Journal: Frontiers in Endocrinology

Article Title: Differential Morphological Diagnosis of Various Forms of Congenital Hyperinsulinism in Children

doi: 10.3389/fendo.2021.710947

Figure Lengend Snippet: Histological and immunohistochemical examination of pancreatic tissue in patients with congenital hyperinsulinism and in controls. Hematoxylin and eosin staining, insulin, NeuroD1 and Nkx2.2, Isl1, chromogranin A, somatostatin and DR1, DR2, DR5, SSTR2 and SSTR5; x50. Note: HEO – hematoxylin and eosin, Somat – somatostatin, ChrA – chromogranin A.

Article Snippet: Immunohistochemical analysis of pancreatic samples from 35 patients with CHI and 10 patients from the control group was carried out according to a standard method ( ) using antibodies to: chromogranin A (rabbit polyclonal antibody, dilution 1:400, Diagnostic BioSystems, The Netherlands); insulin (mouse monoclonal antibody, clone K36aC10, 1:50 dilution, Diagnostic BioSystems, The Netherlands); Isl1 (rabbit polyclonal antibody, 1:1000 dilution, ThermoFisher, USA); Nkx 2.2 (rabbit monoclonal antibody, clone EPR14638, dilution 1:20, Abcam, UK); somatostatin (SST, rabbit polyclonal antibody, dilution 1:1000, Dako, Denmark); NeuroD1 (mouse monoclonal antibody, clone 3H8, dilution 1:1000, Novus Biologicals, USA); somatostatin receptor type 2 (SSTR2, rabbit monoclonal antibody, clone EP149, dilution 1:200, Abcam, UK); somatostatin receptor type 5 (SSTR5, rabbit monoclonal antibody, clone UMB4, dilution 1:100, Abcam, UK); type 1 dopamine receptor (DR1, rabbit polyclonal antibody, dilution 1:50, Novus Biologicals, USA); type 2 dopamine receptor (DR2, mouse monoclonal antibody, clone B10, dilution 1:100, Santa Cruz Biotechnology, USA); and type 5 dopamine receptor (DR5, rabbit polyclonal antibody, 1:100 dilution, ThermoFisher, USA).

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Endocrinology

Article Title: Differential Morphological Diagnosis of Various Forms of Congenital Hyperinsulinism in Children

doi: 10.3389/fendo.2021.710947

Figure Lengend Snippet: Pancreas of a patient with a focal form of congenital hyperinsulinism. (A) – NeuroD1, (B) – insulin; x50.

Article Snippet: Immunohistochemical analysis of pancreatic samples from 35 patients with CHI and 10 patients from the control group was carried out according to a standard method ( ) using antibodies to: chromogranin A (rabbit polyclonal antibody, dilution 1:400, Diagnostic BioSystems, The Netherlands); insulin (mouse monoclonal antibody, clone K36aC10, 1:50 dilution, Diagnostic BioSystems, The Netherlands); Isl1 (rabbit polyclonal antibody, 1:1000 dilution, ThermoFisher, USA); Nkx 2.2 (rabbit monoclonal antibody, clone EPR14638, dilution 1:20, Abcam, UK); somatostatin (SST, rabbit polyclonal antibody, dilution 1:1000, Dako, Denmark); NeuroD1 (mouse monoclonal antibody, clone 3H8, dilution 1:1000, Novus Biologicals, USA); somatostatin receptor type 2 (SSTR2, rabbit monoclonal antibody, clone EP149, dilution 1:200, Abcam, UK); somatostatin receptor type 5 (SSTR5, rabbit monoclonal antibody, clone UMB4, dilution 1:100, Abcam, UK); type 1 dopamine receptor (DR1, rabbit polyclonal antibody, dilution 1:50, Novus Biologicals, USA); type 2 dopamine receptor (DR2, mouse monoclonal antibody, clone B10, dilution 1:100, Santa Cruz Biotechnology, USA); and type 5 dopamine receptor (DR5, rabbit polyclonal antibody, 1:100 dilution, ThermoFisher, USA).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Differential Morphological Diagnosis of Various Forms of Congenital Hyperinsulinism in Children

doi: 10.3389/fendo.2021.710947

Figure Lengend Snippet: The proportion of exocrinocytes expressing NeuroD1 in the field of view in various forms of congenital hyperinsulinism. Data are presented as M ± SD. (*) - the difference compared to the control is statistically significant at p <0.01.

Article Snippet: Immunohistochemical analysis of pancreatic samples from 35 patients with CHI and 10 patients from the control group was carried out according to a standard method ( ) using antibodies to: chromogranin A (rabbit polyclonal antibody, dilution 1:400, Diagnostic BioSystems, The Netherlands); insulin (mouse monoclonal antibody, clone K36aC10, 1:50 dilution, Diagnostic BioSystems, The Netherlands); Isl1 (rabbit polyclonal antibody, 1:1000 dilution, ThermoFisher, USA); Nkx 2.2 (rabbit monoclonal antibody, clone EPR14638, dilution 1:20, Abcam, UK); somatostatin (SST, rabbit polyclonal antibody, dilution 1:1000, Dako, Denmark); NeuroD1 (mouse monoclonal antibody, clone 3H8, dilution 1:1000, Novus Biologicals, USA); somatostatin receptor type 2 (SSTR2, rabbit monoclonal antibody, clone EP149, dilution 1:200, Abcam, UK); somatostatin receptor type 5 (SSTR5, rabbit monoclonal antibody, clone UMB4, dilution 1:100, Abcam, UK); type 1 dopamine receptor (DR1, rabbit polyclonal antibody, dilution 1:50, Novus Biologicals, USA); type 2 dopamine receptor (DR2, mouse monoclonal antibody, clone B10, dilution 1:100, Santa Cruz Biotechnology, USA); and type 5 dopamine receptor (DR5, rabbit polyclonal antibody, 1:100 dilution, ThermoFisher, USA).

Techniques: Expressing, Control

Journal: Frontiers in Endocrinology

Article Title: Differential Morphological Diagnosis of Various Forms of Congenital Hyperinsulinism in Children

doi: 10.3389/fendo.2021.710947

Figure Lengend Snippet: Confocal laser scanning microscopy of the pancreas in congenital hyperinsulinism. (A–D) focal form, (E, F) diffuse form. (A, F) blue fluorescence of cell nuclei (DAPI). (B, F) green fluorescence of NeuroD1 in nuclei. (C, G) red fluorescence of ChrA in cytoplasm. (D, H) greenish-blue/turquoise fluorescence of NeuroD1 and red fluorescence of ChrA in the same cells; x400.

Article Snippet: Immunohistochemical analysis of pancreatic samples from 35 patients with CHI and 10 patients from the control group was carried out according to a standard method ( ) using antibodies to: chromogranin A (rabbit polyclonal antibody, dilution 1:400, Diagnostic BioSystems, The Netherlands); insulin (mouse monoclonal antibody, clone K36aC10, 1:50 dilution, Diagnostic BioSystems, The Netherlands); Isl1 (rabbit polyclonal antibody, 1:1000 dilution, ThermoFisher, USA); Nkx 2.2 (rabbit monoclonal antibody, clone EPR14638, dilution 1:20, Abcam, UK); somatostatin (SST, rabbit polyclonal antibody, dilution 1:1000, Dako, Denmark); NeuroD1 (mouse monoclonal antibody, clone 3H8, dilution 1:1000, Novus Biologicals, USA); somatostatin receptor type 2 (SSTR2, rabbit monoclonal antibody, clone EP149, dilution 1:200, Abcam, UK); somatostatin receptor type 5 (SSTR5, rabbit monoclonal antibody, clone UMB4, dilution 1:100, Abcam, UK); type 1 dopamine receptor (DR1, rabbit polyclonal antibody, dilution 1:50, Novus Biologicals, USA); type 2 dopamine receptor (DR2, mouse monoclonal antibody, clone B10, dilution 1:100, Santa Cruz Biotechnology, USA); and type 5 dopamine receptor (DR5, rabbit polyclonal antibody, 1:100 dilution, ThermoFisher, USA).

Techniques: Confocal Laser Scanning Microscopy, Fluorescence

Journal: Frontiers in Endocrinology

Article Title: Differential Morphological Diagnosis of Various Forms of Congenital Hyperinsulinism in Children

doi: 10.3389/fendo.2021.710947

Figure Lengend Snippet: Confocal laser scanning microscopy of the pancreas in congenital hyperinsulinism. (A–D) focal form, (E, F) diffuse form. (A, F) blue fluorescence of cell nuclei (DAPI). (B, F) green fluorescence of NeuroD1 in nuclei. (C, G) red fluorescence of DR2 in cytoplasm. (D, H) greenish-blue/turquoise fluorescence of NeuroD1 and red fluorescence of DR2 in the same cells; x400.

Article Snippet: Immunohistochemical analysis of pancreatic samples from 35 patients with CHI and 10 patients from the control group was carried out according to a standard method ( ) using antibodies to: chromogranin A (rabbit polyclonal antibody, dilution 1:400, Diagnostic BioSystems, The Netherlands); insulin (mouse monoclonal antibody, clone K36aC10, 1:50 dilution, Diagnostic BioSystems, The Netherlands); Isl1 (rabbit polyclonal antibody, 1:1000 dilution, ThermoFisher, USA); Nkx 2.2 (rabbit monoclonal antibody, clone EPR14638, dilution 1:20, Abcam, UK); somatostatin (SST, rabbit polyclonal antibody, dilution 1:1000, Dako, Denmark); NeuroD1 (mouse monoclonal antibody, clone 3H8, dilution 1:1000, Novus Biologicals, USA); somatostatin receptor type 2 (SSTR2, rabbit monoclonal antibody, clone EP149, dilution 1:200, Abcam, UK); somatostatin receptor type 5 (SSTR5, rabbit monoclonal antibody, clone UMB4, dilution 1:100, Abcam, UK); type 1 dopamine receptor (DR1, rabbit polyclonal antibody, dilution 1:50, Novus Biologicals, USA); type 2 dopamine receptor (DR2, mouse monoclonal antibody, clone B10, dilution 1:100, Santa Cruz Biotechnology, USA); and type 5 dopamine receptor (DR5, rabbit polyclonal antibody, 1:100 dilution, ThermoFisher, USA).

Techniques: Confocal Laser Scanning Microscopy, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Contribution of a mitochondrial tyrosyl-tRNA synthetase mutation to the phenotypic expression of the deafness-associated tRNASer(UCN) 7511A>G mutation

doi: 10.1074/jbc.ra119.010598

Figure Lengend Snippet: Figure 4. Western blot analysis of mitochondrial proteins. A, 5 g of total mitochondrial proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with antibodies specific for ND1, ND4, ND5, ND6, CO1, CYTB, and ATP6 and with VDAC as a loading control, respectively. B, quantification of mitochondrial proteins. The levels of mitochondrial proteins in mutant and control cell lines were determined as described elsewhere (17, 32). The average of three determinations for each cell line is shown. Graph details and symbols are explained in the legend to Fig. 2.

Article Snippet: The antibodies used for this investigation were from Abcam (GAPDH (ab8245), ND1 (ab74257), ND5 (ab92624), ND6 (ab81212), CO1 (ab14705), ATP6 (ab101908), NDUFS3 (ab14711), and total OXPHOS human WB antibody mixture (ab110411)), Novus (ND4 (NBP2-47365)), and Proteintech (VDAC (10866-1-AP), CYTB (55090-1-AP), and COX10 (10611-2-AP)).

Techniques: Western Blot, Control, Mutagenesis

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: NeuroD Expression in Podocytes and Interrelationships with Nephrin at Both Nuclear and Cytoplasmic Sites.

doi: 10.1159/000488818

Figure Lengend Snippet: Fig. 9. NeuroD is associated to nephrin in the cytoplasm of podocytes. The role of NeuroD in the cytoplasm was inves tigated analysing the interactions with some proteins interacting with cytoskel eton. NeuroD binding to nephrin was de tected by WB on podocytes nephrin IPs samples and on total podocytes lysates, used as control, of untreated or NeuroD overexpressed podocytes (A). Thereafter NeuroD and nephrin colocalization was assessed by IF (B) (Scale bar 50μm).On the contrary examination by PLA demon strates that the linking between NeuroD and slit diaphragm protein podocin (C) as well as the association with the interme diate filament nestin (D), is absent.

Article Snippet: The following primary antibodies were used for the study: goat anti-neurod (sc1086) (Santa Cruz), goat antineurod (sc1084) (Santa Cruz), rabbit anti neurod (ab16508) (Abcam), Rabbit anti neurod (Ser 274) phospho specific (TA312014) (Origene, Bologna, Italy) guinea pig anti-nephrin (GP-N2) (Progen), rabbit anti-alphaactinin (sc15335) (Santa Cruz), rabbit anti-cofilin (5175) (Cell Signaling, Euro Clone, Milano, Italy), rabbit anti-histone H3 (ab1791) (Abcam), mouse anti-alpha-tubulin (ab7291) (Abcam), followed by proper HRP conjugated secondary antibody: anti mouse-rabbit-HRP conjugated (by BM Chemiluminescence Western Blotting kit, Roche), anti guinea pig-HRP (ab6771) (Abcam), anti goat-HRP (ab6741) (Abcam).

Techniques: Binding Assay, Control

Journal: Computational and Structural Biotechnology Journal

Article Title: Exploration of gastric neuroendocrine carcinoma (GNEC) specific signaling pathways involved in chemoresistance via transcriptome and in vitro analysis

doi: 10.1016/j.csbj.2020.09.016

Figure Lengend Snippet: Evaluation of the prognostic and chemoresisitance roles of NeuroD1 in GNEC. (A) NeuroD1immunohistochemical verification in MANECs. Compared to GAC part, GNEC part contained high expression level. The top image with 20X magnification is the amplification of the red outlined part in bottom image. (B) Criteria for the degree of gene expression in immunohistochemical experiment. (C) Comparison of overall survival curves for NeuroD1 low and high expression group in GNEC patients. The expression of NeuroD1 was not correlated with overall survival in the GEPIA cohort (D) and the Kaplan–Meier plotter cohort (E). (F and G) The knockdown effect of NeuoD1 in GNEC cell lines. (H and I) Knock down NeuroD1 promoted chemosensitivity of GNEC cell lines to irinotecan (cell viability test), data are presented as mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit anti-human NeuroD1 antibody was purchased from Proteintech.

Techniques: Expressing, Amplification, Gene Expression, Immunohistochemical staining, Comparison, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Hepatitis B X-interacting protein promotes the formation of the insulin gene–transcribing protein complex Pdx-1/Neurod1 in animal pancreatic β-cells

doi: 10.1074/jbc.M117.809582

Figure Lengend Snippet: Hbxip forms a complex with Pdx-1 and Neurod1 in the insulin mini-enhancer in pancreatic β-cells. A, localization of both GFP-Hbxip and Pdx-1 was observed by confocal microscopy in the INS-1 cells. Scale bar, 25 μm for immunofluorescence. B, interaction between Hbxip and Pdx-1 was detected by co-IP assays in INS-1 cells in vivo. C, direct interaction between recombinant GST-Hbxip and His-Pdx-1 proteins was detected by GST pulldown assays followed by Western blot analysis (WB) in vitro. D and E, Pdx-1 is divided into three fragments. GST pulldown assays were performed with GST or GST fusion proteins and His tag fusion proteins containing the indicated amino acid residues of Pdx-1. The proteins were purified from bacteria by glutathione-Sepharose beads. F, INS-1 cells were transfected with Pdx-1 siRNA. ChIP assays were conducted 24 h after transfection. G, schematic showing the Ins mini-enhancer regions with Pdx-1-binding sites or negative control regions without Pdx-1-binding sites. H, interaction between Hbxip, Pdx-1, and Neurod1 was detected by co-IP assays in INS-1 cells in vivo. I, interaction of Pdx-1 with Neurod1 was detected by co-IP assays in INS-1 cells transfected with si-Control or si-Hbxip. J, interaction between Hbxip, Pdx-1, and Neurod1 was detected by co-IP assays in the primary β-cells isolated from floxed Hbxip mice. K, interaction of Pdx-1 with Neurod1 was detected by co-IP assays in the primary β-cells isolated from floxed Hbxip mice transduced with adeno-LacZ or adeno-Cre. L, interaction of Hbxip with Neurod1 was detected by co-IP assays in INS-1 cells transfected with si-Control or si-Pdx-1. M, Venn diagram shows the overlapping target genes of HBXIP and Pdx-1. N, left, real-time PCR analysis was performed to measure 10 of the 149 candidate target genes in pancreas islets of Hbxip-deficient mice and Ins2-cre (wildtype) mice; right, functional assignments for candidate target genes were verified in real-time PCR analysis. Mean ± S.D., n = 5/group. Each experiment was repeated at least three times.

Article Snippet: For Western blot analysis, the following were used: Pdx-1, primary antibody is rabbit anti-Pdx-1 (Proteintech Group, 20989-1-AP); secondary antibody is IPKine HRP mouse anti-rabbit IgG light chain (Abbkine, A25022); Neurod1, primary antibody is mouse anti-Neurod1 (Wuhan Boster Biological Technology Ltd., BM3336); secondary antibody is IPKine HRP goat anti-mouse IgG light chain (Abbkine, A25012); Hbxip, primary antibody is rabbit anti-Hbxip (Santa Cruz Biotechnology, Santa Cruz, CA, sc-373980); and secondary antibody is IPKine HRP goat anti-rabbit IgG heavy chain (Abbkine, {"type":"entrez-nucleotide","attrs":{"text":"A25222","term_id":"904602","term_text":"A25222"}} A25222 ).

Techniques: Confocal Microscopy, Immunofluorescence, Co-Immunoprecipitation Assay, In Vivo, Recombinant, Western Blot, In Vitro, Purification, Bacteria, Transfection, Binding Assay, Negative Control, Control, Isolation, Transduction, Real-time Polymerase Chain Reaction, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Hepatitis B X-interacting protein promotes the formation of the insulin gene–transcribing protein complex Pdx-1/Neurod1 in animal pancreatic β-cells

doi: 10.1074/jbc.M117.809582

Figure Lengend Snippet: Hbxip increases the insulin expression in pancreatic β-cells through activating Pdx-1 and Neurod1. A–C, insulin levels were examined by insulin assay kit in the culture supernatant of INS-1 cells transfected with relative plasmids and siRNAs. D, interference efficiency of Neurod1 siRNA was validated by Western blot analysis in INS-1 cells. The protein levels of Neurod1 from three independent experiments were quantified using ImageJ software as shown. Mean ± S.D. Each experiment was repeated at least three times. Student's t test; *, p < 0.05; **, p < 0.01.

Article Snippet: For Western blot analysis, the following were used: Pdx-1, primary antibody is rabbit anti-Pdx-1 (Proteintech Group, 20989-1-AP); secondary antibody is IPKine HRP mouse anti-rabbit IgG light chain (Abbkine, A25022); Neurod1, primary antibody is mouse anti-Neurod1 (Wuhan Boster Biological Technology Ltd., BM3336); secondary antibody is IPKine HRP goat anti-mouse IgG light chain (Abbkine, A25012); Hbxip, primary antibody is rabbit anti-Hbxip (Santa Cruz Biotechnology, Santa Cruz, CA, sc-373980); and secondary antibody is IPKine HRP goat anti-rabbit IgG heavy chain (Abbkine, {"type":"entrez-nucleotide","attrs":{"text":"A25222","term_id":"904602","term_text":"A25222"}} A25222 ).

Techniques: Expressing, Transfection, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: Hepatitis B X-interacting protein promotes the formation of the insulin gene–transcribing protein complex Pdx-1/Neurod1 in animal pancreatic β-cells

doi: 10.1074/jbc.M117.809582

Figure Lengend Snippet: Model shows that Hbxip contributes to the modulation of insulin transcription. Hbxip acting as a co-activator promotes insulin transcription through binding to the mini-enhancer of insulin and activating transcription factor Pdx-1. Hbxip co-activates Pdx-1 through recruiting Neurod1 to Pdx-1 forming a transcription factor complex of Hbxip/Pdx-1/Neurod1. Thus, Hbxip increases the insulin expression by increasing levels of the Pdx-1/Neurod1 complex in animal pancreatic β-cells.

Article Snippet: For Western blot analysis, the following were used: Pdx-1, primary antibody is rabbit anti-Pdx-1 (Proteintech Group, 20989-1-AP); secondary antibody is IPKine HRP mouse anti-rabbit IgG light chain (Abbkine, A25022); Neurod1, primary antibody is mouse anti-Neurod1 (Wuhan Boster Biological Technology Ltd., BM3336); secondary antibody is IPKine HRP goat anti-mouse IgG light chain (Abbkine, A25012); Hbxip, primary antibody is rabbit anti-Hbxip (Santa Cruz Biotechnology, Santa Cruz, CA, sc-373980); and secondary antibody is IPKine HRP goat anti-rabbit IgG heavy chain (Abbkine, {"type":"entrez-nucleotide","attrs":{"text":"A25222","term_id":"904602","term_text":"A25222"}} A25222 ).

Techniques: Binding Assay, Expressing

Journal: bioRxiv

Article Title: BET Bromodomain Inhibitors Target the NEUROD1-subtype SCLC by Blocking NEUROD1 Transactivation

doi: 10.1101/2021.10.25.465771

Figure Lengend Snippet: A) Western blots showing the loss of NEUROD1 expression in three H446 NEUROD1-KO clones (gRNA #297) versus five control clones and parental H446 cells. B) The aligned Sanger sequencing results showing the indels at the targeted NEUROD1 genomic sequence (complementary strand) in three H446 NEUROD1-KO clones. The protospacer adjacent motif (PAM) sequence was underlined, and the indel sequences were highlighted red. C) A substantial decrease of NEUROD2 expression in the H446 NEUROD1-KO clones, as measured by qRT-PCR. D) Distinct appearance of three H446 NEUROD1-KO clones compared to control cell clones. Scale bar: 100µm. E) Comparison of cell proliferation between the H446 NEUROD1-KO clones and control cell clones. F) Impaired xenograft formation of the H446 NEUROD1-KO cells (#297 cl.15 and cl.12) compared to control cells (Ctr gRNA cl.10). n=10 per clone. Top, three weeks after inoculation. Bottom, eight weeks after injection. G) GSEA plots of neuroendocrine signature, epithelial to mesenchymal transition (EMT) signature, and YAP1 conserved gene signature comparing H446 NEUROD1-KO cells (pooled data of #297 cl. 12, cl. 15, and cl. 2-1) to control cells (pooled data of cl. 3, cl. 10, and cl. 13). H) Relative changes in the expression of select genes (log 10 [Fold Change]), as measured by qRT-PCR, comparing H446 NEUROD1-KO (#297 cl. 15 or #298 cl. S3) to control cells (cl. 10). I) Western blots showing YAP1 protein expression in the H446 NEUROD1-KO cells (#297 cl. 15 and #298 cl. S3). J) Western blots showing knockdown of YAP1 protein in the H446 NEUROD1-KO cells (#297 cl. 12) using two different siRNAs. K) Decreased cell proliferation after knocking down YAP1 in H446 NEUROD1-KO cells (#297 cl. 12). The significance of two-group comparisons was determined using the Student’s t -test in (C), (E), and (H) and the ANOVA test with multi-comparison correction in (K). When indicated, ****, P < 0.0001. Error bars represent SD. Ctr, control; gRNA, guide RNA; ND1, NEUROD1; cl., clone; NES, normalized enrichment score; siRNA, small interfering RNA.

Article Snippet: After blocking the membranes with TBST + 5% non-fat dried milk, membranes were incubated with primary antibodies, NEUROD1 (BD Biosciences, #563000, 1:2,000), YAP1 (Santa Cruz Biotechnology, #sc-101199, 1:200), BRD2 (Santa Cruz Biotechnology, #sc-393720, 1:200), BRD3 (Santa Cruz Biotechnology, #sc-81202, 1:200), BRD4 (Novus, NBP2-52959, 1:1,000), LSAMP (Abcam, #ab89719, 1:1,000), NaK-ATPase (Santa Cruz Biotechnology, #sc-71638, 1:1,000), cleaved caspase 3 (Cell Signaling Technology, #9661, 1:1,000), caspase 3 (Cell Signaling Technology, #9662, 1:1,000), cleaved caspase 9 (Cell Signaling Technology, #7237, 1:1,000), caspase 9 (Cell Signaling Technology, #9502, 1:1,000), γ-tubulin (Sigma, #T6557, 1:10,000).

Techniques: Western Blot, Expressing, Clone Assay, Sequencing, Quantitative RT-PCR, Injection, Small Interfering RNA

Journal: bioRxiv

Article Title: BET Bromodomain Inhibitors Target the NEUROD1-subtype SCLC by Blocking NEUROD1 Transactivation

doi: 10.1101/2021.10.25.465771

Figure Lengend Snippet: A) A pie chart showing the breakdown of the genomic locations of NEUROD1 peaks in H446 control cells. B) A metagene profile of NEUROD1 peaks across the transcriptome of H446 control cells. C) Genome-wide histone H3 lysine 27 acetylation (H3K27Ac) and BRD4 occupancy within 2.5 kilobases of the NEUROD1 peaks in the H446 control cells. D) A flow chart illustrating the identification of NEUROD1-target genes (N=1,216) through an integrated analysis of the RNA- seq (H446 NEUROD1-KO cells vs. control cells) and ChIP-Seq data (H446 control cells). E) The transcriptional activity of NEUROD1 predicted by the Binding and Expression Target Analysis algorithm. F) Ingenuity Pathway Analysis identified multiple neural-related pathways enriched in the NEUROD1-target genes. TSS, transcription start site; UTR, untranslated region; GEPs, gene expression profiles; GPCR, G protein-coupled receptor; ALS, amyotrophic lateral sclerosis; CRH, corticotropin-releasing hormone.

Article Snippet: After blocking the membranes with TBST + 5% non-fat dried milk, membranes were incubated with primary antibodies, NEUROD1 (BD Biosciences, #563000, 1:2,000), YAP1 (Santa Cruz Biotechnology, #sc-101199, 1:200), BRD2 (Santa Cruz Biotechnology, #sc-393720, 1:200), BRD3 (Santa Cruz Biotechnology, #sc-81202, 1:200), BRD4 (Novus, NBP2-52959, 1:1,000), LSAMP (Abcam, #ab89719, 1:1,000), NaK-ATPase (Santa Cruz Biotechnology, #sc-71638, 1:1,000), cleaved caspase 3 (Cell Signaling Technology, #9661, 1:1,000), caspase 3 (Cell Signaling Technology, #9662, 1:1,000), cleaved caspase 9 (Cell Signaling Technology, #7237, 1:1,000), caspase 9 (Cell Signaling Technology, #9502, 1:1,000), γ-tubulin (Sigma, #T6557, 1:10,000).

Techniques: Genome Wide, RNA Sequencing Assay, ChIP-sequencing, Activity Assay, Binding Assay, Expressing

Journal: bioRxiv

Article Title: BET Bromodomain Inhibitors Target the NEUROD1-subtype SCLC by Blocking NEUROD1 Transactivation

doi: 10.1101/2021.10.25.465771

Figure Lengend Snippet: A) Integrated Genomics Viewer (IGV) snapshots showing occupancy of NEUROD1, H3K27Ac, RNA polymerase II (RPII), BRD4, BRD3, and BRD2 at the NEUROD2 (left) and NHLH1 (right) gene loci in the H446 NEUROD1-KO cells (#297 cl.2-1) versus control cells (Ctr gRNA cl.10). B) Genome-wide H3K27Ac, BRD2, BRD3, and BRD4 occupancy within 2.5 kilobases bilateral of the NEUROD1 peaks in the H446 NEUROD1-KO (#297 cl.2-1) versus control cells (Ctr gRNA cl.10). C) Genome-wide H3K27Ac occupancy in the H446 NEUROD1-KO cells (#297 cl.2-1) versus control cells (Ctr gRNA cl.10), within 2.5 kilobases bilateral of the genomic positions of TEAD1, TEAD4, ZEB1, and RBPJ peaks extracted from the literature. D) The KO of NEUROD1 affected only a subset of BRD4 peaks that overlayed with NEUROD1 peaks. BRD4 peaks identified in H446 control cells were divided into two groups based on whether they overlayed with NEUROD1 peaks (heatmap 4 vs. 1). The BRD4 peaks overlaying with NEUROD1 peaks in control cells were abrogated upon NEUROD1 KO (heatmap 3 vs. 2), while those not overlaying with NEUROD1 were minimally affected (heatmap 6 vs. 5). E) Co-IP assays using H446 nuclear extracts. Left, immunoblotting for NEUROD1 after IP of BRD4. Right panel: immunoblotting for BRD2, BRD3, and BRD4 after IP of NEUROD1. Ab, antibody; Ctr, control; KO, knockout; ND1, NEUROD1; IP, immunoprecipitation.

Article Snippet: After blocking the membranes with TBST + 5% non-fat dried milk, membranes were incubated with primary antibodies, NEUROD1 (BD Biosciences, #563000, 1:2,000), YAP1 (Santa Cruz Biotechnology, #sc-101199, 1:200), BRD2 (Santa Cruz Biotechnology, #sc-393720, 1:200), BRD3 (Santa Cruz Biotechnology, #sc-81202, 1:200), BRD4 (Novus, NBP2-52959, 1:1,000), LSAMP (Abcam, #ab89719, 1:1,000), NaK-ATPase (Santa Cruz Biotechnology, #sc-71638, 1:1,000), cleaved caspase 3 (Cell Signaling Technology, #9661, 1:1,000), caspase 3 (Cell Signaling Technology, #9662, 1:1,000), cleaved caspase 9 (Cell Signaling Technology, #7237, 1:1,000), caspase 9 (Cell Signaling Technology, #9502, 1:1,000), γ-tubulin (Sigma, #T6557, 1:10,000).

Techniques: Genome Wide, Co-Immunoprecipitation Assay, Western Blot, Knock-Out, Immunoprecipitation

Journal: bioRxiv

Article Title: BET Bromodomain Inhibitors Target the NEUROD1-subtype SCLC by Blocking NEUROD1 Transactivation

doi: 10.1101/2021.10.25.465771

Figure Lengend Snippet: A) A working model showing that BET bromodomain proteins serve as the transcriptional coactivators of NEUROD1, and BETi could suppress NEUROD1 transactivation by blocking the former proteins. B) GSEA plot of the NEUROD1 gene signature comparing the NEUROD1-subtype SCLC lines (N subtype) to all other SCLC lines (non-N subtype) in the CCLE dataset. C) GSEA plot of NEUROD1 gene signature comparing the JQ1-treated H446 cells (1 µM; 24 hrs) to control cells treated with DMSO. D) GSEA plot of NEUROD1 gene signature comparing the JQ1-treated COR-L279 cells (0.5 µM; 24 hrs) to control cells treated with DMSO. E) A Venn diagram showing overlaps among three superenhancer (SE) groups. F) Among NEUROD1-target genes, the genes with the NEUROD1-loaded SEs in H446 cells were more significantly suppressed by NEUROD1 KO than those without. G) JQ1 (1µM, 24 hrs) differentially affected the expression of the NEUROD1-target genes with or without the NEUROD1-loaded SEs in H446 control cells (left two columns) but not in the NEUROD1-KO cells (right two columns). H) JQ1 treatment (1µM, 24 hrs) caused a similar degree of suppression in the genes with the BRD4-loaded SEs in the NEUROD1-KO cells as in control cells. I) Co-IP using the nuclear extracts from H446 cells treated with JQ1 (1µM, 6 hrs) versus DMSO. Left, detection of NEUROD1 by western blot after IP of BRD4. Right, detection of BRD4 after IP of NEUROD1. The significance of the two-group comparisons was determined using the Student’s t -test in (F-H). The whiskers in (F-H) represent minimum to maximum, and the boxes show the first quartile, median, and third quartile. ****, P<0.0001; Ab, antibody; FC, fold change; ND1, NEUROD1; NES, normalized enrichment score; ns, not significant.

Article Snippet: After blocking the membranes with TBST + 5% non-fat dried milk, membranes were incubated with primary antibodies, NEUROD1 (BD Biosciences, #563000, 1:2,000), YAP1 (Santa Cruz Biotechnology, #sc-101199, 1:200), BRD2 (Santa Cruz Biotechnology, #sc-393720, 1:200), BRD3 (Santa Cruz Biotechnology, #sc-81202, 1:200), BRD4 (Novus, NBP2-52959, 1:1,000), LSAMP (Abcam, #ab89719, 1:1,000), NaK-ATPase (Santa Cruz Biotechnology, #sc-71638, 1:1,000), cleaved caspase 3 (Cell Signaling Technology, #9661, 1:1,000), caspase 3 (Cell Signaling Technology, #9662, 1:1,000), cleaved caspase 9 (Cell Signaling Technology, #7237, 1:1,000), caspase 9 (Cell Signaling Technology, #9502, 1:1,000), γ-tubulin (Sigma, #T6557, 1:10,000).

Techniques: Blocking Assay, Expressing, Co-Immunoprecipitation Assay, Western Blot

Journal: bioRxiv

Article Title: BET Bromodomain Inhibitors Target the NEUROD1-subtype SCLC by Blocking NEUROD1 Transactivation

doi: 10.1101/2021.10.25.465771

Figure Lengend Snippet: A) Comparison of NEUROD1 dependence between the NEUROD1-subtype lines versus all other SCLC lines, using the Achilles Gene Dependency results from the DepMap project (2020 Q2). B) Viability of the H446 NEUROD1-KO cells (gRNA-297 cl. 12 and cl. 2-1; gRNA-298 cl. S3) versus control cells (Ctr gRNA cl. 10 and cl. 13) after JQ1 treatment (72 hrs). C) Viability of the DMS-273 NEUROD1- KO cells versus control and parental cells after JQ1 treatment (72 hrs). D) Comparison of relative NEUROD1 expression between the JQ1-sensitive and -resistant SCLC lines (N=52). An IC 50 to JQ1 = 5 µM was used as a cutoff to define the sensitive and resistant SCLC lines. E) Comparison of JQ1 sensitivity (IC 50 ) among four molecular subtypes of SCLC lines used in this study (N=52). F) Comparison of JQ1 IC 50 values among four molecular subtypes of SCLC lines (N=57) in an NCI drug screen dataset . The significance of the two-group comparisons was determined using the Student’s t -test in (A) and (D) and the ANOVA test with Dunnett’s multiple test correction in (E) and (F). *, P<0.05; **, P<0.01; ***, P<0.001; ns: not significant. The error bars in (B-C) represent SD; the top and bottom whiskers in (A) and (D) represent the first and third quartile, respectively; and the thick black lines in (E) and (F) represent the medians.

Article Snippet: After blocking the membranes with TBST + 5% non-fat dried milk, membranes were incubated with primary antibodies, NEUROD1 (BD Biosciences, #563000, 1:2,000), YAP1 (Santa Cruz Biotechnology, #sc-101199, 1:200), BRD2 (Santa Cruz Biotechnology, #sc-393720, 1:200), BRD3 (Santa Cruz Biotechnology, #sc-81202, 1:200), BRD4 (Novus, NBP2-52959, 1:1,000), LSAMP (Abcam, #ab89719, 1:1,000), NaK-ATPase (Santa Cruz Biotechnology, #sc-71638, 1:1,000), cleaved caspase 3 (Cell Signaling Technology, #9661, 1:1,000), caspase 3 (Cell Signaling Technology, #9662, 1:1,000), cleaved caspase 9 (Cell Signaling Technology, #7237, 1:1,000), caspase 9 (Cell Signaling Technology, #9502, 1:1,000), γ-tubulin (Sigma, #T6557, 1:10,000).

Techniques: Expressing

Journal: bioRxiv

Article Title: BET Bromodomain Inhibitors Target the NEUROD1-subtype SCLC by Blocking NEUROD1 Transactivation

doi: 10.1101/2021.10.25.465771

Figure Lengend Snippet: A) A diagram showing the selection of 45 genes for a custom siRNA screen. B) Results of the siRNA screen in H446 (left) and COR-L279 cells (right). The genes highlighted are the common genes in H446 and COR-L279 cells that showed consistently ≥ 20% growth inhibition upon knockdown by two different siRNAs. C) An inverse correlation in the Kaplan-Meier curve between the LSAMP expression and the overall survival of the SCLC patients . The significance of overall survival difference when comparing the patients with top 50% LSAMP expression versus those with the bottom 50% was determined using the Log-rank test. D-E) Drastic decrease of LSAMP expression in the NEUROD1-KO cells of H446 (D) and DMS-273 (E). NaK-ATPase serves as a loading control for membrane proteins. F) A dose-dependent decrease of membrane LSAMP expression in H446 cells treated with JQ1 (24 hrs at 0.25, 0.5, or 1 µM). G) Knockdown of LSAMP in H446 cells using two siRNAs different from the ones in the initial screen (B). H) Significant decrease in cell growth after knockdown of LSAMP in H446 cells using two siRNAs. I) Stable ectopic expression of LSAMP in COR-L279 cells. For comparison, a tapering amount of H446 membrane protein (20, 10, 5, 2.5 µg protein) and 20 µg COR-L279 membrane protein were loaded. J) Relative growth of the COR-L279 cells with ectopic LSAMP versus cells transfected with an empty vector or parental cells. K) Viability of the COR-L279 cells with ectopic LSAMP versus control cells after JQ1 treatment (72 hrs). L) Comparison of caspase 3 and 9 cleavages after JQ1 treatment (5µM, 24 hrs) in the COR-L279 cells with ectopic LSAMP versus control cells. The significance of the two-group comparisons was determined using the Student’s t -test in (K) and the ANOVA test with Dunnett’s multiple test correction in (H) and (J). Where indicated, **, P<0.01; ****, P<0.0001. The error bars represent SD. Ctr, control; FC, fold change; gRNA, guide RNA; OE, overexpression; siRNA, small interfering RNA.

Article Snippet: After blocking the membranes with TBST + 5% non-fat dried milk, membranes were incubated with primary antibodies, NEUROD1 (BD Biosciences, #563000, 1:2,000), YAP1 (Santa Cruz Biotechnology, #sc-101199, 1:200), BRD2 (Santa Cruz Biotechnology, #sc-393720, 1:200), BRD3 (Santa Cruz Biotechnology, #sc-81202, 1:200), BRD4 (Novus, NBP2-52959, 1:1,000), LSAMP (Abcam, #ab89719, 1:1,000), NaK-ATPase (Santa Cruz Biotechnology, #sc-71638, 1:1,000), cleaved caspase 3 (Cell Signaling Technology, #9661, 1:1,000), caspase 3 (Cell Signaling Technology, #9662, 1:1,000), cleaved caspase 9 (Cell Signaling Technology, #7237, 1:1,000), caspase 9 (Cell Signaling Technology, #9502, 1:1,000), γ-tubulin (Sigma, #T6557, 1:10,000).

Techniques: Selection, Inhibition, Expressing, Transfection, Plasmid Preparation, Over Expression, Small Interfering RNA

Journal: bioRxiv

Article Title: BET Bromodomain Inhibitors Target the NEUROD1-subtype SCLC by Blocking NEUROD1 Transactivation

doi: 10.1101/2021.10.25.465771

Figure Lengend Snippet: A) NHWD-870 (3mg/kg; oral gavage on a 5-day-on/2-day-off schedule) suppressed tumor growth in a NEUROD1-subtype SCLC PDX model - LX22. n=13 per group. B) The Kaplan-Meier survival curve of (A). C) GSEA plot of the NEUROD1 gene signature comparing the LX22 PDX tumors treated with NHWD-870 (3mg/kg; daily for five days) to control treated with the vehicle. n=6 per group. D) Comparison of NEUROD1 and LSAMP transcript abundance in LX22 tumors treated with NHWD-870 versus vehicle. E) NHWD-870 (3mg/kg; oral gavage daily) suppressed tumor growth in LX33, another NEUROD1-subtype SCLC PDX model. n=6 per group. F) The Kaplan- Meier survival curve of (E). G) A dose-dependent decrease in LSAMP expression in the LX33 SCLC line after exposure to NHWD-870 for 24 hrs. H) Knockdown of LSAMP in the LX33 SCLC line using two different siRNAs. I) Relative growth of the LX33 SCLC cells after knockdown of LSAMP. The significance of the two-group comparison was determined using the Student’s t -test in (A), (D), and (E), the Log-rank test in (B) and (F), and the ANOVA test with Dunnett’s multiple test correction in (G) and (I). Where indicated, *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. The error bars represent SD. Ctr, control; FPKM, Fragments Per Kilobase of transcript per Million mapped reads; NES, normalized enrichment score.

Article Snippet: After blocking the membranes with TBST + 5% non-fat dried milk, membranes were incubated with primary antibodies, NEUROD1 (BD Biosciences, #563000, 1:2,000), YAP1 (Santa Cruz Biotechnology, #sc-101199, 1:200), BRD2 (Santa Cruz Biotechnology, #sc-393720, 1:200), BRD3 (Santa Cruz Biotechnology, #sc-81202, 1:200), BRD4 (Novus, NBP2-52959, 1:1,000), LSAMP (Abcam, #ab89719, 1:1,000), NaK-ATPase (Santa Cruz Biotechnology, #sc-71638, 1:1,000), cleaved caspase 3 (Cell Signaling Technology, #9661, 1:1,000), caspase 3 (Cell Signaling Technology, #9662, 1:1,000), cleaved caspase 9 (Cell Signaling Technology, #7237, 1:1,000), caspase 9 (Cell Signaling Technology, #9502, 1:1,000), γ-tubulin (Sigma, #T6557, 1:10,000).

Techniques: Expressing