Journal: Journal of Biomedical Science

Article Title: PPM1D is directly degraded by proteasomes in a ubiquitination-independent manner through its carboxyl-terminal region

doi: 10.1186/s12929-025-01185-z

Figure Lengend Snippet: Proteasome activators PSMD14 and PSME3 promote proteasomal degradation of PPM1D. A Schematic representation of the binding protein identification workflow. FLAG-PPM1D-C-interacting proteins were immunoprecipitated from HEK293T cells treated with 10 μM MG132 for 6 h. The immunoprecipitated proteins were subsequently identified using LC–MS/MS analysis, followed by label-free quantification. B The 19S proteasome regulatory subunit PSMD14 and the proteasome activator PSME3 were identified as putative interaction partners of FLAG-PPM1D-C. PPM1D-C-interacting proteins associated with proteasome subunits are presented. The heatmap depicts the relative abundance of each protein compared to control cells. C In vivo interaction of PSMD14 and PSME3 with FLAG-PPM1D-C was assessed by co-immunoprecipitation (n = 3). Whole cell lysates from HEK293T cells stably expressing FLAG-PPM1D-C were subjected to immunoprecipitation using an anti-FLAG antibody. The immunoprecipitates were then analyzed by immunoblotting with antibodies against FLAG, PSMD14, and PSME3. D , E HEK293T cells were transfected with siRNAs targeting PSME3 (n = 6) and PSMD14 (n = 3). After 48 h, cells were harvested and subjected to immunoblot analysis using antibodies against PPM1D, PSME3, PSMD14, NCOA3/SRC3, CDKN1A/p21, c-Myc, and TP53. Representative immunoblots ( D ) and quantification ( E ) of PPM1D protein levels are shown. Protein levels were normalized to vinculin. Data represent the mean ± SD

Article Snippet: The following primary antibodies were used: anti-FLAG M2 antibody (Sigma-Aldrich Cat# A8592, RRID:AB_439702, 1:2000 dilution), anti-PPM1D (Cell Signaling Technology, Danvers, MA, USA, 1:2000 dilution), anti-PSMD14 (Proteintech Cat# 12059-1-AP, RRID:AB_2170454, Rosemont, IL, USA, 1:2000 dilution), anti-PSME3 (Proteintech Cat# 14907-1-AP, RRID:AB_2171098, 1:2000 dilution), anti-c-Myc (Cell Signaling Technology Cat# 5605, RRID:AB_1903938, 1:2000 dilution), anti-NCOA4 (Bethyl Cat# A302-272A, RRID:AB_1850160, Montgomery, TX, USA, 1:2000 dilution), anti-vinculin (Sigma-Aldrich Cat# V9131, RRID:AB_477629, 1:50,000 dilution), anti-GAPDH (Thermo Fisher Scientific Cat# AM4300, RRID:AB_2536381, 1:100,000 dilution), anti-NCOA3/SRC3 (Cell Signaling Technology Cat# 2126, RRID:AB_823642, 1:2000 dilution), anti-CDKN1A/p21 (Cell Signaling Technology Cat# 2947, RRID:AB_823586, 1:2000 dilution), anti-TP53 (Cell Signaling Technology Cat# 2524, RRID:AB_331743, 1:2000 dilution), and anti-NQO1 (Proteintech Cat# 11451-1-AP, RRID:AB_2298729, 1:2000 dilution).

Techniques: Binding Assay, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Control, In Vivo, Stable Transfection, Expressing, Western Blot, Transfection

Journal: bioRxiv

Article Title: p53Y220C-BET-bifunctionals (tPRIMEs) drive p53Y220C-mutant cancer cells into apoptosis

doi: 10.1101/2025.08.13.669398

Figure Lengend Snippet: p53Y220C-tPRIMEs markedly induce p53 target genes. A. Hierarchical cluster analysis of genes identified as significantly upregulated in RNA-sequencing by 24h treatment with tPRIME-2, tPRIME-3 or control ligands compared to DMSO. Pathway analysis of cluster of genes significantly upregulated by tPRIME-2/3 compared to control ligands. B. qPCR analysis of the p53 target genes CDKN1A/p21, MDM2, BBC3, ATF3, TP53I3 in NUGC3 cells treated with serial dilution of tPRIME-3, tPRIME-5 or control ligands. C. Western Blot analysis of p53-dependent and apoptotic proteins in NUGC3 cells treated for 24h with 1μM of tPRIME-5 or control ligands. D. LC-MS/MS analysis of proteomic changes across multiple p53Y220C-mutant cell lines treated for 24h with 1μM of tPRIME-5 compared to p53Y220C+BET-ligand combination.

Article Snippet: The following list of primary antibodies was used: anti-p53 (SantaCruz, #sc-126), anti-p21 (SantaCruz, #sc- 53870), anti-NQO1 (CST, #3187), anti-TP53I3 (Origene, #TA503656), anti-PUMA (CST, #12450), anti-cleaved Parp (CST, #5625), anti-Parp (CST, #9542), anti-MDM2 (CST, #86934), anti-BRD2 (CST, #5848), anti-BRD3 (CST, #94032), anti-BRD4 (CST, #13440), anti-GAPDH (CST, #97166), anti-β-actin (CST, #3700).

Techniques: RNA Sequencing, Control, Serial Dilution, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

Journal: bioRxiv

Article Title: p53Y220C-BET-bifunctionals (tPRIMEs) drive p53Y220C-mutant cancer cells into apoptosis

doi: 10.1101/2025.08.13.669398

Figure Lengend Snippet: tPRIME-5 induces p53 target genes and tetramer formation at slower kinetics than p53Y220C-ligands. A. qPCR analysis of the p53 target genes CDKN1A, MDM2, BBC3, ATF3, TP53I3 in NUGC3 cells treated with 0.1 or 1μM of tPRIME-5 or p53Y220C-ligand over a time course of 2-24h. B. Western Blot analysis and quantification of p53 tetramer after DSG crosslinking in NUGC3 cells treated for 2-24h with 0.1μM, 1μM or 10μM p53Y220C-ligand or tPRIME-5.

Article Snippet: The following list of primary antibodies was used: anti-p53 (SantaCruz, #sc-126), anti-p21 (SantaCruz, #sc- 53870), anti-NQO1 (CST, #3187), anti-TP53I3 (Origene, #TA503656), anti-PUMA (CST, #12450), anti-cleaved Parp (CST, #5625), anti-Parp (CST, #9542), anti-MDM2 (CST, #86934), anti-BRD2 (CST, #5848), anti-BRD3 (CST, #94032), anti-BRD4 (CST, #13440), anti-GAPDH (CST, #97166), anti-β-actin (CST, #3700).

Techniques: Western Blot

Journal: bioRxiv

Article Title: p53Y220C-BET-bifunctionals (tPRIMEs) drive p53Y220C-mutant cancer cells into apoptosis

doi: 10.1101/2025.08.13.669398

Figure Lengend Snippet: tPRIME-5 induces p53 target genes in vivo and achieves tumor growth inhibition and regression in NUGC3 xenografts. A. Plasma pharmacokinetics and NUGC3 tumor pharmacodynamics measured post a single dose. Left y-axis shows plasma concentration of tPRIME-5 or p53Y220C-ligand measured 0.5h, 1h, 4h, 8h or 24h post dosing mice IV, IP or PO. Right y-axis shows gene expression changes of CDKN1A, BBC3 and TP53I3 in NUGC3 xenografts measured at 4h, 8h and 24h post a single dose. B. Schematic of 21-day efficacy study in NUGC3 xenograft model. C. NUGC3 tumor growth inhibition and body weight changes of individual animals across the ten treatment groups at the end of study.

Article Snippet: The following list of primary antibodies was used: anti-p53 (SantaCruz, #sc-126), anti-p21 (SantaCruz, #sc- 53870), anti-NQO1 (CST, #3187), anti-TP53I3 (Origene, #TA503656), anti-PUMA (CST, #12450), anti-cleaved Parp (CST, #5625), anti-Parp (CST, #9542), anti-MDM2 (CST, #86934), anti-BRD2 (CST, #5848), anti-BRD3 (CST, #94032), anti-BRD4 (CST, #13440), anti-GAPDH (CST, #97166), anti-β-actin (CST, #3700).

Techniques: In Vivo, Inhibition, Clinical Proteomics, Drug discovery, Concentration Assay, Gene Expression

Journal: bioRxiv

Article Title: p53Y220C-BET-bifunctionals (tPRIMEs) drive p53Y220C-mutant cancer cells into apoptosis

doi: 10.1101/2025.08.13.669398

Figure Lengend Snippet: tPRIME-5 induces marked expression of multiple p53 target genes in NUGC3 xenografts post a single dose. A. qPCR analysis of the p53 target genes CDKN1A, GDF15, MDM2, ATF3, BBC3, RRAD, NQO1, TP53I3 and ALDH1A3 in NUGC3 xenografts treated with a single dose of vehicle, 100mpk p53Y220C-ligand (PO), 30mpk BET-ligand (PO), 100mpk p53Y220C-ligand + 30mpk BET-ligand (PO), 100mpk tPRIME-5 (IV) or 100mpk tPRIME-5 (IP) and harvested 4h, 8h and 24h post dosing. B. Analysis of p53 dependent target proteins via Western Blot in NUGC3 xenograft samples harvested 4h, 8h and 24h post a single dose of treatment with vehicle, 100mpk p53Y220C-ligand (PO) or 100mpk tPRIME-5 (IV or IP).

Article Snippet: The following list of primary antibodies was used: anti-p53 (SantaCruz, #sc-126), anti-p21 (SantaCruz, #sc- 53870), anti-NQO1 (CST, #3187), anti-TP53I3 (Origene, #TA503656), anti-PUMA (CST, #12450), anti-cleaved Parp (CST, #5625), anti-Parp (CST, #9542), anti-MDM2 (CST, #86934), anti-BRD2 (CST, #5848), anti-BRD3 (CST, #94032), anti-BRD4 (CST, #13440), anti-GAPDH (CST, #97166), anti-β-actin (CST, #3700).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: p53Y220C-BET-bifunctionals (tPRIMEs) drive p53Y220C-mutant cancer cells into apoptosis

doi: 10.1101/2025.08.13.669398

Figure Lengend Snippet: tPRIME-5 induces marked expression of multiple p53 target genes in NUGC3 xenografts harvested at the end of the efficacy study. A. qPCR analysis of the p53 target genes CDKN1A, GDF15, MDM2, ATF3, BBC3, RRAD, NQO1, TP53I3 and ALDH1A3 in NUGC3 xenografts treated for 21 days with vehicle, 100mpk p53Y220C-ligand (PO QD), 30mpk BET-ligand (PO QD), 100mpk p53Y220C-ligand + 30mpk BET-ligand (PO QD), 100mpk tPRIME-5 (IV BIW) or 100mpk tPRIME-5 (IP QD) and harvested 4h, 8h and 24h post last dose. B. Analysis of p53 dependent target proteins via Western Blot in NUGC3 xenograft samples harvested 4h, 8h and 24h post last treatment with vehicle, 100mpk p53Y220C-ligand (PO QD) or 100mpk tPRIME-5 (IV BIW or IP QD).

Article Snippet: The following list of primary antibodies was used: anti-p53 (SantaCruz, #sc-126), anti-p21 (SantaCruz, #sc- 53870), anti-NQO1 (CST, #3187), anti-TP53I3 (Origene, #TA503656), anti-PUMA (CST, #12450), anti-cleaved Parp (CST, #5625), anti-Parp (CST, #9542), anti-MDM2 (CST, #86934), anti-BRD2 (CST, #5848), anti-BRD3 (CST, #94032), anti-BRD4 (CST, #13440), anti-GAPDH (CST, #97166), anti-β-actin (CST, #3700).

Techniques: Expressing, Western Blot