2-way ANOVA (C), 1-way ANOVA of Pearson's coefficients (D). In the whole figure significance is represented as *P < 0.05, **P < 0.01, and ***P < 0.001 vs control or as indicated by straight lines. DLK1, delta-like noncanonical Notch ligand 1; HIF, hypoxia-inducible factor; HRE, hypoxia-responsive element; N, necrosis; V, vessel.
Supplier Page from Abcam for Recombinant Human DLK-1 protein