Journal: Alcohol (Fayetteville, N.Y.)

Article Title: Profiling the Oxylipidome in Aged Mice after Chronic Ethanol Feeding: Identifying Lipid Metabolites as Drivers of Hepatocyte Stress

doi: 10.1016/j.alcohol.2022.08.012

Figure Lengend Snippet: Young and aged adult WT mice were allowed free access to ethanol (32%,d25) or pair-fed control diets. A) Liver RNA was isolated and expression of senescence markers, p16 and p21 mRNA was detected in mouse livers using qRT-PCR. B/C) Paraffin-embedded liver sections were deparaffinized followed by immunodetection of PCNA and p53 and nuclei were counterstained with hematoxylin. Frozen liver sections were stained for β-galactosidase activity. Images were acquired using a ×10 objective and positive staining was quantified using Image-J. Data are from two independent trials; individual values are reported and represented as means ± SEM, n=8 pair-fed and 12 EtOH-fed mice/group. *Values were significantly different from each other (P<0.05).

Article Snippet: Paraffin-embedded liver sections were deparaffinized and stained with antibodies against NIMP-R14 (Novus Biologicals, cat NB600–1387), PCNA (Millipore, cat# MAB424R) and p53 (Novus Biologicals, cat# NB200–103).

Techniques: Control, Isolation, Expressing, Quantitative RT-PCR, Immunodetection, Staining, Activity Assay

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Signature of chemokine ligands and (B) chemokine receptors in human ovarian cancer cell lines. After isolating total RNA from each cell line, PCR array was performed using a customized PCR array plate containing complementary sequences for human chemokine genes. Different colors indicate average cycle threshold with expression ranges from >35 to <25. (C) Protein expression of p53 and Mdm2 in ovarian cancer cell lines. Whole cell lysates were prepared and Western blot was carried out using antibodies specific to p53, Mdm2 and β-actin as loading control. Experiments were performed in duplicate and a representative result is shown. OV, OVCAR-3 cells; SK, SKOV-3 cells; A, A2780 cells; Ca, CaOV-3 cells; TOV, TOV-21G cells.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) TNF-induced chemokines in SKOV-3 cells. After isolating total RNA, PCR array was performed using a human chemokine PCR array plate. Dotted line indicates 2-fold increase; chemokines with a greater than 2-fold increase are recognized as TNF-induced chemokines. (B) Confirmation of p53 protein expression after transient transfection in SKOV-3 cells. After transfection of empty vector (EM) and p53 expression vector (p53), whole cell lysates were prepared and p53 expression was confirmed by Western blot. β-actin is used as a loading control. (C) Effect of p53 on TNF-induced chemokines. After overnight transfection of vectors, cells were treated with TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Different letters indicate significant differences (P≤0.05) within each chemokine group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Nucleotide sequences of promoters for TNF-induced chemokines such as CCL20, CXCL1, 2, 3 and 8. These chemokine promoters contain one NF-κB site at the proximal region, except for CCL20, which has two NF-κB sites at the distal and proximal region. (B) Effect of p53 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p65, cells were treated with TNF (10 ng/ml) for 4 h. (C) Effect of p53 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 (p53 null), OVCAR-3 (p53 mutant) and A2780 (p53 wild-type), cells were treated with TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown; numbers below are relative density values.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Mutagenesis, Control

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Accumulated effect of p53 on ubiquitylated proteins. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to ubiquitin, p21, IκB, p53 and β-actin (as loading control). Experiments were performed in duplicate and a representative result is shown. (B) Confirmation of p53 activity after transient transfection of p53. ELISA was performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t -test) within each cell line. (C) The effect of p53 on proteasome activity. Assays were performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t -test) within each cell line. (D) Effects of p53 on ubiquitination of IκB. Immunoprecipitated IκB was immunoblotted using ubiquitin antibody. Experiments were performed in duplicate and a representative result is shown.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: Effect of p53 on ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3) obtained from comparison between empty vector and p53 vector transfected ovarian cancer cells.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Ubiquitin Proteomics, Comparison, Plasmid Preparation, Transfection

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Effect of p53 on Mdm2 expression. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to Mdm2; and β-actin served as loading control. (B) Effects of p53 expression on p53 binding to p65 and IκB. After transient transfection of p53, immunoprecipitated (IP) p53 was immunoblotted (IB) using p65 or IκB antibody. (C) Effect of p53 on expression of various IKK isoforms. After transient transfection of p53, whole cell lysates were prepared and Western blot was carried out using antibodies specific to IKKα, IKKβ, IKKγ, IKKε; β-actin served as loading control. Experiments were performed in duplicate and a representative result is shown.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Western Blot, Control, Binding Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Effect of nutlin-3 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p53, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 4 h. Different letters indicate significant differences (P≤0.05) within each group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE. (B) Effect of nutlin-3 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 cells, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown. (C) Effect of nutlin-3 on TNF-induced chemokines. After overnight transfection of vectors, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Asterisk indicates significant differences (P≤0.05, paired Student's t -test) when compared to the presence of nutlin-3. Experiments were performed in triplicate and all data are shown as mean ± SE.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: Chronic inflammation promotes ovarian cancer progression via NF-κB signaling. Wild-type p53 reduces activity of the ubiquitin-proteasome system, resulting in low IκB degradation (blue line). This reduces NF-κB activity, inhibiting proinflammatory chemokine expression and attenuating the proinflammatory tumor microenvironment (blue arrow). On the other hand, p53 increases Mdm2 expression (dark arrow) in a feedback loop to compensate for the reduced activity of the ubiquitin-proteasome system. Loss of p53 observed frequently in advanced ovarian cancer triggers high proinflammatory chemokines by increasing NF-κB signaling which is composed of IκB and p65/p50 followed by a high IκB degradation (red arrow). Enhanced NF-κB activity results in potentiation of the proinflammatory tumor microenvironment for ovarian cancer progression such as peritoneal tumor dissemination and massive ascites. The imbalance between p53 and Mdm2 also contributes to increasing NF-κB signaling via the ubiquitin-proteasome system.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Ubiquitin Proteomics, Expressing

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: Western blot analyses of lamin A/C and progerin, full-length p53, Δ133p53α, and p21 Waf1/Cip1 were performed in four 15-week-old Δ133p53α-expressing Group-1 mice ( CAG-133 Tam/+ ; Cre Tg/+ ; Lmna G609G/+ ), along with four each of age-matched, non-expressing control Group-2 ( CAG-133 LSL/+ ; Cre Tg/+ ; Lmna G609G/+ ) and Group-4 mice ( CAG-133 +/+ ; Cre +/+ ; Lmna G609G/+ ), as well as two age-matched wild-type mice ( CAG-133 +/+ ; Cre +/+ ; Lmna +/+ ). Results from skin ( a ), skeletal muscle ( b ), kidney ( c ), spleen ( d ), and lung ( e ) are presented. An inter-blot control (liver from a Group-1 mouse) was included in all blots. F, female; M, male. GAPDH was a loading control and used for normalization of p21 Waf1/Cip1 , progerin, and full-length p53 expression levels. Quantitative data summaries of p21 Waf1/Cip1 , progerin, and full-length p53 in Group-1, -2 and -4 mice are shown as relative values to Group-2 mice (mean ± s.d. from n = 4; open circles indicate two females, and closed circles indicate two males). P values were determined by Welch’s t -test. Two wild-type mice were used only as references and not for statistical comparisons.

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: a,b , Top Hallmark pathways identified by Gene set enrichment analysis (GSEA). The bulk RNA-seq data were obtained from the heart ( a ) and kidney ( b ) in 9-10-month-old Group-1 and Group-3 mice (n = 5 each). Pathways are ranked by normalized enrichment score. False discovery rate < 0.10. c-g, Enrichment plots illustrate significant downregulation of the p53 pathway ( c,d ) and upregulation of the oxidative phosphorylation ( e,f ) in both heart and kidney, as well as upregulation of the glycolysis in the kidney ( g ). Enrichment plots depict running enrichment scores (ES) and the distribution of genes within each Hallmark gene set. All leading edge genes in each pathway are listed in Extended Data Table 2. h,i, qRT-PCR assays of mRNA expression of genes in the oxidative phosphorylation pathway (Ndufs6, Ndufc2, Uqcrq, Hsd17b10, and Gpx4) and an antioxidant gene Prdx1 in the heart ( h ) and kidney ( i ) of 9-10-month-old Group-1 and Group-3 mice (mean ± s.d. from n = 5, each with technical triplicate; open circles, females; closed circles, males). P values were calculated by Welch’s t -test.

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: RNA Sequencing, Phospho-proteomics, Quantitative RT-PCR, Expressing

Journal: Antioxidants

Article Title: Saposhnikovia divaricata Inhibits Inflammation, Oxidative Stress, and Ferroptosis to Alleviate DSS-Induced Ulcerative Colitis

doi: 10.3390/antiox15020258

Figure Lengend Snippet: Network pharmacology analysis identifies p53 as a core ferroptosis-related target of FF in UC. ( A ) Venn diagram illustrating the intersection of FF compound targets with ferroptosis- and UC-related targets. ( B ) Protein–protein interaction (PPI) network of the common targets. Node size and color intensity represent the degree of connectivity, with TP53 (p53) identified as the core target. ( C ) Compound-target-pathway network diagram. The inner pink nodes represent the 38 intersecting targets linking FF, UC, and ferroptosis. ( D ) Gene Ontology (GO) enrichment analysis of the common targets, categorized into Biological Process (BP, red), Cellular Component (CC, green), and Molecular Function (MF, blue). ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Article Snippet: The p53 antibody was obtained from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA, Item #BM0101).

Techniques:

Journal: Antioxidants

Article Title: Saposhnikovia divaricata Inhibits Inflammation, Oxidative Stress, and Ferroptosis to Alleviate DSS-Induced Ulcerative Colitis

doi: 10.3390/antiox15020258

Figure Lengend Snippet: FF modulates the expression of ferroptosis-related proteins in colon tissue via the p53 pathway. ( A ) Representative immunohistochemical (IHC) images of p53, SLC7A11, and GPX4 expression in colon sections (scale bar = 50 μm). ( B – D ) Quantitative analysis of the relative protein expression levels of p53 (B), SLC7A11 (C), and GPX4 (D). Data are presented as the mean ± SD ( n = 3 independent experiments). ### p < 0.001 versus the control (CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus the DSS model group.

Article Snippet: The p53 antibody was obtained from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA, Item #BM0101).

Techniques: Expressing, Immunohistochemical staining, Control

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Growth curves of Cas9-expressing HCT116 in the genome wide CRISPR/Cas9 knockout screen shown in . At each plotted point, cells were treated for 24 hours with vehicle or 1 µM MM17 and allowed to recover without treatment until 80% confluency (n=1). B. Immunoblots of p53 and actin (loading control) in HCT116 mCherry mock, ZsGreen p53 KO guide 1 (p53 KO-1), and ZsGreen p53 KO guide 2 (p53 KO-2) cells. C. Immunoblots of p21 and tubulin (loading control) in HCT116 mCherry mock, and ZsGreen p21 KO cells. D, E. Growth competition assay between mCherry mock and ZsGreen mock, ZsGreen p53 KO-1, ZsGreen p53 KO-2, or ZsGreen p21 KO cells. At each plotted point, cells were treated for 24 hours with MM17 ( D ) or paclitaxel ( E ) and allowed to recover without compound until confluency. (n=3 biological replicates, mean ± sem). F. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem). G. Immunoblot of MDM2 and Ponceau S staining (loading control) in HCT116 p53 KO cells transduced with non-target control or four sgRNAs targeting MDM2. sgMDM2 guide 3 was selected for subsequent experiments. H. Cresyl violet staining after 1 week of antibiotics selection of HCT116 cells expressing empty vector, ectopic MDM2 WT, or MDM2 C311F transduced with mock or MDM2 guide. I. Immunoblots of MDM2 and actin (loading control) of cell lines in (H). J. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem). K. Viability assay of HCT116 cells treated with etoposide for 72 hours. (n = 3 biological replicates, mean ± sem). L. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Expressing, Genome Wide, CRISPR, Knock-Out, Western Blot, Control, Competitive Binding Assay, Quantitation Assay, Staining, Transduction, Selection, Plasmid Preparation, Viability Assay

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Genome wide CRISPR/Cas9 knockout screen in HCT116 cells. The log2 fold change for each gene comparing samples treated with vehicle or 1 µM MM17 was determined by averaging the log2 fold changes of its associated sgRNAs and subtracting the average log2 fold change of non-targeting sgRNAs. The top two most enriched genes, and one of the biogenesis factors removed by Rix7 are indicated in red and labeled. B. Immunoblots of p53, p21, and tubulin (loading control) in HCT116 NVL WT and NVL R403W CRISPR cells treated with 3 µM MM17. C . Cryo-EM structure of the MDM2 299-340 –5S RNP complex (PDB ID - 8BGU; MDM2 - orange, uL5 - turquoise, uL18 - red, 5S rRNA - pink) . MDM2 C311 residue (yellow) and Zn 2+ (purple) at the MDM2-uL5 binding interface are labeled. D, E. Immunoblots of p53, p21 and actin (loading control) in HCT116 MDM2 WT and MDM2 C311F cells treated with MM17 ( D ) or etoposide (E) for 24 hours. F. Cell cycle analysis of HCT116 mock and p53 KO cells treated with 3 µM MM17. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, ns = non-significant). G. Live cell imaging analysis of relative cell area of HCT116 mock and p53 KO cells treated with 3 µM MM17 for 72 hours. (n = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Genome Wide, CRISPR, Knock-Out, Labeling, Western Blot, Control, Cryo-EM Sample Prep, Residue, Binding Assay, Cell Cycle Assay, Two Tailed Test, Live Cell Imaging

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Immunoblots and quantitation of caspase-3 and actin (loading control) in HCT116 cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). B. Flow cytometry-based cell cycle analysis in HCT116 cells treated with vehicle or 3 µM MM17 for 3 days. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, *p<0.05). C. Immunoblots of p53 and actin (loading control) in HCT116 mock engineered or p53 knockout (KO) cells. D. Viability (log 2 fold change relative to vehicle) of 855 cancer cell lines treated with 10 µM MM17, grouped by their p53 mutation or copy number deletion status. Each dot represents a cell line. (two-tailed paired t-test, ****p<0.0001).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Quantitation Assay, Control, Flow Cytometry, Cell Cycle Assay, Two Tailed Test, Knock-Out, Mutagenesis

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A bioavailable analog MM927 inhibits colorectal and leukemia tumor xenograft growth in vivo without overt toxicity. A. Chemical structure of MM927 and the potency (IC50) of its impact on the viability of HCT116. B. Pharmacokinetic analysis of MM927 in mouse plasma after a 10 mg/kg intraperitoneal (IP) injection (n = 3 mice/group, mean ± sem). C, D. Representative microscopy images ( C ) and quantitation ( D ) showing immunofluorescence of p53 (magenta), Ki-67 (green), and nucleus (DAPI, blue) in NVL WT and NVL R403W CRISPR tumor xenografts implanted in either flank of the same mouse. Mice were treated with 35 mg/kg MM927 IP twice daily for 3 days, and tumors were harvested 6 hours after the last dose. (Scale bar = 10 µm; n = 5 mice/group, mean ± sem). E. Effect of MM927 (35 mg/kg IP twice daily for 21 days) on HCT116 NVL WT or NVL R403W CRISPR tumor xenograft growth (n = 10 mice/group, mean ± sem). F . Tumor volume measurements in (E) at the end of 21 days or euthanasia due to tumor diameter exceeding 2 cm (n = 10 mice per group, mean ± sem; two-tailed unpaired t-test, ** p < 0.01, ns = non-significant). G. Body weight % changes in HCT116-tumor bearing mice at the end of MM927 treatment (n = 20 mice/group, mean ± sem; two-tailed unpaired t-test, ns = non-significant). H. Levels of hemoglobin, white blood cells, platelets, liver enzymes AST and ALT, and renal clearance of blood urea nitrogen (BUN) 3 hours after the last dose (n = 10 mice per group, mean ± sem; two-tailed unpaired t-test, **p<0.01, *p<0.01, ns = non-significant). I. Representative images and quantitation of leukemic disease burden by bioluminescent imaging of luciferase expressing MOLM-13 cells. (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, *** p < 0.001). J, K . Flow cytometry-based quantitation of MOLM-13 cells (human CD45 positive) in the mouse bone marrow ( J ) and peripheral blood ( K ) (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, ****p<0.0001). L . Body weight changes at the end of MM927 treatment in mice xenografted with MOLM-13 (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, ns = non-significant).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: In Vivo, Clinical Proteomics, Injection, Microscopy, Quantitation Assay, Immunofluorescence, CRISPR, Two Tailed Test, Imaging, Luciferase, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Polysome profiling of HCT116 NVL WT ( A ) and NVL R403W CRISPR ( B ) cells treated with 0.5 µM MM927 for 24 hours. B, C. Representative microscopy images and quantitation of existing (green) and newly synthesized (magenta) eL36-SNAP in NVL WT ( B ) and NVL R403W CRISPR ( C ) cells treated with 0.5 µM MM927 for 24 hours. (Scale bar = 10 µm; n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, **p<0.01, ns = non-significant). Data from the vehicle group is re-used from these are done in the same experiment. D. Representative microscopy images and quantitation of existing (green) and newly synthesized (magenta) eS17-SNAP in HCT116 cells treated with 0.5 µM MM927 for 24 hours. (Scale bar = 10 µm; n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, **p<0.01, ns = non-significant). Data from the vehicle group is re-used from , as these are done in the same experiment. E. Flow cytometry-based analysis of cell cycle profile in HCT116 mock and p53 KO cells treated with 0.5 µM MM927. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, *p<0.05, ns = non-significant). F. Viability assay of HCT116 NVL R403W CRISPR cells treated with MM927 for 72 hours. (N = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: CRISPR, Microscopy, Quantitation Assay, Synthesized, Two Tailed Test, Flow Cytometry, Viability Assay

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: MM927 studies in HCT116 tumor xenografts. A. Immunoblots of Flag (NVL) and tubulin (loading control) in HCT116 NVL AID/AID cells ectopically expressing empty vector, human NVL, or mouse NVL treated with Ph-IAA for 6 hours. B. Viability assay of cell lines in (A) treated with 0.03 µM Ph-IAA for 72 hours (n = 2 biological replicates, mean ± sem). C. Viability assay of HCT116 NVL AID/AID cells ectopically expressing human NVL or mouse NVL in treated with MM927 and 0.03 µM Ph-IAA for 72 hours (n = 2 biological replicates; mean ± sem). D, E. Immunoblots ( D ) and quantitation ( E ) of p53, p21, and tubulin (loading control) of HCT116 NVL WT and NVL R403W CRISPR tumor xenografts implanted in either flank of the same mouse treated with one 35 mg/kg MM927 IP injection. Right two lanes are in vitro samples of HCT116 cells treated with 10 µM MM17 for 24 hours. (n=2 to 3 biological replicates, mean ± sem). F. Individual tumor volume measurements in . Each line represents a mouse. G. Pharmacokinetic quantitation of MM927 3 hours after the last dose in the mouse plasma and tumors (n = 10 mice/group, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Control, Expressing, Plasmid Preparation, Viability Assay, Quantitation Assay, CRISPR, Injection, In Vitro, Clinical Proteomics

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Immunoblots of p53 and actin (loading control) in MOLM-13 mock engineered or p53 knockout (KO) cells. B. Viability assay of cell lines in (A) treated with MM17 for 72 hours. (n = 3 biological replicates, mean ± sem). C. Immunoblots and quantitation of caspase-3 and actin (loading control) in MOLM-13 mock and p53 KO cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). D. Flow cytometry-based apoptosis analysis and quantitation in MOLM-13 mock and p53 KO cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). E. Schematic illustrating the proposed mechanism of cellular response to MM17. F. Immunoblots of NVL and actin (loading control) in MOLM-13 cells expressing empty vector, NVL WT, or NVL R403W. G. Growth curves of cell lines in (F). n = 3 biological replicates, mean ± sem). H. Viability assay of cell lines in (F) treated with MM927 for 72 hours. (n = 2 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Control, Knock-Out, Viability Assay, Quantitation Assay, Flow Cytometry, Expressing, Plasmid Preparation