Journal: bioRxiv

Article Title: Cardiac neural crest lineage diversity and underlying gene regulatory networks revealed by multimodal analysis

doi: 10.1101/2022.06.23.497419

Figure Lengend Snippet: (a) Lineage tracing of Dlx1-positive cells in the craniofacial region of Dlx1-CreERT2;R26-EYFP mice treated with tamoxifen at E9.5. (b-g) Immunohistochemistry of EYFP ( Dlx1 -lineage) with co-staining for Sox9 (b), SM22α (c, d, g) or Dlk1 (e, f) at E11.5 (b-d), E12.5 (e), E14.5 (f) and E15.5 (g). Boxed areas are magnified in lower panels. Arrows indicate Dlx1 -lineage cells. Nuclei are stained with DAPI. Scale bars, 50 μm.

Article Snippet: Antibodies were below, SM22α (ab14106, Abcam, 1:800), GFP (04404-26, Nacalai Tesque, 1:1000), GFP (600-101-215, Rockland, 1:500), Collagen typeII (600-401-104-0.1, Rockland, 1:400), smooth muscle myosin heavy chain 11 (ab53219, Abcam, 1:200), Jagged 1 (AF599, R&D systems, 1:500), Dlk1 (AF1144, R&D systems, 1:500), Sox9 (A19710, ABclonal, 1:500) and Alexa Fluor (488, 555 and 647)-conjugated secondary antibodies (Abcam, 1:200).

Techniques: Immunohistochemistry, Staining

Journal: iScience

Article Title: The single-cell transcription reveals the aberrant differentiation trajectory of chondrocytes in the intervertebral disc for congenital scoliosis

doi: 10.1016/j.isci.2025.112608

Figure Lengend Snippet: Expressions of the specific genes and transcription factors in CS and CF (A) Immunohistochemistry (IHC) staining of DBP, CCN1, CCN2, and ECRG4 in the cartilage endplate (CEP) of CS, LS, and NC. (B) IHC score (IOD/area) of DBP, CCN1, CCN2, and ECRG4 in 24 CS, 7 LS, and 5 NC patients. (C) The correlation analysis between DBP and CCN1 in the IHC analysis (36 patients). (D) The correlation analysis between DBP and CCN2 in the IHC analysis (36 patients). (E) The correlation analysis between DBP and ECRG4 in the IHC analysis (36 patients). (F) IHC staining of DLK1, MATN1, and HOXC6 in the CEP of CF and normal fetus (NF). (G) IHC score (IOD/area) of DLK1, MATN1, and HOXC6 in 2 CF and 4 NF. (H) The correlation analysis between DBP and DLK1 or MATN1 in the IHC analysis (6 fetuses). (I) Immunofluorescence of the specific genes and transcription factors in CEP of CS and CF. The scale bars in panel A and F indicated 500 μm, and the scale bars in panel I indicated 50 μm. Kruskal-Wallis test was used to compare the data in CS, LS, and NC, followed by Dunn’s multiple comparison test in every two groups for post-hoc analysis. Wilcoxon signed-rank test was employed to compare the data between CF and NF. Spearman test was adopted to analyze the correlation between two indicators. ns, not significant; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. Abbreviations: CS, congenital scoliosis; CF, CS at the fetal stage.

Article Snippet: In each cycle of procedure, the IVD section underwent antigen retrieval and antigen blocking, and were incubated overnight at 4°C with the following primary antibodies including CCN1, CCN2, ECRG4, DBP, DLK1, HOXC6, NCAM1, SEMA5A, SOX9 (Cell Signaling Technology, 82630S), TBXT (Abcam, ab209665), COL10A1, COL1A1, TAGLN (Proteintech, 10493-1-AP), and ACTA2 (Cell Signaling Technology, 48938), and then reacted with the secondary antibodies for 1 hour at room temperature, and colored with a fluorescent dye at 3 different wavelengths (λ 520, λ 570, λ 650) for 15 min at room temperature.

Techniques: Immunohistochemistry, Immunofluorescence, Comparison

Journal: bioRxiv

Article Title: Parthenogenote-Derived Brain Unveils the Critical Role of Paternal Genome in Neural Development

doi: 10.64898/2026.01.06.697935

Figure Lengend Snippet: Notch hyperactivation with reduced Dlk1 in Pg-derived cells CReP brains skews fate toward gliogenesis. ( a ) Schematic of the Notch signaling pathway in neural stem cell fate determination. ( b ) Expression level of Notch1, Dll1, Dlk1 and Hes1 in GFP-positive sorted cells from CReF and CReP brains, as analyzed by RNA-sequencing. WILD-TYPE refers to fertilized embryo-derived cells from CReF embryos, and Pg refers to Pg embryo-derived cells in CReP embryos. Each dot represents an individual replicate. * P <0.05. ( c ) Representative immunofluorescence images of Notch intracellular domain (NICD) of brain cells cultured for five days. Scale bar, 100 μm. ( d ) Quantification of NICD-positive nuclei as a proportion of DAPI-positive cells in CReP and CReF cultures. Each dot represents an individual replicate. * P <0.05. ( e ) Representative immunofluorescence images of Tubulin beta III (TUBB3)-positive neurons in the brain cells cultured for five days. Scale bar, 100 μm. ( f ) Quantification of TUBB3-positive neurons as a proportion of DAPI-positive cells of CReP and CReF. Each dot represents an individual replicate. * P <0.05. ( g ) Representative immunofluorescence images of PDGFRα-positive oligodendrocyte precursor cells (OPCs) in the brain cells cultured for five days. Scale bar, 100 μm. ( h ) Quantification of PDGFRα-positive OPCs as a proportion of DAPI-positive cells of CReP and CReF. Each dot represents an individual replicate. * P <0.05. ( i ) Representative immunofluorescence images of TUBB3 in cultured CReP brain cells after 72 h of recombinant Dlk1 (rDlk1, 200 ng/mL) or PBS control treatment. Scale bar, 100 μm. ( j ) Quantification of TUBB3-positive neurons as a proportion of DAPI-positive cells in each condition. Each dot represents an individual replicate. * P <0.05. ( k ) Representative immunofluorescence images of PDGFRα in cultured CReP brain cells after 72 h of rDlk1 (200 ng/mL) or PBS control treatment. Scale bar, 100 μm. ( l ) Quantification of PDGFRα-positive OPCs as a proportion of DAPI-positive cells in each condition. Each dot represents an individual replicate. * P <0.05.

Article Snippet: For Dlk1 supplementation, brain cells were treated for 72 h with recombinant Dlk1 (R&D systems, 1144-PR-025/CF, 200ng/ml) or PBS, then fixed in 4% PFA for 10 min.

Techniques: Derivative Assay, Expressing, RNA Sequencing, Immunofluorescence, Cell Culture, Recombinant, Control

Journal: bioRxiv

Article Title: Parthenogenote-Derived Brain Unveils the Critical Role of Paternal Genome in Neural Development

doi: 10.64898/2026.01.06.697935

Figure Lengend Snippet: Schematic model: Dlk1 dosage constrains Notch signaling to maintain neural cell fate balance in the developing mouse brain.

Article Snippet: For Dlk1 supplementation, brain cells were treated for 72 h with recombinant Dlk1 (R&D systems, 1144-PR-025/CF, 200ng/ml) or PBS, then fixed in 4% PFA for 10 min.

Techniques:

Journal: Nature Communications

Article Title: Identification of the Notch ligand DLK1 as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma

doi: 10.1038/s41467-025-60649-w

Figure Lengend Snippet: A DLK1 mRNA expression across adult refractory metastatic cancers ( n = 948). Tumor types with high DLK1 expression are highlighted in the colors shown. The percentage of each tumor type with high DLK1 expression is shown on the right. B DLK1 mRNA expression in the TCGA PanCancer dataset. C Expression of Notch ligands from two independent ACC bulk RNA-seq datasets. D Quantification of DLK1 IHC staining among ACC NCI tumors (n = 38). Vertical bars with more than one tumor represent the mean H-score. E IHC images from four representative ACC NCI tumors with varying levels of DLK1 expression. Scale bars represent 200 μm. IHC immunohistochemistry. Source data are provided as a Source Data file.

Article Snippet: Then, attached cells were incubated with APC-conjugated DLK1 antibody (R&D Systems, a bio-techne brand, #FAB1144A) or isotype control antibody (R&D Systems, a bio-techne brand, #IC0041A) for 1 h in 2 ml of cell culture media at 37 °C.

Techniques: Expressing, RNA Sequencing, Immunohistochemistry

Journal: Nature Communications

Article Title: Identification of the Notch ligand DLK1 as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma

doi: 10.1038/s41467-025-60649-w

Figure Lengend Snippet: A Schematic structure of ADCT-701, a DLK1 targeting antibody drug conjugate. B Representative surface expression of DLK1 among ACC cell lines: CU-ACC1, CU-ACC2, and H295R. C DLK1 molecules/cell in ACC cell lines (n = 3 independent experiments). D ADCT-701 cytotoxicity among CU-ACC1, CU-ACC2, and H295R cells. Cells were treated with ADCT-701 and B12-PL1601 (non-targeted control ADC) for 7 days (data representative of n = 3 independent experiments). E Representative imaging flow cytometry images and signal intensity analysis ( n = 3 biological replicates) showing cellular internalization of DLK1 antibodies in CU-ACC1, CU-ACC2, and H295R cells. F Cytotoxic activity of ADCT-701 responsive ( n = 6) and ( G ) ADCT-701 non-responsive ( n = 6) ACC short-term patient-derived organoids (PDOs). Cells were treated with ADCT-701 and B12-PL1601 for 7 days. Data representative of n = 1 (ACC17, ACC44, ACC56, and ACC49) or n = 2 (ACC47, ACC51, ACC52, ACC40, ACC42, ACC48, and ACC54) independent experiments. Flow cytometry histograms assessing DLK1 among ADCT-701 H responsive and I non-responsive ACC PDOs. Error bars represent mean values ± S.E.M. Drug response curve data are presented as mean values ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Then, attached cells were incubated with APC-conjugated DLK1 antibody (R&D Systems, a bio-techne brand, #FAB1144A) or isotype control antibody (R&D Systems, a bio-techne brand, #IC0041A) for 1 h in 2 ml of cell culture media at 37 °C.

Techniques: Expressing, Control, Imaging, Flow Cytometry, Activity Assay, Derivative Assay

Journal: Nature Communications

Article Title: Identification of the Notch ligand DLK1 as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma

doi: 10.1038/s41467-025-60649-w

Figure Lengend Snippet: A CU-ACC1 and H295R xenograft tumor growth curves after treatment with saline (CU-ACC1: n = 3; H295R: n = 4), B12-PL1601 (CU-ACC1: n = 3; H295R: n = 4), or ADCT-701 (CU-ACC1: n = 3; H295R: n = 3) (1 mg/kg) (initial tumor size reached an average of 100–150 mm 3 ). Additional doses of ADCT-701 indicated by arrows. Error bars represent mean values ± S.E.M . B ACC PDXs 164165, 592788, and POBNCI_ACC004 tumor growth curves after treatment with saline (164165: n = 8; 592788: n = 7; POBNCI_ACC004: n = 5), B12-PL1601 (164165: n = 7; 592788: n = 7; POBNCI_ACC004: n = 5) or ADCT-701 (164165: n = 8; 592788: n = 6; POBNCI_ACC004: n = 5) (1 mg/kg) (initial tumor size reached an average of 100–200 mm 3 ). Each line shown represents an individual mouse within a given experiment. The X symbol indicates the administration of ADCT-701 re-dosing. Arrow indicates unexpected death of 1 POBNCI_ACC004 tumor-bearing mouse prior to endpoint. DLK1 immunohistochemistry with H-scores shown above each individual xenograft or PDX tumor. PR partial response, CR complete response. Scale bars represent 200 μm. Source data are provided as a Source Data file.

Article Snippet: Then, attached cells were incubated with APC-conjugated DLK1 antibody (R&D Systems, a bio-techne brand, #FAB1144A) or isotype control antibody (R&D Systems, a bio-techne brand, #IC0041A) for 1 h in 2 ml of cell culture media at 37 °C.

Techniques: Saline, Immunohistochemistry

Journal: Nature Communications

Article Title: Identification of the Notch ligand DLK1 as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma

doi: 10.1038/s41467-025-60649-w

Figure Lengend Snippet: A Cytotoxicity of SG3199 among ADCT-701 responsive (NCI-ACC44, NCI-ACC51, and NCI-ACC56) and non-responsive (NCI-ACC40, NCI-ACC48, and NCI-ACC54) DLK1 + ACC short-term patient-derived organoids (PDOs). Cells were treated with SG3199 for 3 days ( n = 3 independent experiments for ACC44, ACC51, ACC56, and ACC48 PDOs; n = 5 independent experiments for ACC40 and ACC54 PDOs). B Drug transporter mRNA expression in DLK1 + ADCT-701 responder and non-responder PDOs as measured by quantitative RT-PCR. Results were presented as Δcycle threshold (ΔCt). Each color represents an individual PDO. R responder, NR non-responder ( n = 3 independent experiments). Unpaired t tests were used to calculate two-tailed p-values. C Flow cytometry histograms assessing ABCB1 and the number of ABCB1 molecules per cell of DLK1 + ADCT-701 responsive (NCI-ACC44, NCI-ACC51, and NCI-ACC56) and non-responsive (NCI-ACC40, NCI-ACC48, and NCI-ACC54) ACC PDOs. D ADCT-701 cytotoxicity in the NCI-ACC40 and NCI-ACC48 PDOs with and without treatment with ABCB1 inhibitors (1 μM valspodar, 10 μM elacridar and 1 μM tariquidar). Cells were treated with ADCT-701 combined with or without ABCB1 inhibitors for 7 days (data representative of n = 3 independent experiments). E Flow cytometry histograms assessing ABCB1 among 164165, 592788 and POBNCI_ACC004 PDXs (data representative of n = 2 independent experiments). F ADCT-701 cytotoxicity in 164165, 592788 and POBNCI_ACC004 PDX-derived organoids. Cells were treated with ADCT-701 for 7 days (data representative of n = 3 independent experiments). G ADCT-701 cytotoxicity in the 164165 and 592788 PDX-derived organoids treated with or without ABCB1 inhibitors (1 μM valspodar, 10 μM elacridar and 1 μM tariquidar). Cells were treated with ADCT-701 combined with or without ABCB1 inhibitors for 7 days (data representative of n = 3 independent experiments). H Volcano plot of differentially expressed genes in control tumors ( n = 4) versus post-ADCT-701 acquired resistant tumors ( n = 3) in POBNCI_ACC004 PDX. Wald test negative log 10 p-values are shown on the y -axis. I Flow cytometry histograms assessing ABCB1 among ADCT-701 resistant and control POBNCI_ACC004 PDX tumors. J ADCT-701 cytotoxicity in the ADCT-701 resistant POBNCI_ACC004 PDX-derived organoid treated with or without ABCB1 inhibitors (1 μM valspodar, 10 μM elacridar and 1 μM tariquidar). Cells were treated with ADCT-701 combined with or without ABCB1 inhibitors for 7 days (data representative of n = 3 independent experiments). Error bars represent mean values ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Then, attached cells were incubated with APC-conjugated DLK1 antibody (R&D Systems, a bio-techne brand, #FAB1144A) or isotype control antibody (R&D Systems, a bio-techne brand, #IC0041A) for 1 h in 2 ml of cell culture media at 37 °C.

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Two Tailed Test, Flow Cytometry, Control

Journal: Nature Communications

Article Title: Identification of the Notch ligand DLK1 as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma

doi: 10.1038/s41467-025-60649-w

Figure Lengend Snippet: A Cell surface DLK1 expression by flow cytometry in 3 small cell lung cancer (SCLC) cell lines (H524, H146, and H1436) (data representative of n = 3 independent experiments). B DLK1 molecules/cell relative to DLL3 among SCLC cell lines ( n = 3 independent experiments). Error bars represent mean values ± S.E.M . C Flow cytometry histograms assessing ABCB1 in SCLC cell lines (data representative of n = 3 independent experiments). D SG3199 cytotoxicity in SCLC cell lines. Cells were treated with SG3199 for 3 days (data representative of n = 4 independent experiments). E ADCT-701 cytotoxicity among SCLC cell lines. Cells were treated with ADCT-701 or B12-PL1601 for 7 days (data representative of n = 4 independent experiments). F Tumor growth curves of SCLC xenograft models after treatment (1 mg/kg) with saline (H524: n = 5; H146: n = 5; H1436: n = 6), B12-PL1601 (H524: n = 5; H146: n = 4; H1436: n = 7) or ADCT-701 (H524: n = 4; H146: n = 6; H1436: n = 7) when tumor size reached an average of 100–150 mm 3 . Arrows indicate re-treatment with ADCT-701. Scale bars represent 200 μm. Error bars represent mean values ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: Then, attached cells were incubated with APC-conjugated DLK1 antibody (R&D Systems, a bio-techne brand, #FAB1144A) or isotype control antibody (R&D Systems, a bio-techne brand, #IC0041A) for 1 h in 2 ml of cell culture media at 37 °C.

Techniques: Expressing, Flow Cytometry, Saline

Journal: Nature Communications

Article Title: Identification of the Notch ligand DLK1 as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma

doi: 10.1038/s41467-025-60649-w

Figure Lengend Snippet: A Immunoblot analysis of DLK1 and loading control (α-tubulin) proteins in CU-ACC1 cells with and without DLK1 KO. Four single-cell KO clones are shown. B Immunoblot analysis of NOTCH1 signaling, total NOTCH1 and NOTCH1 intracellular domain (ICD), NE marker synaptophysin (SYP), and loading control (α-tubulin) proteins with and without DLK1 KO in CU-ACC1 cells. Two single-cell KO clones are shown. C Correlation between NOTCH1 and DLK1 expression among TCGA ACC tumors. Pearson correlation coefficients with two-tailed p-values are shown. D DLK1 and NOTCH1 expression in TCGA ACC tumors and normal adrenals. Unpaired t tests were used to calculate two-tailed p -values. Error bar represents mean values ± 95% C.I. E SG3199 cytotoxicity in CU-ACC1 parental and DLK1 KO clones. Cells were treated with SG3199 for 3 days (data representative of n = 4 independent experiments). Error bars represent mean values ± S.E.M. F Flow cytometry histograms assessing ABCB1 in CU-ACC1 cells with and without DLK1 KO. G Immunoblot analysis of DLK1, total NOTCH1 and NOTCH1-ICD, SYP, and α-tubulin proteins in DLK1 + NCI-ACC40, DLK1 + NCI-ACC48 and DLK1 - ACC49 PDOs. H Flow cytometry histograms assessing ABCB1 in DLK1 negative NCI-ACC49 PDOs. I Immunoblot analysis of total NOTCH1 (to detect the NOTCH1-ICD plasmid expression), SYP, and α-tubulin proteins in CU-ACC1 cells with and without NOTCH1-ICD overexpression. J Flow cytometry histograms assessing ABCB1 in CU-ACC1 cells with and without N1ICD overexpression. K Correlation between NOTCH1 and ABCB1 expression among TCGA ACC tumors and ( L ) among GTEx normal adrenal tissues. Pearson correlation coefficients with two-tailed p-values are shown. M Single cell RNA-seq data of ABCB1 expression comparing high NOTCH1 to low NOTCH1 expressing cells from 18 ACC metastatic tumors. Unpaired t tests were used to calculate two-tailed p -values. N Model summarizing the findings of the current study. For immunoblots, experiments were performed two times with similar results. Source data are provided as a Source Data file.

Article Snippet: Then, attached cells were incubated with APC-conjugated DLK1 antibody (R&D Systems, a bio-techne brand, #FAB1144A) or isotype control antibody (R&D Systems, a bio-techne brand, #IC0041A) for 1 h in 2 ml of cell culture media at 37 °C.

Techniques: Western Blot, Control, Clone Assay, Marker, Expressing, Two Tailed Test, Flow Cytometry, Plasmid Preparation, Over Expression, RNA Sequencing

Journal: Oncotarget

Article Title: Imprinting defects at human 14q32 locus alters gene expression and is associated with the pathobiology of osteosarcoma.

doi: 10.18632/oncotarget.6965

Figure Lengend Snippet: Figure 3: Downregulation of genes by imprinting defects at the 14q32-locus. (A) 14q index (14q-I) of four normal bone tissue samples, six 14q-I(−) osteosarcoma and eight 14q-I(+) osteosarcoma samples. (B) mRNA levels of six imprinted genes by qRT-PCR with GAPDH used as control. The right column indicates the average and standard error of the imprinted gene expression levels in 3 different groups, including normal bone tissues, 14q-I(−) osteosarcoma and 14q-I(+) osteosarcoma groups, respectively. *P < 0.05. (C) Relative expression levels of representative DLK1-DIO3 cluster mRNAs and 14q32 miRNAs from an independent cohort of 43 osteosarcoma patient samples.

Article Snippet: Human DLK1 overexpression plasmid was obtained from Origene.

Techniques: Quantitative RT-PCR, Control, Gene Expression, Expressing

Journal: Oncotarget

Article Title: Imprinting defects at human 14q32 locus alters gene expression and is associated with the pathobiology of osteosarcoma.

doi: 10.18632/oncotarget.6965

Figure Lengend Snippet: Figure 4: Histone modifications around imprinted genes and their potential tumor suppressor functions at the 14q32- locus. (A) Enrichment of H3, H3K4-me3, H3K27-me3, H3K9-me2 and H3K9-me3 at the promoter regions of imprinted genes in normal bone tissues. Input was used as internal control. Up-5kb: site ~5 kb upstream of the promoter of DLK1; SNO: small non coding RNA region; CIT: miRNA cluster 3 region. (B) H3K4-me3, H3K27-me3, H3K9-me2 and H3K9-me3 enrichment normal bone tissues, 14q-I(−) and 14q-I(+) osteosarcoma samples. H3 was used as internal control. *P < 0.05. (C) DNA methylation levels of the ChIP samples pulled down by H3, H3K4-me3, and H3K27-me3 antibodies in normal bone tissues, 14q-I(−) osteosarcoma and 14q-I(+) osteosarcoma samples. Input sample was used as control. (D) Significant decrease of cell viability by overexpression of DIO3, DLK1 and RTL1 in SaOS2 cells. Paired t-test was performed. *P < 0.05. (E) Increased apoptosis by overexpression of DIO3, DLK1 and RTL1 in SaOS2 cells. pCDNA3.1 empty vector was used as control. Sham represents SaOS2 cells without plasmid transfection. Results represent the average of three independent transfections. *P < 0.05.

Article Snippet: Human DLK1 overexpression plasmid was obtained from Origene.

Techniques: Control, DNA Methylation Assay, Over Expression, Plasmid Preparation, Transfection

Journal: Cell reports

Article Title: Tcf21 marks visceral adipose mesenchymal progenitors and functions as a rate-limiting factor during visceral adipose tissue development

doi: 10.1016/j.celrep.2023.112166

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: APC Rabbit anti-mouse Dlk1 antibody , R & D Systems , Cat# FAB8634A, RRID:AB_2890004.

Techniques: Virus, Recombinant, Plasmid Preparation, SYBR Green Assay, Electron Microscopy, cDNA Synthesis, Flow Cytometry, Multiplex Assay, Single Cell, Sequencing, Isolation, shRNA, Software