Journal: bioRxiv

Article Title: Menin inhibition impairs metastatic colonization of Ewing sarcoma

doi: 10.1101/2025.11.10.687648

Figure Lengend Snippet: ( A ) MYC transcript levels in control and MEN1-KO cells as determined by RT-qPCR. ns p > 0.05, * p ≤ 0.05. ( B ) MYC transcript counts from RNA-seq of A673 control and MEN1-KO tumors. ( C ) Western blot of MYC and Vinculin levels in A673 and TC32 control and MEN1-KO cells ( D ) Western blot of MYC and Vinculin in tumors derived from A673 control and MEN1-KO cells. ( E ) Co-immunoprecipitations with an anti-Menin antibody were performed on A673, CHLA10 and U2OS nuclear extracts and immunoblotted for Menin, MYC, MLL2 and WDR5. ( F ) Co-immunoprecipitations with an anti-MYC antibody were performed on A673 and CHLA10 nuclear extracts and immunoblotted for MYC, Menin, MAX, MLL2 and WDR5.

Article Snippet: Membranes were blocked with Intercept (TBS) Blocking Buffer (LICORbio 927-60001) and probed for the primary antibodies GAPDH Rabbit mAb (Cell Signaling 2118), MAX Rabbit pAb (Cell Signaling 4739), Menin Goat pAb (Bethyl A300-106A), MLL1-C (C-terminal) Rabbit pAb (Bethyl A300-374A), MLL2-C (C-terminal) Rabbit mAb (Cell Signaling 63735), MYC Rabbit mAb (Cell Signaling 18583), Vinculin Rabbit mAb (Cell Signaling 13901) or WDR5 Rabbit pAb (Bethyl A302-430A) followed by the secondary antibody Goat anti-Rabbit 800CW (Licor 926-32211), Donkey anti-Rabbit 800CW (Licor 926-32213) or Donkey anti-Goat 680RD (Licor 926-68074).

Techniques: Control, Quantitative RT-PCR, RNA Sequencing, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Menin inhibition impairs metastatic colonization of Ewing sarcoma

doi: 10.1101/2025.11.10.687648

Figure Lengend Snippet: ( A ) Incucyte proliferation assay plotting percent confluence for A673 and TC32 cells treated with 0.1% DMSO or 10 µM VTP50469 starting at time 0. Representative of n=3-4). ( B and C ) Invasion of cells from a sphere of A673 cells embedded in rat tail collagen and treated with 0.1% DMSO or 10 µM VTP50469 for 5 days. ( B ) Representative phase and phalloidin (red)/DAPI (blue) stained images are shown (scale bars=100 µm). ( C ) Plot of circularity quantified from replicate spheroids treated as in ( B ). ( D ) A673 and TC32 cells were treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours and genes that were significantly downregulated in VTP50469-treated cells (padj<0.05) were overlapped with genes downregulated in MEN1-KO cells. The top 5 enriched Hallmark pathways for the overlapping downregulated genes in each cell line are shown along with the overlap/total genes for each pathway. The green arrows indicate pathways that were reactivated in A673 MEN1-KO tumors (see ). ( E ) MYC transcript levels in A673, CHLA10 and TC32 cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours as determined by RT-qPCR (ns p > 0.05). ( F ) Western of MYC and Vinculin in cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours. ( G ) Co-immunoprecipitations with an anti-Menin antibody were performed on nuclear extracts from A673 and CHLA10 cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours and immunoblotted for Menin, MYC, MLL2 and WDR5.

Article Snippet: Membranes were blocked with Intercept (TBS) Blocking Buffer (LICORbio 927-60001) and probed for the primary antibodies GAPDH Rabbit mAb (Cell Signaling 2118), MAX Rabbit pAb (Cell Signaling 4739), Menin Goat pAb (Bethyl A300-106A), MLL1-C (C-terminal) Rabbit pAb (Bethyl A300-374A), MLL2-C (C-terminal) Rabbit mAb (Cell Signaling 63735), MYC Rabbit mAb (Cell Signaling 18583), Vinculin Rabbit mAb (Cell Signaling 13901) or WDR5 Rabbit pAb (Bethyl A302-430A) followed by the secondary antibody Goat anti-Rabbit 800CW (Licor 926-32211), Donkey anti-Rabbit 800CW (Licor 926-32213) or Donkey anti-Goat 680RD (Licor 926-68074).

Techniques: Proliferation Assay, Staining, Quantitative RT-PCR, Western Blot

Journal: Science Advances

Article Title: Condensin II is anchored by TFIIIC and H3K4me3 in the mammalian genome and supports the expression of active dense gene clusters

doi: 10.1126/sciadv.1700191

Figure Lengend Snippet: ( A ) H3K4me3 and H3K27ac, but not H3K9me3 or H3K27me3, colocalized with CTS. ( B and C ) Pull-down assays were performed by mixing nuclear extracts from HEK293T cells with biotinylated histone peptides (HP) modified as indicated. Condensin II and TFIIIC-220 were pulled down with H3K4me3, but not H3K27ac, histone peptides. ( D ) Pull-down assays were performed by mixing full-length proteins of condensin II generated by in vitro translation with biotinylated histone peptides with and without H3K4me3 modification. NCAPD3 and SMC2 interacted with the H3K4me3 histone peptide. ( E and F ) Co-IP with anti-SMC2 and anti-NCAPD3 antibodies pulled down chromatin with H3K4me3, but not H3K27ac. ( G ) HEAT repeat domains from NCAPD3 or NCAPG2 and the C terminus of NCAPD3 were identified and cloned out as described . Pull-down assays were performed by mixing HEAT repeat domains from NCAPD3 or NCAPG2 or the C terminus of NCAPD3 with biotinylated H3K4me3 histone peptides. HEAT repeat domains from NCAPD3 interacted with the H3K4me3 peptide. Three independent experiments were performed for each assay.

Article Snippet: Antibodies recognizing H3K4me3 (39915), H3K27ac (39133), and WDR5 (61485) were purchased from Active Motif.

Techniques: Modification, Generated, In Vitro, Co-Immunoprecipitation Assay, Clone Assay

Journal: Science Advances

Article Title: Condensin II is anchored by TFIIIC and H3K4me3 in the mammalian genome and supports the expression of active dense gene clusters

doi: 10.1126/sciadv.1700191

Figure Lengend Snippet: ( A ) CTS, but not CFTS, were enriched at TAD boundaries in mESCs. ( B ) Twenty-eight percent of CTS were within 50 kb of a TAD boundary in mESCs, in contrast to 13% TAD boundary association of random control peaks by chance ( P = 2.1 × 10 −16 , χ 2 test). ( C ) CTS, but not CFTS, were enriched at TAD boundaries in human HEK293 cells. ( D ) Representative image from a genome browser showing tracks of in situ Hi-C and ChIP-seq of NCAPH2, TFIIIC-220, and H3K4me3. ( E ) Fifty-five percent of CTS were within 50 kb of a TAD boundary in mESCs, in contrast to 41% TAD boundary association of random control peaks by chance ( P = 2.11 ×10 −8 , χ 2 test). ( F ) The total number of genes that are significantly down-regulated upon condensin knockdown is 1516 in mESCs. Eighty-five percent (1293) of genes that were significantly down-regulated upon NCAPH2 knockdown were within 50 kb of a TAD boundary. ( G ) A total of 2415 genes are significantly down-regulated upon condensin knockdown in HEK293 cells. Sixty-five percent (1570) of genes that were significantly down-regulated upon NCAPH2 knockdown were within 50 kb of a TAD boundary.

Article Snippet: Antibodies recognizing H3K4me3 (39915), H3K27ac (39133), and WDR5 (61485) were purchased from Active Motif.

Techniques: In Situ, Hi-C, ChIP-sequencing

Journal: Science Advances

Article Title: Condensin II is anchored by TFIIIC and H3K4me3 in the mammalian genome and supports the expression of active dense gene clusters

doi: 10.1126/sciadv.1700191

Figure Lengend Snippet: ( A ) Contact profiles of control and NCAPH2 knockdown mESCs at histone gene loci measured by 4C-seq. Knockdown of NCAPH2 reduced interactions between two histone gene loci (1.86 ± 0.25–fold change, P = 7.81 × 10 −8 ). The green dotted line indicates the anchor point. ( B ) Knockdown of NCAPH2 by short hairpin RNA (shRNA) in mESCs significantly reduced the expression of histone genes in the loci shown in (A). Error bars, SEM. ( C ) Knockdown of NCAPH2 in HEK293 cells by siRNA significantly reduced the expression of histone genes. Error bars, SEM. ( D ) The size of the histone clusters in NCAPH2 knockdown mESCs is significantly smaller than the sizes of control cells, indicating that the formation of the clusters is disrupted in NCAPH2 knockdown cells. Fluorescent staining was performed with an antibody to NPAT, a protein that labels the histone clusters . Error bars, SD. FWHM, full width at half maximum. ( E ) Working model for how condensin II and TFIIIC complexes tether dense active promoters in the mammalian genome: Condensin II and TFIIIC complexes (CTS) are colocalized at densely clustered active promoters at TAD boundaries. Condensin II may support the expression of the genes within the boundaries. Some TADs associated with the lamina are less transcriptionally active. The lower right inset shows interaction between CTS that are associated with transcriptionally active promoters. The upper right inset depicts details of the CTS, including condensin II (H, NCAPH2; D, NCAPD3; G, NCAPG2), the TFIIIC complex, the B box–like sequence, and the H3K4me3 histone modification. Condensin II may be recruited to the chromatin, in part, by the HEAT domain of NCAPD3 binding to the H3K4me3 histone modification and, in part, by the interaction with TFIIIC.

Article Snippet: Antibodies recognizing H3K4me3 (39915), H3K27ac (39133), and WDR5 (61485) were purchased from Active Motif.

Techniques: shRNA, Expressing, Staining, Sequencing, Modification, Binding Assay

Journal: bioRxiv

Article Title: Menin inhibition impairs metastatic colonization of Ewing sarcoma

doi: 10.1101/2025.11.10.687648

Figure Lengend Snippet: ( A ) CRISPR guides in exons 2 (guide 1) and 3 (guide 5) used to knock out MEN1 . ( B ) MEN1 expression in control and MEN1-KO clones. RT-qPCR determined expression levels and p-values plotted relative to control cell lines. ( C) Western blot of Menin, MLL1, MLL2 and GAPDH in control and MEN1-KO clones. ( D ) Incucyte proliferation assays of control and MEN1-KO clones plotting percent confluence over time. Representative of n=3-4. ( E ) Subcutaneous tumor growth of A673 and TC32 control and MEN1-KO cells in NSG mice. Tumor volume is plotted over time for each mouse. The mean tumor size in mice from each cell line at endpoint was used to determine p values. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Membranes were blocked with Intercept (TBS) Blocking Buffer (LICORbio 927-60001) and probed for the primary antibodies GAPDH Rabbit mAb (Cell Signaling 2118), MAX Rabbit pAb (Cell Signaling 4739), Menin Goat pAb (Bethyl A300-106A), MLL1-C (C-terminal) Rabbit pAb (Bethyl A300-374A), MLL2-C (C-terminal) Rabbit mAb (Cell Signaling 63735), MYC Rabbit mAb (Cell Signaling 18583), Vinculin Rabbit mAb (Cell Signaling 13901) or WDR5 Rabbit pAb (Bethyl A302-430A) followed by the secondary antibody Goat anti-Rabbit 800CW (Licor 926-32211), Donkey anti-Rabbit 800CW (Licor 926-32213) or Donkey anti-Goat 680RD (Licor 926-68074).

Techniques: CRISPR, Knock-Out, Expressing, Control, Clone Assay, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Menin inhibition impairs metastatic colonization of Ewing sarcoma

doi: 10.1101/2025.11.10.687648

Figure Lengend Snippet: ( A ) MYC transcript levels in control and MEN1-KO cells as determined by RT-qPCR. ns p > 0.05, * p ≤ 0.05. ( B ) MYC transcript counts from RNA-seq of A673 control and MEN1-KO tumors. ( C ) Western blot of MYC and Vinculin levels in A673 and TC32 control and MEN1-KO cells ( D ) Western blot of MYC and Vinculin in tumors derived from A673 control and MEN1-KO cells. ( E ) Co-immunoprecipitations with an anti-Menin antibody were performed on A673, CHLA10 and U2OS nuclear extracts and immunoblotted for Menin, MYC, MLL2 and WDR5. ( F ) Co-immunoprecipitations with an anti-MYC antibody were performed on A673 and CHLA10 nuclear extracts and immunoblotted for MYC, Menin, MAX, MLL2 and WDR5.

Article Snippet: Membranes were blocked with Intercept (TBS) Blocking Buffer (LICORbio 927-60001) and probed for the primary antibodies GAPDH Rabbit mAb (Cell Signaling 2118), MAX Rabbit pAb (Cell Signaling 4739), Menin Goat pAb (Bethyl A300-106A), MLL1-C (C-terminal) Rabbit pAb (Bethyl A300-374A), MLL2-C (C-terminal) Rabbit mAb (Cell Signaling 63735), MYC Rabbit mAb (Cell Signaling 18583), Vinculin Rabbit mAb (Cell Signaling 13901) or WDR5 Rabbit pAb (Bethyl A302-430A) followed by the secondary antibody Goat anti-Rabbit 800CW (Licor 926-32211), Donkey anti-Rabbit 800CW (Licor 926-32213) or Donkey anti-Goat 680RD (Licor 926-68074).

Techniques: Control, Quantitative RT-PCR, RNA Sequencing, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Menin inhibition impairs metastatic colonization of Ewing sarcoma

doi: 10.1101/2025.11.10.687648

Figure Lengend Snippet: ( A ) Incucyte proliferation assay plotting percent confluence for A673 and TC32 cells treated with 0.1% DMSO or 10 µM VTP50469 starting at time 0. Representative of n=3-4). ( B and C ) Invasion of cells from a sphere of A673 cells embedded in rat tail collagen and treated with 0.1% DMSO or 10 µM VTP50469 for 5 days. ( B ) Representative phase and phalloidin (red)/DAPI (blue) stained images are shown (scale bars=100 µm). ( C ) Plot of circularity quantified from replicate spheroids treated as in ( B ). ( D ) A673 and TC32 cells were treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours and genes that were significantly downregulated in VTP50469-treated cells (padj<0.05) were overlapped with genes downregulated in MEN1-KO cells. The top 5 enriched Hallmark pathways for the overlapping downregulated genes in each cell line are shown along with the overlap/total genes for each pathway. The green arrows indicate pathways that were reactivated in A673 MEN1-KO tumors (see ). ( E ) MYC transcript levels in A673, CHLA10 and TC32 cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours as determined by RT-qPCR (ns p > 0.05). ( F ) Western of MYC and Vinculin in cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours. ( G ) Co-immunoprecipitations with an anti-Menin antibody were performed on nuclear extracts from A673 and CHLA10 cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours and immunoblotted for Menin, MYC, MLL2 and WDR5.

Article Snippet: Membranes were blocked with Intercept (TBS) Blocking Buffer (LICORbio 927-60001) and probed for the primary antibodies GAPDH Rabbit mAb (Cell Signaling 2118), MAX Rabbit pAb (Cell Signaling 4739), Menin Goat pAb (Bethyl A300-106A), MLL1-C (C-terminal) Rabbit pAb (Bethyl A300-374A), MLL2-C (C-terminal) Rabbit mAb (Cell Signaling 63735), MYC Rabbit mAb (Cell Signaling 18583), Vinculin Rabbit mAb (Cell Signaling 13901) or WDR5 Rabbit pAb (Bethyl A302-430A) followed by the secondary antibody Goat anti-Rabbit 800CW (Licor 926-32211), Donkey anti-Rabbit 800CW (Licor 926-32213) or Donkey anti-Goat 680RD (Licor 926-68074).

Techniques: Proliferation Assay, Staining, Quantitative RT-PCR, Western Blot

Journal: Nucleic Acids Research

Article Title: Three classes of response elements for human PRC2 and MLL1/2–Trithorax complexes

doi: 10.1093/nar/gky595

Figure Lengend Snippet: Enrichment of H3 marks and PRC2 at the endogenous loci of PRE-like DNA fragments. ( A ) ChIP-qPCR of H3 (open bars), H3K27me3 (dotted open bars) and H3K4me3 (filled solid bars) at the endogenous loci of PRE-like DNA fragments in HeLa cells shown as % input. ( B ) ChIP-qPCR of EZH2 (open bars) and EED (filled solid bars) shown as % input. ( C ) The expression of SUZ12 in SUZ12 shRNA knockdown cell line was significantly reduced as compared to that in control cell line treated with scrambled shRNA. ( D ) The luciferase activity of PRE-like DNA fragments in SUZ12 knockdown cells (filled solid bars) compared to control cell line treated with scrambled shRNA (open bars). The RLU at Gene Desert was considered as 100%. ( E ) ChIP-qPCR of EED at the endogenous loci of PRE-like DNA fragments in SUZ12 knockdown cell line shown as % input. In a, b, d, e , data were represented as mean ± SD from at least three biological replicates that were performed on different samples on different days. The −1, −2 labels in a, b, e reflect the use of multiple primers in qPCR as listed in . The P values of Student's t -test comparing SUZ12 shRNA and scrambled shRNA ( D, E ) were showed as * P < 0.05, ** P < 0.005 and *** P < 0.001.

Article Snippet: The following antibodies were used for chromatin immunoprecipitation (ChIP): normal mouse IgG from Millipore (Catalog # 17-662), normal rabbit IgG from Santa Cruz (Catalog # sc-2027), anti-EZH2 from Millipore (Catalog # 17-10044), anti-EED from Millipore (Catalog # 05-1320), anti-WDR5 from Bethyl Lab (Catalog # 429A), anti-MLL1 from Santa Cruz (Catalog # sc-20153), anti-H3 from Active Motif (Catalog # 61277), anti-H3K4me3 from Active Motif (Catalog # 39915), anti-H3K4me3 from Active Motif (Catalog # 61017), and anti-YY1 from Santa Cruz (Catalog # sc-7341 X).

Techniques: Expressing, shRNA, Luciferase, Activity Assay

Journal: Nucleic Acids Research

Article Title: Three classes of response elements for human PRC2 and MLL1/2–Trithorax complexes

doi: 10.1093/nar/gky595

Figure Lengend Snippet: Enrichment of H3 marks and MLL1/2-TrxG complex at the endogenous loci of TRE-like DNA fragments. ( A ) ChIP-qPCR of H3 (open bars), H3K27me3 (dotted open bars) and H3K4me3 (filled solid bars) at the endogenous loci of TRE-like DNA fragments in HeLa cells shown as % input. ( B ) ChIP-qPCR of MLL1 (open bars) and WDR5 (filled solid bars) shown as % input. ( C ) The expression of WDR5 in WDR5 shRNA knockdown cell nuclear extract was significantly reduced as compared to that in control cell line treated with scrambled shRNA. ( D ) The luciferase activity of TRE-like DNA fragments in WDR5 knockdown cells (filled solid bars) compared to control cell line treated with scrambled shRNA (open bars). The RLU at Gene Desert was considered as 100%. ( E ) ChIP-qPCR of MLL1 at the endogenous loci of TRE-like DNA fragments in WDR5 knockdown cell line shown as % input. In a, b, d, e , data were represented as mean ± SD from at least three biological replicates that were performed on different samples on different days. The −1, −2 labels in a, b, e reflect the use of multiple primers in qPCR as listed in . The P values of Student's t -test comparing WDR5 shRNA and scrambled shRNA ( D, E ) were showed as * P < 0.05, ** P < 0.005 and *** P < 0.001.

Article Snippet: The following antibodies were used for chromatin immunoprecipitation (ChIP): normal mouse IgG from Millipore (Catalog # 17-662), normal rabbit IgG from Santa Cruz (Catalog # sc-2027), anti-EZH2 from Millipore (Catalog # 17-10044), anti-EED from Millipore (Catalog # 05-1320), anti-WDR5 from Bethyl Lab (Catalog # 429A), anti-MLL1 from Santa Cruz (Catalog # sc-20153), anti-H3 from Active Motif (Catalog # 61277), anti-H3K4me3 from Active Motif (Catalog # 39915), anti-H3K4me3 from Active Motif (Catalog # 61017), and anti-YY1 from Santa Cruz (Catalog # sc-7341 X).

Techniques: Expressing, shRNA, Luciferase, Activity Assay

Journal: Nucleic Acids Research

Article Title: Three classes of response elements for human PRC2 and MLL1/2–Trithorax complexes

doi: 10.1093/nar/gky595

Figure Lengend Snippet: Effects of putative PREs and TREs on transcription in an identical genomic environment. A) . Schematic illustration of the strategy that integrated the core sequences of the putative PREs and TREs (2FRT-PRE/TRE-YY1pLuc donor) into AAVS1 locus by CRISPR-Cas9. B) . Dual luciferases assay in HeLa cells carrying stably integrated core sequences comparing FLP(-) samples (open bars) and FLP(+) samples (filled solid bars). Data are represented as mean ± SD from at least three replicates. The P value of Student's t -test comparing FLP(+) and FLP(-) for each integrated core sequence was shown as * for P <0.05, ** for P <0.005 and *** for P <0.001. C) . ChIP-qPCR of H3 (dotted open bars), H3K27me3 (open bars) and H3K4me3 (filled solid bars) at the promoter region downstream of the integrated core sequences shown as % input. D) . ChIP-qPCR results of EZH2 (open bars) and WDR5 (filled solid bars) at the promoter region downstream of the integrated core sequences shown as % input.

Article Snippet: The following antibodies were used for chromatin immunoprecipitation (ChIP): normal mouse IgG from Millipore (Catalog # 17-662), normal rabbit IgG from Santa Cruz (Catalog # sc-2027), anti-EZH2 from Millipore (Catalog # 17-10044), anti-EED from Millipore (Catalog # 05-1320), anti-WDR5 from Bethyl Lab (Catalog # 429A), anti-MLL1 from Santa Cruz (Catalog # sc-20153), anti-H3 from Active Motif (Catalog # 61277), anti-H3K4me3 from Active Motif (Catalog # 39915), anti-H3K4me3 from Active Motif (Catalog # 61017), and anti-YY1 from Santa Cruz (Catalog # sc-7341 X).

Techniques: CRISPR, Stable Transfection, Sequencing