Journal: Cancer research

Article Title: Mutational landscape of pediatric acute lymphoblastic leukemia

doi: 10.1158/0008-5472.CAN-16-1303

Figure Lengend Snippet: A, mutational diagram illustrating the mutations of WHSC1. B, representative Sanger sequence tracings that show somatic E1099K mutation in 4 patients (upper and middle panel, diagnosis and the matched remission sample, respectively) and 2 ALL cell lines (RS4;11, SEM, lower panel). C, elevation of WHSC1 mRNA in B-ALL samples or B-ALL cell lines compared with normal B-cells (data were retrieved from GSE48558). ****, p<0.0001. D, patients whose ALL cells at diagnosis had higher levels of WHSC1, also had a higher likelihood to undergo relapse (GSE11877, 207 Pediatric ALL. Censored: Patients in clinical remission, or with a second malignancy, or with a toxic death as a first event were censored at the date of last contact). **, p<0.01. E, aberrant WHSC1 expression was associated with high risk leukemia and early occurrence of relapse: very early relapse (within 18 months after initial diagnosis); early relapse (>18 months after initial diagnosis but <6 months after cessation of frontline treatment); late relapse (>6 months after cessation of frontline treatment). Data were retrieved from GSE4698 (60 childhood ALL (28)). **, p<0.01. F, western blot shows the silencing effect of CRISPR-Cas9 sgRNA targeting WHSC1. G, H, silencing of WHSC1 by either shRNA or CRISPR-Cas9 guide RNAs markedly reduced the clonogenic growth of B-ALL cells RS4;11 (cell line carrying WHSC1 E1099K mutation) in methylcellulose assay. Non-target, Non-target shRNA Control. EV, empty vector control. **, p<0.01, ***, p<0.001. I, J, silencing of WHSC1 impairs in vivo cell growth of RS4;11. *, p<0.05.

Article Snippet: SDS-PAGE and Western blot analysis was performed according to standard protocol, using the following primary antibodies: WHSC1 (Santa Cruz, sc-365627), ARID1A (Millipore, 04-080), c-MYC (Cell Signaling, 9402S), CTCF (Santa Cruz, sc-271474) and GAPDH (Cell Signaling, 2118S).

Techniques: Sequencing, Mutagenesis, Biomarker Discovery, Expressing, Western Blot, CRISPR, shRNA, Methylcellulose Assay, Control, Plasmid Preparation, In Vivo

Journal: bioRxiv

Article Title: Targeting high-risk multiple myeloma genotypes with optimized anti-CD70 CAR-T cells

doi: 10.1101/2024.02.24.581875

Figure Lengend Snippet: A. Cell surface capture proteomics of myeloma (AMO-1 and MM.1S) cell lines in comparison to acute myeloid leukemia (AML) cell lines indicated surface expression of CD70. Re-analysis of primary patient sample surface proteomic datasets (NatComm = Ferguson et al, Nature Communications (2022); Blood = Anderson et al, Blood (2022)) also indicates detectable CD70 in some samples. Plot shows normalized abundance based on mass spectrometric intensity. B. Example mass spectra from MM.1S cell line demonstrating peptides from CD70 identified by cell surface proteomics. C. CoMMpass transcript data ( n = 776) demonstrating CD70 expression as a function of Revised International Staging System (R-ISS) stage (1, 2, 3) at diagnosis. D. Box plot of NSD2 (left) and CD70 (right) expression grouped by gene expression subtype in newly diagnosed multiple myeloma (NDMM) CoMMpass samples ( n = 764). Gene expression subtypes are from Zhan et al. (CD-1: Cyclin D1; CD-2: Cyclin D1 and CD20; HP: Hyperdiploid; LB: Low Bone Disease; MF: MAF; MS: MMSET; PR: Proliferation) E. Volcano plot of genes associated with CD70 expression in CoMMpass NDMM samples ( n = 764). Genes significantly associated with CD70 (FDR <0.01) are shown in red (positive) and blue (negative) with the numbers listed above and select genes labelled. F. Top Gene Set Enrichment Analysis (GSEA) Hallmark pathways associated with CD70 expression. Top 5 pathways positively (red) and negatively (blue) associated with CD70 expression are shown, and only pathways with an FDR < 0.01 are included. G. Top GSEA positional gene sets associated with CD70 expression. Only the top 5 pathways positively and negatively associated with CD70 expression are shown.

Article Snippet: Primary antibodies targeting NSD2 (Abcam Cat# ab75359), HDAC2 (Millipore Cat# 05-814), H3 (Cell Signaling Technology Cat# 14269) and H3K36me2 (Abcam Cat# ab9049) were diluted in blocking buffer at 1:1000.

Techniques: Comparison, Expressing

Journal: bioRxiv

Article Title: Targeting high-risk multiple myeloma genotypes with optimized anti-CD70 CAR-T cells

doi: 10.1101/2024.02.24.581875

Figure Lengend Snippet: A. Knockout of the driver oncogene NSD2 in t(4;14) myeloma cell lines KMS-11 and KMS-34 led to decreased surface CD70 expression by flow cytometry. n = 3 biological replicates. p -value by t -test. *p<0.05, **p<0.01. B. MM.1S or AMO.1 myeloma cells were treated with the indicated dose of azacytidine or 0.1% DMSO for 4 days, and surface CD70 assessed by flow cytometry 3 days later. p -value by 2-way ANOVA. * p <0.05, ** p <0.01. n = 3 technical replicates. C. An eXtreme Gradient Boosting (XGB) model (see Methods) extracts features of transcription factor gene expression from CoMMpass ( n = 776) that best-model CD70 expression in patient tumors. 80% of data was used as a test set with 20% left out as a training set, with subsequent 5-fold cross validation (see Methods). n = 776 total samples included in the analysis. R 2 value corresponds to Pearson correlation between model-predicted CD70 expression in the test set and measured tumor CD70 expression in CoMMpass. D. Shapley Additive Explanations (SHAP) analysis suggest expression of transcription factors most strongly impacting CD70 expression levels in CoMMpass tumors. E. Correlation of CD70 with TFAP2A and PAX5 in the CoMMpass dataset. F. Surface CD70 by flow cytometry on LP-1 cells measured after Cas9 RNP nucleofection with sgRNA targeting TFAP2A , CD70 , or scramble control. Flow cytometry mean fluorescence intensity shown as fold-change versus isotype control. p -value by t -test. *** p <0.005, ****p<0.001. n = 3 technical replicates, representative of 2 biological replicates. Mean +/− S.D. shown.

Article Snippet: Primary antibodies targeting NSD2 (Abcam Cat# ab75359), HDAC2 (Millipore Cat# 05-814), H3 (Cell Signaling Technology Cat# 14269) and H3K36me2 (Abcam Cat# ab9049) were diluted in blocking buffer at 1:1000.

Techniques: Knock-Out, Expressing, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Targeting high-risk multiple myeloma genotypes with optimized anti-CD70 CAR-T cells

doi: 10.1101/2024.02.24.581875

Figure Lengend Snippet: A. Immunoblots confirming NSD2 knockout in KMS11 and KMS34 cell lines. H3K36me2 signal decrease confirms functional loss of histone methylation in the context of NSD2 loss. Total HDAC2 and Histone H3 levels are unchanged. B. Called peaks from ATAC-seq (data from Jin et al (2018) at the CD70 locus, across normal memory B-cells (MB), plasmablasts, normal plasma cells, and malignant plasma cells derived from multiple myeloma (MM) patients (ten illustrated here; 24 total included in study). Dashed red lines highlight major ATAC-seq peak present only in myeloma patient samples. Transcription factor motifs (listed in ) extracted from this peak.

Article Snippet: Primary antibodies targeting NSD2 (Abcam Cat# ab75359), HDAC2 (Millipore Cat# 05-814), H3 (Cell Signaling Technology Cat# 14269) and H3K36me2 (Abcam Cat# ab9049) were diluted in blocking buffer at 1:1000.

Techniques: Western Blot, Knock-Out, Functional Assay, Methylation, Derivative Assay

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 expression is decreased in the proliferative-phase endometrium of patients with RIF compared with that of fertile controls (FER). (A) Heatmap of histone methyltransferases (HMT) and histone demethylase (HDM) gene levels in the RIF and FER groups. (B) EZH2 and NSD2 expression levels in endometrial stromal cells during the menstrual cycle (GES111976). The median menstrual cycle is 28 days in length. The details of staging in the menstrual cycle are described in Supplementary Table 3. M, menstrual phase; EP, early-proliferative; MP, mid-proliferative; LP, late-proliferative; ES, early-secretory; MS, mid-secretory; LS, late-secretory. (C) NSD2 mRNA levels in RIF ( n = 12) and FER ( n = 12) proliferative endometrium as measured using qRT-PCR. (D, E) Protein levels of NSD2 in the proliferative endometrium of patients with RIF ( n = 5) and FER controls ( n = 5) as measured using western blotting. (F, G) Representative immunohistochemical images depicting the expression of NSD2 in the proliferative endometrium from patients with RIF ( n = 10) and FER controls ( n = 10). The negative control was normal rabbit IgG. GE, glandular epithelium; S, stroma; * P < 0.05; ** P < 0.01.

Article Snippet: The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Negative Control

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 regulates the proliferation of HESCs. (A, B, C) Flow cytometry showing the proportion of HESCs in different cell cycle phases in the shNSD2 group compared with that in the shNC group, the mean percentage of HESCs in G0/G1 phase increased from 80.91 ± 0.69% to 93.89 ± 0.63% ( P < 0.01), the mean percentage of HESCs in S phase decreased from 15.58 ± 0.35% to 3.87 ± 0.32% ( P < 0.01), and the mean percentage of HESCs in G2/M phase decreased from 3.51 ± 0.96% to 2.24 ± 0.96% ( P = 0.11). (D) Cell Counting Kit-8 assay showing that decreased NSD2 level impaired HESCs proliferation at 72 h (the mean OD 450 decreased from 0.54 ± 0.03 to 0.47 ± 0.01, P < 0.01) and 96 h (the mean OD 450 decreased from 0.92 ± 0.06 to 0.78 ± 0.02, P < 0.01) compared with that in the shNC group. (E, F) EdU assay showing that the incorporation of the EdU label was diminished in shNSD2 cells (red). The proportion of EdU-positive cells decreased from 14.19 ± 0.70% to 4.48 ± 1.40% after NSD2 knockdown ( P < 0.01). (G, H, I) Flow cytometry showing the proportion of HESCs in different cell cycle phases in the NSD2 overexpressing group compared with that in the pLV group, the mean percentage of HESCs in G0/G1 phase decreased from 82.81 ± 0.08% to 74.18 ± 0.32% ( P < 0.01), the mean percentage of HESCs in S phase increased from 15.85 ± 0.22% to 24.24 ± 0.55% ( P < 0.01), and the mean percentage of HESCs in G2/M phase decreased from 1.34 ± 0.14% to 1.57 ± 0.67% ( P = 0.94). (J) Cell Counting Kit-8 assay showing that NSD2 overexpression promotes HESC proliferation at 96 h (the mean OD 450 increased from 1.07 ± 0.11 to 1.28 ± 0.12, P < 0.01). (K, L) EdU assay showing that the incorporation of EdU-label increased in NSD2 overexpressing cells (red). The proportion of EdU-positive cells increased from 2.95 ± 1.10% to 6.24 ± 1.67% after NSD2 overexpression ( P = 0.047). Each set of the experiments was replicated at least three times. shNC, pLV-Neo-U6; shNSD2, pLV[shRNA]-Neo-U6>hNSD2; pLV, pLV-Neo-CMV; NSD2, pLV[Exp]-Neo-CMV>hNSD2; * P < 0.05; ** P < 0.01.

Article Snippet: The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C.

Techniques: Flow Cytometry, Cell Counting, EdU Assay, Over Expression, shRNA

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 mainly regulates the enrichment of H3K36me2 in HESCs. (A, B) Downregulation of NSD2 leads to decreased H3K36me2 levels. (C, D) Upregulation of NSD2 leads to increased H3K36me2 levels. Each set of experiments was replicated at least three times. shNC, pLV-Neo-U6; shNSD2, pLV[shRNA]-Neo-U6>hNSD2; pLV, pLV-Neo-CMV; NSD2, pLV [Exp]-Neo-CMV>hNSD2; * P < 0.05; ** P < 0.01).

Article Snippet: The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C.

Techniques: shRNA

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: Reduced H3K36me2 levels in the proliferative endometrium of patients with RIF compared with those in the FER group. (A, B) Western blot showing H3K36me2 protein levels in the proliferative endometrium of patients with RIF ( n = 5) and FER controls ( n = 5). (C, D) Representative immunohistochemical images depicting the expression of NSD2 in the endometrium from patients with RIF ( n = 10) and FER controls ( n = 10). The negative control was normal rabbit IgG. GE, glandular epithelium; S, stroma; ns: not statistically different; * P < 0.05.

Article Snippet: The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C.

Techniques: Western Blot, Immunohistochemistry, Expressing, Negative Control

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 knockdown in HESCs leads to downregulated cell cycle-related gene sets. (A) Volcano plot showing differentially expressed genes after knockdown of NSD2 . (B) Enriched GO terms and KEGG pathways of DEGs between the siNSD2 group and the siNC group. (C) Enriched GO terms and KEGG pathways of upregulated DEGs between the siNSD2 group and the siNC group. (D) Enriched GO terms and KEGG pathways of downregulated DEGs between the siNSD2 group and the siNC group. (E) GSEA showing genes enriched by NSD2 knockdown in HESCs. (F) GSEA showing genes enriched between patients with RIF and the FER group.

Article Snippet: The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C.

Techniques:

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 knockdown in HESCs leads to reduced H3K36me2 modifications and the corresponding functional changes. (A) Pie chart depicting the genomic distribution of H3K36me2 peaks. (B) Heatmap showing the occupancy for H3K36me2 across the genome in shNSD2 cells and shNC cells. (C) Chromosomal views showing broadly reduced H3K36me2 peaks after NSD2 knockdown. (D) Venn diagram illustrating the overlap of CUT&Tag-Seq peaks in shNC cells. (E) Venn diagram illustrating the overlap of CUT&Tag-Seq peaks in shNSD2 cells. (F) Venn diagram of downregulated genes after knockdown of NSD2 and decreased genes in patients with RIF and the common peaks in (D), (E). (G) GO terms and KEGG pathways for genes in the overlap of genes downregulated after NSD2 knockdown, genes decreased in patients with RIF, and H3K36me2 peaks in shNC cells but not in shNSD2 cells. (H) Connection between genes, GO terms, and Kegg_Cell Cycle. (I) MCM7 and TTK mRNA levels in endometrial stromal cells during the menstrual cycle (GES111976). The details of the staging of the menstrual cycle are described in Supplementary Table 3. M, menstrual phase; EP, early-proliferative; ≤MP, mid-proliferative; LP, late-proliferative; ES, early-secretory; MS, mid-secretory; LS, late-secretory.

Article Snippet: The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C.

Techniques: Functional Assay

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 regulates MCM7 expression through H3K36me2 modifications on promoter regions. (A) IGV presentation of the enrichment of H3K36me2 in shNC and shNSD2 cells on MCM7 promoter regions. (B) ChIP-qPCR validation of the CUT&Tag-Seq in (A). (C) MCM7 is downregulated by knockdown of NSD2 . (D, E) Western blot showing that knockdown NSD2 reduces MCM7 protein levels. (F) MCM7 expression decreased in the proliferative endometrium of patients with RIF ( n = 10) compared with FER controls ( n = 10). (G, H) Protein levels of MCM7 in the proliferative endometrium of patients with RIF ( n = 5) and FER controls ( n = 5). (I, J) Representative immunohistochemical images depicting the expression of MCM7 in the endometrium from patients with RIF ( n = 10) and FER controls ( n = 10). The negative control was normal rabbit IgG. GE, glandular epithelium; S, stroma; ns: not statistically different; * P < 0.05; ** P < 0.01.

Article Snippet: The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C.

Techniques: Expressing, Western Blot, Immunohistochemistry, Negative Control

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: Working model showing how decreased NSD2 impairs the proliferation of endometrial stromal cells in patients with RIF.

Article Snippet: The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Recruitment of FBXO22 for Targeted Degradation of NSD2

doi: 10.1101/2023.11.01.564830

Figure Lengend Snippet: (A) RCH-ACV cells were treated for 11 days with 0-10 µM UNC8732 or UNC8884. Cells were collected and analyzed by immunoblotting. H3 and HDAC2 are loading controls. NSD2 protein level is normalized to HDAC2. H3K36me2 and H3K27me3 levels are normalized to H3. (B) Viability of isogenic RCH-ACV ALL cell lines determined by CellTiter-Glo after treatment with varying concentrations of UNC8732 and UNC8884 for 18 days. (C) Apoptosis of isogenic RCH-ACV cell lines detected using Annexin V/PI staining by flow cytometry after treatment with varying concentrations of UNC8732 and UNC8884 for 18 days. (D) Viability of NSD2 mutant RCH-ACV cells determined by CellTiter-Glo after pretreatment with varying concentrations of UNC8732 and UNC8884 for 18 days, followed by dexamethasone (1 µM) for 72 hours. (E) Apoptosis of NSD2 mutant RCH-ACV cells detected using Annexin V/PI staining by flow cytometry after pretreatment with varying concentrations of UNC8732 and UNC8884 for 18 days followed by dexamethasone (1 µM) for 72 hours. Error bars represent mean ± SEM from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. WT, NSD2 WT; Mut, NSD2 p.E1099K; Dex, Dexamethasone.

Article Snippet: After blocking for one hour, the membrane was incubated with primary antibodies including anti-NSD2 (Abcam, RRID:AB_1310816), anti-HDAC2 (Millipore, RRID:AB_11212215), anti-H3K36me2 (Abcam, RRID:AB_1280939), anti-H3K27me3 (Cell Signaling, RRID:AB_2616029), anti-H3 (Cell Signaling, RRID:AB_2756816) at 4°C overnight.

Techniques: Western Blot, Staining, Flow Cytometry, Mutagenesis

Journal: bioRxiv

Article Title: Recruitment of FBXO22 for Targeted Degradation of NSD2

doi: 10.1101/2023.11.01.564830

Figure Lengend Snippet: (A): Compound structures of UNC8153 (top) and UNC8732 (bottom) prodrugs and their corresponding aldehyde metabolites. (B): MS peak area ratio representing the relative levels of UNC8732 and its aldehyde metabolite in lysates generated from U2OS cells treated with UNC8732 and cultured with DMEM and 10% FBS at indicated time points. *For , 0 hours denotes mass spectrometric analysis performed immediately after compound treatment, which includes a very brief sample preparation time to process for MS. (C): MS peak area ratio representing the relative levels of UNC8732 and its associated aldehyde species in cell-free DMEM and 10% FBS at indicated time points. (D): MS peak area ratio representing the relative levels of UNC8732 and its associated aldehyde species in cell-free DMEM without FBS at indicated time points. (E): U2OS cells were treated with 2 µM UNC8732 and varying concentrations of aminoguanidine for 6h. NSD2 levels were measured by immunoblot. The experiment was performed 3 times with consistent results. Vinculin is a loading control. (F): U2OS cells were cultured in DMEM + 10% FBS or Opti-MEM with no FBS while being treated with the indicated concentrations of UNC8732 for 24h, followed by immunoblotting analysis for NSD2 levels. The experiment was performed 3 times with consistent results. Vinculin is a loading control. (G): Compound structures of UNC9801 (top) and UNC10088 (bottom) prodrugs and their corresponding hydrolyzed aldehyde products. (H): U2OS cells were treated with the specified compounds in a dose-response format for 24h. NSD2 levels were measured by an ICW assay. The data presented are from one representative experiment, including 4 technical replicates. (I): U2OS cells were treated for the indicated amount of time with UNC8732 (2 µM), UNC10088 (2 µM), UNC8153 (10 µM), or UNC9801 (10 µM), and were subsequently analyzed by immunoblotting. Vinculin is a loading control. The experiment was performed 3 times with consistent results. The data shown are representative immunoblots. (J): Cells were cultured in DMEM + 10% FBS or Opti-MEM with no FBS and were subsequently treated with indicated concentrations of UNC10088 for 24h, followed by immunoblotting analysis for NSD2 levels. The experiment was performed 3 times with consistent results. Vinculin is a loading control.

Article Snippet: After blocking for one hour, the membrane was incubated with primary antibodies including anti-NSD2 (Abcam, RRID:AB_1310816), anti-HDAC2 (Millipore, RRID:AB_11212215), anti-H3K36me2 (Abcam, RRID:AB_1280939), anti-H3K27me3 (Cell Signaling, RRID:AB_2616029), anti-H3 (Cell Signaling, RRID:AB_2756816) at 4°C overnight.

Techniques: Generated, Cell Culture, Sample Prep, Western Blot

Journal: bioRxiv

Article Title: Recruitment of FBXO22 for Targeted Degradation of NSD2

doi: 10.1101/2023.11.01.564830

Figure Lengend Snippet: (A): Schematic showing a representation of the NSD2-miniTurbo fusion protein (brown and green), putative UNC8732 mediated interaction with an unknown E3 ubiquitin ligase (blue), and the approximate radius (10 nm) of biotinylation events generated by the NSD2-miniTurbo (pink). (B): Volcano plot of BioID results showing log 2 fold change (FC) and -log 10 adjusted p-values derived from normalized protein spectral counts. Proteins were previously filtered for SAINT BFDR ≤ 0.01 in either DMSO control or UNC8732 treated conditions. Red data points represent proteins in the GO:BP protein ubiquitination term (GO:0016567), and blue data points represent proteins included in the GO:CC proteasome core complex term (GO:0005839). Significance cut-off set at adj. p-value <0.05 and fold change >1.5. (C): Histogram showing the spectral counts of NSD2 and FBXO22. Data presented are the mean ± SD of 3 independent experiments. (D): A NanoBRET protein-protein interaction assay with NanoLuc-NSD2 and FBXO22-HT in U2OS cells reveals ternary complex formation. U2OS cells were treated with 0.05-40 µM of UNC8732 24h post-transfection for 3 hours. BRET ratios (mBU) are shown relative to DMSO treatment control (= 1.00). The data presented are the mean ± SD of 3 independent experiments. (E): Knockdown of SCF FBXO22 components by siRNA in U2OS cells. UNC8732 and UNC10088 degrader treatments were performed 4 days post-siRNA transfection for 4h. The immunoblot presented is representative of three independent experiments. (F): Representative BLI sensorgrams upon the addition of increasing concentrations of UNC10088 and a fixed concentration of NSD2-PWWP1 (2 μM). SKP1-FBXO22 was loaded on SA biosensors to an average response of 1 nm. Curves are shown as the average of three independent experiments. (G): Binding curve with the average BLI response calculated based on steady-state approximation. The data presented are the mean ± SEM of three independent experiments. (H): In vitro NSD2-PWWP1 ubiquitination with the indicated sets of substrate priming machinery, UBCH5B or UBCH7 with HHARI. Each reaction also contained CDC34B. The reaction mixture was analyzed by SDS-PAGE and fluorescence scanning. NSD2-PWWP1 was subject to a sortase reaction for fluorescent labeling. The gel presented is representative of three independent experiments.

Article Snippet: After blocking for one hour, the membrane was incubated with primary antibodies including anti-NSD2 (Abcam, RRID:AB_1310816), anti-HDAC2 (Millipore, RRID:AB_11212215), anti-H3K36me2 (Abcam, RRID:AB_1280939), anti-H3K27me3 (Cell Signaling, RRID:AB_2616029), anti-H3 (Cell Signaling, RRID:AB_2756816) at 4°C overnight.

Techniques: Generated, Derivative Assay, Protein Protein Interaction Assay, Transfection, Western Blot, Concentration Assay, Binding Assay, In Vitro, SDS Page, Fluorescence, Labeling

Journal: bioRxiv

Article Title: Recruitment of FBXO22 for Targeted Degradation of NSD2

doi: 10.1101/2023.11.01.564830

Figure Lengend Snippet: (A): In-cell western quantification of NSD2 levels in U2OS cells co-treated with 1 µM of UNC8732 and 6.25-100 µM of UNC9360 (E3-recruiting handle of UNC8732) or UNC9631 (monomethylated E3-recruiting handle of UNC8732) for 6h. Data represents the average ± SEM of 4 technical replicates from 2 independent experiments. (B): Normalized fluorescence data from DSF upon incubation of co-expressed SKP1/FBXO22 with 0-100 µM UNC10088. Data represents the average ± SD of 3 independent experiments. (C): Change in melting temperature (Tm) as measured by DSF of co-expressed SKP1/FBXO22 treated with 0-100 µM UNC8732 and UNC10088. Data represents the average ±SD of 3 independent experiments. (D): AlphaFold predicted structure of FBXO22 highlighted to indicate the cumulative HDX difference signal in an HDX-MS experiment. Blue = reduced uptake, red = increased uptake, black = no FBXO22 sequence coverage. For clarity, SKP1 is not shown in the AlphaFold structure, and no changes in deuterium uptake were observed for SKP1 (Extended Data Figure 4B). (E): Normalized fluorescence data from DSF upon incubation of co-expressed SKP1/FBXO22 with 0.1-100 µM UNC10088. Error bars represent the SD of 4 technical replicates from one independent experiment. (F): Binding curve of the average BLI response for both SKP1-FBXO22 WT and C326A calculated based on steady-state approximation. Data represents the average ± SEM of three independent experiments. (G): NanoBRET assay results with NanoLuc-NSD2 and FBXO22-HT wildtype or C326A mutant performed in U2OS cells. U2OS cells were treated with the indicated concentration of UNC8732 24h post-transfection for 3 hours. BRET ratios (mBU) are shown relative to DMSO treatment control (= 1.00). Data represents the average ± SD of 3 independent experiments.

Article Snippet: After blocking for one hour, the membrane was incubated with primary antibodies including anti-NSD2 (Abcam, RRID:AB_1310816), anti-HDAC2 (Millipore, RRID:AB_11212215), anti-H3K36me2 (Abcam, RRID:AB_1280939), anti-H3K27me3 (Cell Signaling, RRID:AB_2616029), anti-H3 (Cell Signaling, RRID:AB_2756816) at 4°C overnight.

Techniques: In-Cell ELISA, Fluorescence, Incubation, Sequencing, Binding Assay, Mutagenesis, Concentration Assay, Transfection

Journal: bioRxiv

Article Title: Recruitment of FBXO22 for Targeted Degradation of NSD2

doi: 10.1101/2023.11.01.564830

Figure Lengend Snippet: (A): Chemical structure of primary amine-containing XIAP degrader, Compound 10, reported by den Besten et al. (B): MCF7 cells were treated with Compound 10 and varying concentrations of aminoguanidine (AG) for 24 hours, followed by immunoblotting analysis for XIAP levels. Actin is a loading control. (C): MCF7 cells were cultured in DMEM + 10% FBS or Opti-MEM with no FBS and subsequently treated with the indicated concentrations of Compound 10 for 24h, followed by immunoblotting analysis for XIAP levels. Vinculin is a loading control. (D): MCF7 cells were treated with Compound 10 and varying concentrations of UNC10088 and UNC8732 for 24 hours, followed by immunoblotting for XIAP and NSD2. Actin is a loading control. (E): MCF7 cells were treated with Compound 10 and varying concentrations of MLN4924 (a selective neddylation inhibitor for 24 hours, followed by immunoblotting for XIAP levels. CUL1 was blotted as a representative Cullin. Vinculin is a loading control. (F): siRNA knockdown of core SCF FBXO22 components in MCF7 cells was performed. Compound 10 treatments were performed 72h post-siRNA transfection for 24h. Vinculin is a loading control.

Article Snippet: After blocking for one hour, the membrane was incubated with primary antibodies including anti-NSD2 (Abcam, RRID:AB_1310816), anti-HDAC2 (Millipore, RRID:AB_11212215), anti-H3K36me2 (Abcam, RRID:AB_1280939), anti-H3K27me3 (Cell Signaling, RRID:AB_2616029), anti-H3 (Cell Signaling, RRID:AB_2756816) at 4°C overnight.

Techniques: Western Blot, Cell Culture, Transfection

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 expression is decreased in the proliferative-phase endometrium of patients with RIF compared with that of fertile controls (FER). (A) Heatmap of histone methyltransferases (HMT) and histone demethylase (HDM) gene levels in the RIF and FER groups. (B) EZH2 and NSD2 expression levels in endometrial stromal cells during the menstrual cycle (GES111976). The median menstrual cycle is 28 days in length. The details of staging in the menstrual cycle are described in Supplementary Table 3. M, menstrual phase; EP, early-proliferative; MP, mid-proliferative; LP, late-proliferative; ES, early-secretory; MS, mid-secretory; LS, late-secretory. (C) NSD2 mRNA levels in RIF ( n = 12) and FER ( n = 12) proliferative endometrium as measured using qRT-PCR. (D, E) Protein levels of NSD2 in the proliferative endometrium of patients with RIF ( n = 5) and FER controls ( n = 5) as measured using western blotting. (F, G) Representative immunohistochemical images depicting the expression of NSD2 in the proliferative endometrium from patients with RIF ( n = 10) and FER controls ( n = 10). The negative control was normal rabbit IgG. GE, glandular epithelium; S, stroma; * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Negative Control

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 regulates the proliferation of HESCs. (A, B, C) Flow cytometry showing the proportion of HESCs in different cell cycle phases in the shNSD2 group compared with that in the shNC group, the mean percentage of HESCs in G0/G1 phase increased from 80.91 ± 0.69% to 93.89 ± 0.63% ( P < 0.01), the mean percentage of HESCs in S phase decreased from 15.58 ± 0.35% to 3.87 ± 0.32% ( P < 0.01), and the mean percentage of HESCs in G2/M phase decreased from 3.51 ± 0.96% to 2.24 ± 0.96% ( P = 0.11). (D) Cell Counting Kit-8 assay showing that decreased NSD2 level impaired HESCs proliferation at 72 h (the mean OD 450 decreased from 0.54 ± 0.03 to 0.47 ± 0.01, P < 0.01) and 96 h (the mean OD 450 decreased from 0.92 ± 0.06 to 0.78 ± 0.02, P < 0.01) compared with that in the shNC group. (E, F) EdU assay showing that the incorporation of the EdU label was diminished in shNSD2 cells (red). The proportion of EdU-positive cells decreased from 14.19 ± 0.70% to 4.48 ± 1.40% after NSD2 knockdown ( P < 0.01). (G, H, I) Flow cytometry showing the proportion of HESCs in different cell cycle phases in the NSD2 overexpressing group compared with that in the pLV group, the mean percentage of HESCs in G0/G1 phase decreased from 82.81 ± 0.08% to 74.18 ± 0.32% ( P < 0.01), the mean percentage of HESCs in S phase increased from 15.85 ± 0.22% to 24.24 ± 0.55% ( P < 0.01), and the mean percentage of HESCs in G2/M phase decreased from 1.34 ± 0.14% to 1.57 ± 0.67% ( P = 0.94). (J) Cell Counting Kit-8 assay showing that NSD2 overexpression promotes HESC proliferation at 96 h (the mean OD 450 increased from 1.07 ± 0.11 to 1.28 ± 0.12, P < 0.01). (K, L) EdU assay showing that the incorporation of EdU-label increased in NSD2 overexpressing cells (red). The proportion of EdU-positive cells increased from 2.95 ± 1.10% to 6.24 ± 1.67% after NSD2 overexpression ( P = 0.047). Each set of the experiments was replicated at least three times. shNC, pLV-Neo-U6; shNSD2, pLV[shRNA]-Neo-U6>hNSD2; pLV, pLV-Neo-CMV; NSD2, pLV[Exp]-Neo-CMV>hNSD2; * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight.

Techniques: Flow Cytometry, Cell Counting, EdU Assay, Knockdown, Over Expression, shRNA

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 mainly regulates the enrichment of H3K36me2 in HESCs. (A, B) Downregulation of NSD2 leads to decreased H3K36me2 levels. (C, D) Upregulation of NSD2 leads to increased H3K36me2 levels. Each set of experiments was replicated at least three times. shNC, pLV-Neo-U6; shNSD2, pLV[shRNA]-Neo-U6>hNSD2; pLV, pLV-Neo-CMV; NSD2, pLV [Exp]-Neo-CMV>hNSD2; * P < 0.05; ** P < 0.01).

Article Snippet: The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight.

Techniques: shRNA

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: Reduced H3K36me2 levels in the proliferative endometrium of patients with RIF compared with those in the FER group. (A, B) Western blot showing H3K36me2 protein levels in the proliferative endometrium of patients with RIF ( n = 5) and FER controls ( n = 5). (C, D) Representative immunohistochemical images depicting the expression of NSD2 in the endometrium from patients with RIF ( n = 10) and FER controls ( n = 10). The negative control was normal rabbit IgG. GE, glandular epithelium; S, stroma; ns: not statistically different; * P < 0.05.

Article Snippet: The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight.

Techniques: Western Blot, Immunohistochemical staining, Expressing, Negative Control

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 knockdown in HESCs leads to downregulated cell cycle-related gene sets. (A) Volcano plot showing differentially expressed genes after knockdown of NSD2 . (B) Enriched GO terms and KEGG pathways of DEGs between the siNSD2 group and the siNC group. (C) Enriched GO terms and KEGG pathways of upregulated DEGs between the siNSD2 group and the siNC group. (D) Enriched GO terms and KEGG pathways of downregulated DEGs between the siNSD2 group and the siNC group. (E) GSEA showing genes enriched by NSD2 knockdown in HESCs. (F) GSEA showing genes enriched between patients with RIF and the FER group.

Article Snippet: The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight.

Techniques: Knockdown

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 knockdown in HESCs leads to reduced H3K36me2 modifications and the corresponding functional changes. (A) Pie chart depicting the genomic distribution of H3K36me2 peaks. (B) Heatmap showing the occupancy for H3K36me2 across the genome in shNSD2 cells and shNC cells. (C) Chromosomal views showing broadly reduced H3K36me2 peaks after NSD2 knockdown. (D) Venn diagram illustrating the overlap of CUT&Tag-Seq peaks in shNC cells. (E) Venn diagram illustrating the overlap of CUT&Tag-Seq peaks in shNSD2 cells. (F) Venn diagram of downregulated genes after knockdown of NSD2 and decreased genes in patients with RIF and the common peaks in (D), (E). (G) GO terms and KEGG pathways for genes in the overlap of genes downregulated after NSD2 knockdown, genes decreased in patients with RIF, and H3K36me2 peaks in shNC cells but not in shNSD2 cells. (H) Connection between genes, GO terms, and Kegg_Cell Cycle. (I) MCM7 and TTK mRNA levels in endometrial stromal cells during the menstrual cycle (GES111976). The details of the staging of the menstrual cycle are described in Supplementary Table 3. M, menstrual phase; EP, early-proliferative; ≤MP, mid-proliferative; LP, late-proliferative; ES, early-secretory; MS, mid-secretory; LS, late-secretory.

Article Snippet: The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight.

Techniques: Knockdown, Functional Assay

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: NSD2 regulates MCM7 expression through H3K36me2 modifications on promoter regions. (A) IGV presentation of the enrichment of H3K36me2 in shNC and shNSD2 cells on MCM7 promoter regions. (B) ChIP-qPCR validation of the CUT&Tag-Seq in (A). (C) MCM7 is downregulated by knockdown of NSD2 . (D, E) Western blot showing that knockdown NSD2 reduces MCM7 protein levels. (F) MCM7 expression decreased in the proliferative endometrium of patients with RIF ( n = 10) compared with FER controls ( n = 10). (G, H) Protein levels of MCM7 in the proliferative endometrium of patients with RIF ( n = 5) and FER controls ( n = 5). (I, J) Representative immunohistochemical images depicting the expression of MCM7 in the endometrium from patients with RIF ( n = 10) and FER controls ( n = 10). The negative control was normal rabbit IgG. GE, glandular epithelium; S, stroma; ns: not statistically different; * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight.

Techniques: Expressing, ChIP-qPCR, Biomarker Discovery, Knockdown, Western Blot, Immunohistochemical staining, Negative Control

Journal: Reproduction (Cambridge, England)

Article Title: Decreased NSD2 impairs stromal cell proliferation in human endometrium via reprogramming H3K36me2

doi: 10.1530/REP-23-0254

Figure Lengend Snippet: Working model showing how decreased NSD2 impairs the proliferation of endometrial stromal cells in patients with RIF.

Article Snippet: The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight.

Techniques: