Journal: Molecular Medicine

Article Title: ONECUT2 reprograms neuroendocrine fate and is an actionable therapeutic target in small cell lung cancer

doi: 10.1186/s10020-025-01267-6

Figure Lengend Snippet: Inducible expression of OC2 in NCI-H510 cells leads to a phenotypic transdifferentiation towards a non-NE subtype. A Scheme of the experimental protocol of induction/off-phase/induction of OC2 expression in the NCI-H510 cell line. Cells were cultured with 50 ng/mL doxycycline (dox) for 5 days (first induction). Subsequently, the medium was replaced with complete RPMI for 6 days (off-phase). Finally, doxycycline was added again for 5 days (second induction). B Heatmap showing DEGs (adjusted P -value < 0.001) during the first induction (samples 1A and 1B), off-phase (samples 2A and 2B), and second induction (samples 3A and 3B) analyzed by hierarchical clustering. Three independent RNA-Seq experiments were performed per condition. C) Venn diagram of the overlapping DEGs during the induction/off-phase/induction experiment. D Plots showing upregulated (purple bars) and downregulated (green bars) SCLC subtype-specific gene signatures (Ireland et al. ) in NCI-H510 cells. E Immunoblot showing OC2 and ASCL1 expression during the induction/off-phase/induction experiment. F YAP1, ASCL1 and NEUROD1 mRNA expression after constitutive OC2 overexpression in NCI-H510 cells. Results were normalized using β-actin. The mean + SEM from at least three independent experiments is shown. G MSigDB Hallmark Gene Sets enriched in OC2 upregulated (purple bars) and downregulated (green bars) genes in OC2 overexpressing NCI-H510 and DMS53 cells

Article Snippet: The empty vector pLenti-C-mGFP-P2A-Puro (Origene, PS100093) was used as a control and the OC2 plasmid Lenti-ORF clone of ONECUT2 (mGFP-tagged)-Human one cut homeobox 2 (Origene, RC211951L4) was used for constitutive expression of this protein.

Techniques: Expressing, Cell Culture, RNA Sequencing, Western Blot, Over Expression

Journal: Molecular Medicine

Article Title: ONECUT2 reprograms neuroendocrine fate and is an actionable therapeutic target in small cell lung cancer

doi: 10.1186/s10020-025-01267-6

Figure Lengend Snippet: OC2 induction alters NE transdifferentiation drivers. A GSEA plots showing enrichment of Notch signaling signatures (Vilimas et al. ; Milacic et al. ) in NCI-H510 and DMS53 cells after OC2 inducible and constitutive overexpression, respectively. Three independent RNA-Seq experiments were performed. B REST mRNA expression after OC2 enforced expression in the DMS53 and NCI-H510 cell lines. The mean + SEM from three independent experiments is shown. Unpaired two-tailed Student’s t-test, ** P < 0.01. Results were normalized using β-actin. C Cell proliferation curve showing OC2 induced vs. control NCI-H510 cells. Proliferation was calculated relative to T0 number of cells. The values shown are the mean ± SEM from five independent experiments. D Heatmap showing DEPs ( P -value < 0.05) after OC2 induction in NCI-H510 cells. E Heatmap of transcription factors generated with Metascape employing the TRRUST method using DEPs between control and OC2 overexpressing NCI-H510 cells. F c-MYC and l-MYC mRNA expression after OC2 constitutive overexpression in the NCI-H510 cell line. The mean + SEM from four independent experiments is shown. Unpaired two-tailed Student’s t-test, ** P < 0.01. Results were normalized using β-actin. G c-MYC protein expression levels detected by western blot after OC2 overexpression in the NCI-H510 and DMS53 cell lines. β-actin was used as control. H Protein networks and their cellular localization obtained in response to OC2 induction in the NCI-H510 cell line. Networks are arranged in the form of nodes (proteins) and lines (biological relationships between nodes). The proteins in green are overexpressed and those in red are repressed. The proteins in white do not belong to our dataset but are included in the database. In orange, the program predicts protein activity activation, and in blue, inhibition. The solid lines connecting the nodes represent direct interactions, while the dashed lines represent indirect interactions. The color of the lines indicates activation if orange, inhibition if blue, inconsistent findings if yellow, and unpredictable effects if gray. The shape of the nodes denotes the protein function: enzymes (diamond), transcriptional regulators (oval), kinases (inverted triangle), peptidases (horizontally oriented diamond), nuclear receptors (rectangle), transporters (trapezoid), others (circle). Image obtained from Ingenuity

Article Snippet: The empty vector pLenti-C-mGFP-P2A-Puro (Origene, PS100093) was used as a control and the OC2 plasmid Lenti-ORF clone of ONECUT2 (mGFP-tagged)-Human one cut homeobox 2 (Origene, RC211951L4) was used for constitutive expression of this protein.

Techniques: Over Expression, RNA Sequencing, Expressing, Two Tailed Test, Control, Generated, Western Blot, Activity Assay, Activation Assay, Inhibition

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ONECUT2 reprograms neuroendocrine fate and is an actionable therapeutic target in small cell lung cancer.

doi: 10.1186/s10020-025-01267-6

Figure Lengend Snippet: Fig. 2 OC2 modulates plasticity from NE to non-NE phenotypes in the DMS53 cell line. A Heatmap showing DEGs (adjusted P-value < 0.001) after OC2 enforced expression in DMS53 cells analyzed by hierarchical clustering. Three independent RNA-Seq experiments were performed per condition. B Plot showing upregulated (red bars) and downregulated (blue bars) SCLC subtype-specific gene signatures (Ireland et al. 2020) after OC2 enforced expres sion. C Immunohistochemical staining of SYP in OC2 overexpressing DMS53 cells. The boxes show the 25–75th percentile range, and the center line is the median. Whiskers extend from the minimum and maximum values. P-values were obtained from Wilcoxon two-tailed rank-sum test. D, E mRNA (D) and protein levels (E) of OC2, ASCL1, NEUROD1 and YAP1 after OC2 enforced expression in DMS53 cells. For (D) qRT-PCR results were normalized using β-actin. The mean + SEM from three independent experiments is shown. Unpaired two-tailed Student’s t-test, *P < 0.05, **P < 0.01. For (E) representative blots from three independent experiments are shown. F Plot showing upregulated (red bars) non-NE gene signatures and downregulated (blue bars) NE gene signatures (Cai et al. 2021; Zhang et al. 2018)

Article Snippet: The empty vector pLenti-C-mGFP-P2A-Puro (Origene, PS100093) was used as a control and the OC2 plasmid Lenti-ORF clone of ONECUT2 (mGFP-tagged)-Human one cut homeobox 2 (Origene, RC211951L4) was used for constitutive expression of this protein.

Techniques: Expressing, RNA Sequencing, Immunohistochemical staining, Staining, Two Tailed Test, Quantitative RT-PCR

Journal: Nature Communications

Article Title: The homeodomain regulates stable DNA binding of prostate cancer target ONECUT2

doi: 10.1038/s41467-024-53159-8

Figure Lengend Snippet: a Overall structure of the OC2- PEG10 DNA complex. The position of CUT and HOX domains on DNA and their respective helices are labeled (α1- α8) while the unmodelled loop is depicted as a dashed line. CUT and HOX domains are shown in green and blue, respectively. b , c Arrangement of DNA interacting residues in α3 helix of CUT domain and α8 helix of HOX domain of OC2 are shown. d Schematic representation of the protein-DNA contacts in the complex. Hydrogen bonds are shown as dashed lines and water molecules are depicted as cyan spheres. DNA interacting residues of CUT and HOX domains are shown in green and blue, respectively.

Article Snippet: The OC2 overexpression construct was generated by cloning the full-length OC2 cDNA (NM_004852) into the pLenti-C-Myc-DDK-IRES-Puro (Origene PS100069) lentivirus system.

Techniques: Labeling

Journal: Nature Communications

Article Title: The homeodomain regulates stable DNA binding of prostate cancer target ONECUT2

doi: 10.1038/s41467-024-53159-8

Figure Lengend Snippet: a , b PEG10 and TTR DNA sequences and corresponding conserved base-specific interactions with OC2 and OC1, respectively. The core sequence is shown in blue except the bases at which PEG10 and TTR vary, that are in red. Black triangles depict interaction sites; the respective base-interacting residues are indicated. The difference in interaction of the first arginine (OC2 R479 or OC1 R438) of RR pair is shown with a black rectangular outline. c – f Interaction of OC2 S364, equivalent OC1 S323, and OC2 R479 and equivalent OC1 R438, to DNA. Hydrogen bonds in panels ( c – f ) are shown as yellow dashed lines. g Interaction of OC2 R480 and equivalent OC1 R439 (yellow and orange dashed lines, respectively). h Interactions of the POU residues corresponding to the OC arginine pair [PDB 1E3O [10.2210/pdb1E3O/pdb] (OCT1) and 1AU7 [10.2210/pdb1AU7/pdb] (PIT1)]. Hydrogen bonds are shown in the same color as respective proteins. i The relative orientations of the OC arginine pair and corresponding OCT1 and PIT1 residues. j , k Structure-based sequence alignment of CUT ( j ) and HOX ( k ) domains of OC2, OC1, OCT1, PIT1 and SATB1. The amino acid ranges are indicated. The conserved serine (S364) in CUT is highlighted (in green) while the arginine pair (RR motif; R479/R480) in HOX are shown with a red border. The conserved glutamine (Q365) in CUT and asparagine (N476) in HOX are also highlighted (in green). The color of highlighted residues is based on Clustal scheme.

Article Snippet: The OC2 overexpression construct was generated by cloning the full-length OC2 cDNA (NM_004852) into the pLenti-C-Myc-DDK-IRES-Puro (Origene PS100069) lentivirus system.

Techniques: Sequencing

Journal: Nature Communications

Article Title: The homeodomain regulates stable DNA binding of prostate cancer target ONECUT2

doi: 10.1038/s41467-024-53159-8

Figure Lengend Snippet: ITC analysis of OC2, CUT and HOX binding to the PEG10 DNA

Article Snippet: The OC2 overexpression construct was generated by cloning the full-length OC2 cDNA (NM_004852) into the pLenti-C-Myc-DDK-IRES-Puro (Origene PS100069) lentivirus system.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The homeodomain regulates stable DNA binding of prostate cancer target ONECUT2

doi: 10.1038/s41467-024-53159-8

Figure Lengend Snippet: a – c ITC binding analysis of intact OC2, CUT domain and CUT + HOX, to PEG10 DNA. The raw heats (differential power, DP; top) and binding isotherms (bottom) are shown and representative of three independent experiments ( n = 3; technical replicates). Source data are provided as a Source Data file. d – g Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) characterization of DNA binding by OC2. Structure of OC2 bound to PEG10 DNA showing the helix α3 (purple) which appears to become much more structured upon PEG10 binding ( d ). The CUT and HOX are in green and gold, respectively, while the PEG10 is in wheat. The protein N- and C-termini are indicated. Relative deuterium uptake into OC2 with and without bound PEG10 is rendered on the structure using a blue-white-red gradient scale ( e ); regions of the protein not covered by the HDX-MS experiment are in black. No regions of increased uptake were observed but many parts of the protein experienced decreased (blue) deuterium uptake upon PEG10 binding with the maximum decrease being 0.28. Relative deuterium uptake into the CUT domain alone with and without bound PEG10 is plotted on the structure according to the same scale and color scheme as in panel e ( f ). There were no significant differences in deuterium uptake upon PEG10 binding of the CUT domain alone (consequently, mostly white except the black regions that showed no coverage). Note that the CUT(-DNA) structure is extracted from the OC2- PEG10 structure for demonstration only. Deuterium uptake plot for the helix α3 containing peptide (residues 358-371) in the four different conditions as indicated ( g ). The y -axis corresponds to the total number of amides in the peptide. The HDX-MS data shown is representative of three independent experiments ( n = 3; technical replicates); P -values based on One-way ANOVA as provided by the software were 0.001 for OC2- PEG10 vs apo-OC2; 0.9 for CUT- PEG10 vs apo-CUT.

Article Snippet: The OC2 overexpression construct was generated by cloning the full-length OC2 cDNA (NM_004852) into the pLenti-C-Myc-DDK-IRES-Puro (Origene PS100069) lentivirus system.

Techniques: Binding Assay, Mass Spectrometry, Software

Journal: Nature Communications

Article Title: The homeodomain regulates stable DNA binding of prostate cancer target ONECUT2

doi: 10.1038/s41467-024-53159-8

Figure Lengend Snippet: Heat capacity analysis of OC2 and CUT binding to the PEG10 DNA

Article Snippet: The OC2 overexpression construct was generated by cloning the full-length OC2 cDNA (NM_004852) into the pLenti-C-Myc-DDK-IRES-Puro (Origene PS100069) lentivirus system.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The homeodomain regulates stable DNA binding of prostate cancer target ONECUT2

doi: 10.1038/s41467-024-53159-8

Figure Lengend Snippet: ITC analysis of the binding of OC2 mutants to the PEG10 DNA

Article Snippet: The OC2 overexpression construct was generated by cloning the full-length OC2 cDNA (NM_004852) into the pLenti-C-Myc-DDK-IRES-Puro (Origene PS100069) lentivirus system.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The homeodomain regulates stable DNA binding of prostate cancer target ONECUT2

doi: 10.1038/s41467-024-53159-8

Figure Lengend Snippet: a – d Kinetics of PEG10 DNA binding by OC2, OC2SQ, OC2N and OC2RR. All proteins are titrated at concentrations 75, 125, 175, 225 and 275 nM, and representative curves are shown ( n = 3; technical replicates). The association and dissociation phases are separated by a dotted line. e Plot showing the proliferation of LNCaP cells upon stable expression of ectopic wild-type and mutant OC2 compared to cells with endogenous OC2 (vector control) at 48 and 72 h. Data are presented as mean values ± SD. Two-sample t -test was used for statistical analysis ( n = 4; biological replicates). At 48 h, **** P (OC2) < 0.0001, P (OC2SQ) = 0.64, P (OC2N) = 0.67, P (OC2RR) = 0.26, and at 72 h, *** P (OC2) = 0.00011, P (OC2SQ) = 0.09, P (OC2N) = 0.31, P (OC2RR) = 0.33. f - g Relative mRNA levels of AR target genes KLK3 , NKX3-1 and TMPRSS2 and NEPC marker genes NSE , PEG10 and SYP , upon stable overexpression of ectopic wild-type and mutant OC2 compared to cells with endogenous OC2 (vector control). Data are presented as mean values ± SD. Two-sample t -test was used for statistical analysis ( n = 3; biological replicates). In case of AR targets, for KLK3, ** P (OC2) = 0.008, P (OC2SQ) = 0.53, ** P (OC2N) = 0.009, ** P (OC2RR) = 0.007; for NKX3-1, *** P (OC2) = 0.0007, ** P (OC2SQ) = 0.008, P (OC2N) = 0.19, P (OC2RR) = 0.62; and for TMPRSS2, **** P (OC2) < 0.0001, **** P (OC2SQ) < 0.0001, P (OC2N) = 0.93, P (OC2RR) = 0.16. In case of NE markers, for NSE, * P (OC2) = 0.02, * P (OC2SQ) = 0.02, P (OC2N) = 0.45, * P (OC2RR) = 0.04; for PEG10, ** P (OC2) = 0.003, * P (OC2SQ) = 0.01, P (OC2N) = 0.14, *** P (OC2RR) = 0.0008; and for SYP, * P (OC2) = 0.01, P (OC2SQ) = 0.09, P (OC2N) = 0.2, P (OC2RR) = 0.09. Source data are provided as a Source Data file.

Article Snippet: The OC2 overexpression construct was generated by cloning the full-length OC2 cDNA (NM_004852) into the pLenti-C-Myc-DDK-IRES-Puro (Origene PS100069) lentivirus system.

Techniques: Binding Assay, Expressing, Mutagenesis, Plasmid Preparation, Control, Marker, Over Expression

Journal: Nature Communications

Article Title: The homeodomain regulates stable DNA binding of prostate cancer target ONECUT2

doi: 10.1038/s41467-024-53159-8

Figure Lengend Snippet: Kinetics analysis of wild-type and mutant OC2 proteins to the PEG10 DNA

Article Snippet: The OC2 overexpression construct was generated by cloning the full-length OC2 cDNA (NM_004852) into the pLenti-C-Myc-DDK-IRES-Puro (Origene PS100069) lentivirus system.

Techniques: Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: Hyperglycemia-independent neonatal streptozotocin-induced retinopathy (NSIR) in rats

doi: 10.3389/fphar.2024.1395887

Figure Lengend Snippet: Intravitreal STZ injection on P1 inhibits the proliferation of neonatal rat retinal progenitors and delays their cell cycle exit and differentiation. (A) Horizontal P4 retinal sections of indicated locations and groups were stained for nuclear (DAPI, blue) and cell proliferation (Ki67, green). (B) Quantification of Ki67+ cells per mm 2 area of P4 and P8 retinas. (C) Horizontal P8 retinal sections of indicated locations and groups were stained for nuclear (DAPI, blue) and cell proliferation (Ki67, green). (D) Flow cytometry analysis of P4 retinal cells. (E) Whole-mount or horizontal sections of P8 retinas of indicated groups were stained for nuclear (DAPI, blue) and cell type markers, including Ganglion cells (Brn3, green, whole-mount), Cone (ARR3, green), Horizontal cells (OC2, green), Amacrine cells (Ap2a, green), Rod (Rho, green), Bipolar cells (Chx10, green) and Müller glia (Sox9, green). (F) Quantifying all seven retinal cell types (relative to the control group, %). Error bars represent SD of measurements from three animals or three retinas of three animals (n = 3), and asterisks indicate significant differences between control and STZ-treated groups (* p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni’s correction). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; Ivit, Intravitreal injection; PI, Propidium iodide. Scale bar: 50 μm.

Article Snippet: Slides were incubated with blocking solution (1% donkey serum and 0.1% Triton X-100 in PBS) for 1 h, and then with primary antibodies including Ap2a (Santa Cruz, SC-8975); Chx10 (Bremner lab, University of Toronto); Cleaved caspase-3 (Cell Signaling 9661); Cone arrestin or ARR3 (Millipore, AB15282); γ-H2ax (Millipore, 05–636); Onecut2 or OC2 (R&D system AF6294); Ki67 (BD science Pharmingen 550609); PH3 (Upstate 05–598); Rhodopsin (Santa Cruz SC-57433) and Sox9 (Millipore AB5535), overnight at 4°C.

Techniques: Injection, Staining, Flow Cytometry, Control