Journal: Cancer cell

Article Title: Re-programing Chromatin with a Bifunctional LSD1/HDAC Inhibitor Induces Therapeutic Differentiation in DIPG.

doi: 10.1016/j.ccell.2019.09.005

Figure Lengend Snippet: Figure 6. Corin Reduces DIPG Xenograft Size and Alters Tumor Phenotypes (A and B) Representative bioluminescent images (BLI) (A) and quantification (B) of mice xenografted with HSJD-DIPG007-luciferase cells immediately before or 7 days after treatment with 0.03 mg Corin or saline/DMSO control by CED. The boxplot in (B) shows the 25th to 75th percentile and median (center line) with error bars indicating the minimum and maximum values. (C) BLI signal from SU-DIPGXIIIP* xenografts taken at various time points before and after CED (teal arrow) with either 0.03 mg Corin or DMSO. Error bars show SEM and statistical significance was determined by ANOVA. (D) H&E-stained sections of the pons of mice xenografted with HSJD-DIPG007-luciferase/Zs-green cells treated with vehicle or Corin in (C). Scale bars, 200 mm (top images) and 20 mm (bottom images). (E) Images of SU-DIPGXIIIP* pontine xenografts or adjacent regions of the mouse pons stained to monitor H3K27ac and H3K27me3 levels 3 days after CED with either vehicle or Corin. Scale bar, 50 mm. (F and G) Immunofluorescent images of NP2 and SYNI/II/III staining (F) or immunohistochemistry for Ki67 and cleaved Caspase-3 (G) in SU-DIPGXIIIP* xenografts from vehicle- and Corin-treated mice. Scale bars, 20 mm. See also Figure S6.

Article Snippet: For staining, slides were permeabilized with 0.25%Triton-X-100 in PBS for 20minutes, blocked with 4%Bovine Serum Albumin (BSA) in PBS plus 0.1% Tween-20 for 45 minutes, and then stained overnight with primary antibodies: H3K27ac (Active Motif, #39135), 1:2000), H3K27me3 (Millipore, 07-449, 1:1000), SYNII/II/II (Biolegend a17080a, 1:1000), or NP2 (1:500, Santa Cruz Biotechnology, sc-166035).

Techniques: Luciferase, Saline, Control, Staining, Immunohistochemistry

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: A. Percentage of nuclear NRP2-positive cells across the Gleason score. B. Representative images of immunohistochemical staining for nuclear NRP2 in human prostate cancer tissues. Arrows indicate the nuclear positive NRP2. C. Endogenous NRP2 staining (Alexa 488) in prostate cancer cell line C4–2B. INM was stained with lamin A/C (Alexa 594). DAPI indicates nuclear staining. Using Zen Blue lite software, cells were cut through the middle plane and then rotated across the x-axis. Inset is the magnified image of the nucleus of the cells. D. Schematic diagram of NRP2 isomers. E. TEM images of immunogold staining of untagged NRP2A in C4–2B cells ectopically expressing NRP2A. Cells were stained with NRP2 antibody and counterstained with gold-labeled secondary antibody. NRP2 staining appears as black punctate structures inside the cells. The nuclear and plasma membranes are indicated by the arrow and arrowhead, respectively. Inset is magnified. In the magnified image, ONM and INM are represented by opaque and black arrowheads, respectively. Scale Bar 500 nm. F. Representative TEM images of immunogold staining of HA-tagged NRP2B ectopically expressed in C4–2B. Cells were stained with HA-antibody and counterstained with gold-labeled secondary antibody. The nuclear membrane and plasma membrane are indicated by arrows and arrowheads, respectively. Insets is magnified. ONM and INM are represented by opaque and black arrowheads. Scale Bar 200 nm.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Immunohistochemical staining, Staining, Software, Expressing, Labeling, Membrane

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: A. Immunofluorescence image of ectopically expressed untagged NRP2A (Alexa 488) in C4–2B cells. NRP2A staining was carried out with NRP2 antibody. The nuclear pore was stained with pore protein Nup98 (Alexa 594). Inset is magnified. Green arrowhead indicates NRP2A, whereas red arrows indicate Nup98. Green and red peaks in the line graph represent the relative position of NRP2A and Nup98 around the nucleus. B. Immunofluorescence image of ectopically expressed HA-tagged NRP2B (Alexa 488) in C4–2B cells. NRP2B staining was carried out with HA-antibody. The nuclear pore was stained with central pore protein Nup98 (Alexa 594). Inset is magnified. NRP2B and Nup98 are indicated by green arrowheads and red arrows, respectively. Red and green peaks in the line graph represent the relative positions of NRP2B and Nup98 around the nucleus. GFP-tagged NRP2A was ectopically co-expressed with HA-tagged NRP2B in C. Hek293T and D. C4–2B cells. Inset is magnified. NRP2B was stained with HA (Alexa 594). The INM and ONM are indicated by the red and green arrows. Scale Bar 10 µm. The nucleus was stained with DAPI. E-F. NRP2A and HA-tagged NRP2B were ectopically over-expressed in C4–2B cells and were counterstained with PDI (Alexa 594). Insets are magnified views. Red and green arrows indicate the relative localization of NRP2 and PDI around the nucleus. Yellow arrows indicate colocalization. Scale Bar 10 µm. G. Bar graph indicates NRP2 and PDI colocalization quantitation.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Immunofluorescence, Staining, Quantitation Assay

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: A. HA-tagged NRP2B was ectopically expressed in C4–2B cells. Immunofluorescence images representing the nuclear translocation of NRP2B under various conditions. NRP2B was detected using HA primary antibody and counterstained with Alexa 488-tagged secondary antibody. Arrowheads indicate invagination of the nuclear envelope. Nup98 was co-stained with NRP2. Inset is magnified. DAPI was used for nuclear staining. Nuclei are marked with dotted lines. Scale Bar 10 µm. B. Bar graph shows quantification of nuclear NRP2B. C. Immunoprecipitation with SUMO1 was carried out in C4–2B cells. HA-tagged wild type NRP2B and HA-tagged NRP2B K892A mutant were ectopically expressed in C4–2B cells. Immunoblots were carried out with HA-antibody. IgG bands show equal pulldown. D. Immunostaining of HA-tagged wild type and mutant K892A NRP2B in the C4–2B cell line. NRP2B was stained with HA-antibody and counterstained with secondary antibody labeled with Alexa 488. Nup98 was stained with Alexa 594. Arrowhead represents nuclear invagination in C4–2B cells expressing wild type NRP2B. Insets are magnified views. Nuclei are demarcated with dotted lines. DAPI indicates nucleus. Scale Bar 20 µm.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Immunofluorescence, Translocation Assay, Staining, Immunoprecipitation, Mutagenesis, Western Blot, Immunostaining, Labeling, Expressing

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: Following ectopic expression of untagged NRP2B in C4–2B cells, nuclear and post-nuclear fractions were separated, and a pull-down assay was performed on the nuclear fraction with NRP2 antibody. Mass-Spectrometry was carried out with the pull-down samples. Using the genes detected in NRP2 Mass-Spectrometry. A. Sum of all the spectra associated with NRP2B pulldown sample is determined by the spectral graph. B. Venn-diagram represent the overlapping genes identified in two independent NRP2B mass-spectrometry assay. C. Group of nuclear pore proteins (Nups) identified through NRP2B Mass-Spectrometry. Schematic diagram indicating the relative positions of Nups around the nuclear pore. D. Validation of NRP2-Nup93 interactions in C4–2B. C4–2B cells were transfected with HA-tagged NRP2B. IP was carried out with HA-antibody and immunoblots were carried out with anti-Nup93. E. Endogenously, NRP2 and Nup93 interaction was validated in C4–2B and 22Rv1 cell lines. NRP2 was pull down with NRP2-specific antibody and IB with Nup93 antibody. F. NRP2B and AR interaction was carried out in C4–2B following ectopic interaction of HA-tagged NRP2B. Pulldown was carried out with HA-antibody and IP was carried out against NRP2 and AR. G. NRP2 and AR interaction was also monitored in PC3 and 22Rv1 following ectopic expression of HA-tagged NRP2B. IB was carried out against HA and AR. H. Co-IP for AR with NRP2 was carried out with pulldown of NRP2 by HA-antibody in C4–2B cells expressing wild type and mutant K892A NRP2B isoforms. NRP2B immunoblot was carried out with HA-antibody. I. IP with AR antibody was carried out to test the interaction among AR, Nup93 and NRP2 under the presence or absence of NRP2 and Nup 93 from the nuclear fraction of LNCaP C4–2B cells. An immunoblot was performed with anti-AR, anti-Nup93 and anti-NRP2 antibody. The Co-IP was carried out in the presence of 50ng/ml VEGF-C. J. PLA was carried out to validating the NRP2-AR interaction within the nucleus. Following ectopic expression of HA-tagged NRP2B, PLA was carried out with HA and endogenous AR antibodies under the presence or absence of NUP93. As a negative control, only HA-antibody was used for PLA reaction. Arrowhead indicates the immune-reactive PLA puncta. Nucleus was counter-stained with NUP98 to demarcate the nuclear periphery. DAPI used for nuclear staining. PLA quantification was shown in Bar diagram.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Expressing, Pull Down Assay, Mass Spectrometry, Transfection, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Negative Control, Staining

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet:

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques:

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: A. Percentage of nuclear NRP2-positive cells across the Gleason score. B. Representative images of immunohistochemical staining for nuclear NRP2 in human prostate cancer tissues. Arrows indicate the nuclear positive NRP2. C. Endogenous NRP2 staining (Alexa 488) in prostate cancer cell line C4–2B. INM was stained with lamin A/C (Alexa 594). DAPI indicates nuclear staining. Using Zen Blue lite software, cells were cut through the middle plane and then rotated across the x-axis. Inset is the magnified image of the nucleus of the cells. D. Schematic diagram of NRP2 isomers. E. TEM images of immunogold staining of untagged NRP2A in C4–2B cells ectopically expressing NRP2A. Cells were stained with NRP2 antibody and counterstained with gold-labeled secondary antibody. NRP2 staining appears as black punctate structures inside the cells. The nuclear and plasma membranes are indicated by the arrow and arrowhead, respectively. Inset is magnified. In the magnified image, ONM and INM are represented by opaque and black arrowheads, respectively. Scale Bar 500 nm. F. Representative TEM images of immunogold staining of HA-tagged NRP2B ectopically expressed in C4–2B. Cells were stained with HA-antibody and counterstained with gold-labeled secondary antibody. The nuclear membrane and plasma membrane are indicated by arrows and arrowheads, respectively. Insets is magnified. ONM and INM are represented by opaque and black arrowheads. Scale Bar 200 nm.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Immunohistochemical staining, Staining, Software, Expressing, Labeling, Membrane

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: A. Immunofluorescence image of ectopically expressed untagged NRP2A (Alexa 488) in C4–2B cells. NRP2A staining was carried out with NRP2 antibody. The nuclear pore was stained with pore protein Nup98 (Alexa 594). Inset is magnified. Green arrowhead indicates NRP2A, whereas red arrows indicate Nup98. Green and red peaks in the line graph represent the relative position of NRP2A and Nup98 around the nucleus. B. Immunofluorescence image of ectopically expressed HA-tagged NRP2B (Alexa 488) in C4–2B cells. NRP2B staining was carried out with HA-antibody. The nuclear pore was stained with central pore protein Nup98 (Alexa 594). Inset is magnified. NRP2B and Nup98 are indicated by green arrowheads and red arrows, respectively. Red and green peaks in the line graph represent the relative positions of NRP2B and Nup98 around the nucleus. GFP-tagged NRP2A was ectopically co-expressed with HA-tagged NRP2B in C. Hek293T and D. C4–2B cells. Inset is magnified. NRP2B was stained with HA (Alexa 594). The INM and ONM are indicated by the red and green arrows. Scale Bar 10 µm. The nucleus was stained with DAPI. E-F. NRP2A and HA-tagged NRP2B were ectopically over-expressed in C4–2B cells and were counterstained with PDI (Alexa 594). Insets are magnified views. Red and green arrows indicate the relative localization of NRP2 and PDI around the nucleus. Yellow arrows indicate colocalization. Scale Bar 10 µm. G. Bar graph indicates NRP2 and PDI colocalization quantitation.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Immunofluorescence, Staining, Quantitation Assay

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: A. HA-tagged NRP2B was ectopically expressed in C4–2B cells. Immunofluorescence images representing the nuclear translocation of NRP2B under various conditions. NRP2B was detected using HA primary antibody and counterstained with Alexa 488-tagged secondary antibody. Arrowheads indicate invagination of the nuclear envelope. Nup98 was co-stained with NRP2. Inset is magnified. DAPI was used for nuclear staining. Nuclei are marked with dotted lines. Scale Bar 10 µm. B. Bar graph shows quantification of nuclear NRP2B. C. Immunoprecipitation with SUMO1 was carried out in C4–2B cells. HA-tagged wild type NRP2B and HA-tagged NRP2B K892A mutant were ectopically expressed in C4–2B cells. Immunoblots were carried out with HA-antibody. IgG bands show equal pulldown. D. Immunostaining of HA-tagged wild type and mutant K892A NRP2B in the C4–2B cell line. NRP2B was stained with HA-antibody and counterstained with secondary antibody labeled with Alexa 488. Nup98 was stained with Alexa 594. Arrowhead represents nuclear invagination in C4–2B cells expressing wild type NRP2B. Insets are magnified views. Nuclei are demarcated with dotted lines. DAPI indicates nucleus. Scale Bar 20 µm.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Immunofluorescence, Translocation Assay, Staining, Immunoprecipitation, Mutagenesis, Western Blot, Immunostaining, Labeling, Expressing

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: Following ectopic expression of untagged NRP2B in C4–2B cells, nuclear and post-nuclear fractions were separated, and a pull-down assay was performed on the nuclear fraction with NRP2 antibody. Mass-Spectrometry was carried out with the pull-down samples. Using the genes detected in NRP2 Mass-Spectrometry. A. Sum of all the spectra associated with NRP2B pulldown sample is determined by the spectral graph. B. Venn-diagram represent the overlapping genes identified in two independent NRP2B mass-spectrometry assay. C. Group of nuclear pore proteins (Nups) identified through NRP2B Mass-Spectrometry. Schematic diagram indicating the relative positions of Nups around the nuclear pore. D. Validation of NRP2-Nup93 interactions in C4–2B. C4–2B cells were transfected with HA-tagged NRP2B. IP was carried out with HA-antibody and immunoblots were carried out with anti-Nup93. E. Endogenously, NRP2 and Nup93 interaction was validated in C4–2B and 22Rv1 cell lines. NRP2 was pull down with NRP2-specific antibody and IB with Nup93 antibody. F. NRP2B and AR interaction was carried out in C4–2B following ectopic interaction of HA-tagged NRP2B. Pulldown was carried out with HA-antibody and IP was carried out against NRP2 and AR. G. NRP2 and AR interaction was also monitored in PC3 and 22Rv1 following ectopic expression of HA-tagged NRP2B. IB was carried out against HA and AR. H. Co-IP for AR with NRP2 was carried out with pulldown of NRP2 by HA-antibody in C4–2B cells expressing wild type and mutant K892A NRP2B isoforms. NRP2B immunoblot was carried out with HA-antibody. I. IP with AR antibody was carried out to test the interaction among AR, Nup93 and NRP2 under the presence or absence of NRP2 and Nup 93 from the nuclear fraction of LNCaP C4–2B cells. An immunoblot was performed with anti-AR, anti-Nup93 and anti-NRP2 antibody. The Co-IP was carried out in the presence of 50ng/ml VEGF-C. J. PLA was carried out to validating the NRP2-AR interaction within the nucleus. Following ectopic expression of HA-tagged NRP2B, PLA was carried out with HA and endogenous AR antibodies under the presence or absence of NUP93. As a negative control, only HA-antibody was used for PLA reaction. Arrowhead indicates the immune-reactive PLA puncta. Nucleus was counter-stained with NUP98 to demarcate the nuclear periphery. DAPI used for nuclear staining. PLA quantification was shown in Bar diagram.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Expressing, Pull Down Assay, Mass Spectrometry, Transfection, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Negative Control, Staining

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: A. TFs’ binding probability within the consensus sequence derived from the NRP2-ChIP-sequencing was predicted through the heat map analysis. Consensus sequence map was generated using the indicated regions of promoter sequence from the NRP2 ChIP-Seq. B. AR motifs was analyzed using the TOMTOM-MEME-motif analyzer. C. Heat map for RNA-sequencing analysis of 200 differentially downregulated and 100 upregulated genes following depletion of NRP2B. D. Heat map of gene expression profile following the depletion of AR by siRNA. E. Box diagram representing the common up and down-regulated gene expression following AR and NRP2B depletion. F. GSEA analysis using AR-regulated genes.

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques: Binding Assay, Sequencing, Derivative Assay, ChIP-sequencing, Generated, RNA Sequencing Assay, Expressing

Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet:

Article Snippet: Wild type NRP2A (Origene, RG220706 and RC220706) and NRP2B (Origene, RG210928 and RC210928) plasmids were purchased from Origene (Rockville, MD).

Techniques:

Journal: Cell

Article Title: Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance

doi: 10.1016/j.cell.2018.12.009

Figure Lengend Snippet:

Article Snippet: Human Neuropilin 2 expression plasmid , OriGene , CAT#: RC220920.

Techniques: Subcloning, Recombinant, Isolation, Expressing, Plasmid Preparation, Software