mouse expression 430a array  (Thermo Fisher)

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