Journal: bioRxiv

Article Title: Mutant p53 Directs PARP to Regulate Replication Stress and Drive Breast Cancer Metastasis

doi: 10.64898/2026.03.26.713220

Figure Lengend Snippet: Whole cell lysate (WCE), soluble and chromatin fractions were prepared from MCF7 (A) and MDA-MB-468 (B) treated with either vehicle (DMSO), 10 μM talazoparib (TAL), 1 mM temozolomide (TMZ), or combination (TAL + TMZ) for 15 h. Protein levels of PAR,MDMX, MDM2, γ-H2AX, p53, p21 and PARP were determined by Western blot analysis. Representative of two independent experiments.

Article Snippet: Proteintech: Cat# 84534-5-RR MDMX/MDM4 Rabbit; Cat# 10442-1-AP P53 Rabbit PolyAb; Cat# 10355-1-AP P21 Rabbit PolyAb; Cat# SA00001-2 HRP-conjugated Goat Anti-Rabbit IgG(H+L), Cell Signaling: Cat# 9718S Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb; Cat# 89190S Poly/Mono-ADP Ribose (D9P7Z) Rabbit mAb; Cat# 4717S NF-κB1 p105 Antibody; Cat# 7074S Anti-rabbit IgG, HRP-linked Antibody; Cat# 3879S Snail (C15D3) Rabbit mAb; Cat# 2661S Phospho-Chk2 (Thr68) Antibody; Cat# 2662S Chk2 Antibody, Abcam: Cat# AB6326-1001 Anti-BrdU antibody; Cat# AB259265-1001 Anti-MDM2 antibody; Cat# AB191217-1001 Anti-PARP1 antibody; Cat# AB46154-1001 Anti-VEGFA antibody.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Mutant p53 Directs PARP to Regulate Replication Stress and Drive Breast Cancer Metastasis

doi: 10.64898/2026.03.26.713220

Figure Lengend Snippet: (A) MDA-MB-468 cells were subcutaneous implanted in the flank of female NSG mice. Once tumor volumes reached approximately 150 mm 3 , mice were randomized to treatment via oral gavage either with vehicles (Control), or in combination of talazoparib 0.2mg/kg daily and temozolomide 6 mg/kg every 4 days (TMZ + TAL) treatment group. The CTCs from endpoint MDA-MB-468 xenografts mice were stained with human specific PE-conjugated EGFR antibody and the number of CTCs was determined by flow cytometry. The number of CTCs per milliliter was obtained by dividing the number of positive events by blood volume from individual animals (left). In the box-and-whisker plots, each dot represents one mouse, median values are represented by horizontal lines. + indicated the mean. *P < 0.05, **P < 0.01, ***P <0.001 Mann-Whitney test. Representative fluorescence-activated cell sorting plots showing PE-positive events in Control and TMZ +TAL mouse groups (right). (B) Whole-cell proteins were extracted from NSG mice xenograft tumors and 10 μg of protein was loaded on 10% SDS-PAGE gel. Protein levels of PAR, MDMX, Snail, mtp53 and PARP in endpoint MDA-MB-468 (Control) and MDA-MB-468 (TMZ + TAL) snap-frozen tumors were determined by Western blot analysis. (C) Soluble and Chromatin fractions were prepared from endpoint MDA-MB-468 (Control) and MDA-MB-468 (TMZ +TAL) snap-frozen tumors. Protein levels of p53, PARP, γ-H2AX, and Snail were determined by Western blot analysis.

Article Snippet: Proteintech: Cat# 84534-5-RR MDMX/MDM4 Rabbit; Cat# 10442-1-AP P53 Rabbit PolyAb; Cat# 10355-1-AP P21 Rabbit PolyAb; Cat# SA00001-2 HRP-conjugated Goat Anti-Rabbit IgG(H+L), Cell Signaling: Cat# 9718S Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb; Cat# 89190S Poly/Mono-ADP Ribose (D9P7Z) Rabbit mAb; Cat# 4717S NF-κB1 p105 Antibody; Cat# 7074S Anti-rabbit IgG, HRP-linked Antibody; Cat# 3879S Snail (C15D3) Rabbit mAb; Cat# 2661S Phospho-Chk2 (Thr68) Antibody; Cat# 2662S Chk2 Antibody, Abcam: Cat# AB6326-1001 Anti-BrdU antibody; Cat# AB259265-1001 Anti-MDM2 antibody; Cat# AB191217-1001 Anti-PARP1 antibody; Cat# AB46154-1001 Anti-VEGFA antibody.

Techniques: Control, Staining, Flow Cytometry, Whisker Assay, MANN-WHITNEY, Fluorescence, FACS, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Mutant p53 Directs PARP to Regulate Replication Stress and Drive Breast Cancer Metastasis

doi: 10.64898/2026.03.26.713220

Figure Lengend Snippet: (A) MCF7 cells were orthotopic implanted into the mammary fat pad of female NSG mice. Once tumor volumes reached approximately 150 mm 3 , mice were randomized to treatment via oral gavage either with vehicles (Control), or in combination of talazoparib 0.2mg/kg daily and temozolomide 6 mg/kg every 4 days (TMZ + TAL) treatment group. Lung metastases were measured by IHC staining with human specific mitochondrial antigen on the sections of lungs from orthotopically engrafted MCF7. Quantification of IHC positive cells was assessed with QuPath software (right). (B) Whole-cell proteins were extracted from NSG mice xenograft tumors and 10 μg of protein was loaded on 10% SDS-PAGE gel. Protein levels of PAR, MDMX, MDM2, p53, PARP and, γ-H2AX in endpoint MCF7 Control and TMZ + TAL snap-frozen tumors were determined by Western blot analysis. (C) MDA-MB-468 cells were orthotopic implanted in female NSG mice. Once tumor volumes reached approximately 150 mm 3 , mice were randomized to treatment via oral gavage vehicles (Control), or in combination of talazoparib 0.2mg/kg daily and temozolomide 6 mg/kg every 4 days (TMZ + TAL) treatment group. Lung metastases were measured by IHC staining with human specific mitochondrial antigen on the sections of lungs from orthotopically engrafted MDA-MB-468 Control and TMZ + TAL mouse group. Quantification of IHC positive cells was assesed with QuPath software (right). (D)The CTCs from endpoint MDA-MB-468 xenografts mice were stained with human specific PE-conjugated EGFR antibody and the number of CTCs was determined by flow cytometry. The number of CTCs per milliliter was obtained by dividing the number of positive events by blood volume from individual animals (left). Representative fluorescence-activated cell sorting plots showing PE-positive events in Control and TMZ +TAL mouse groups (right). (E) RNA-seq-based pathway enrichment analysis of MDA-MB-468 (mtp53 R273H) cells treated with temozolomide and talazoparib (TMZ + TAL) compared with control (Method). Gene Set Enrichment Analysis (GSEA) using MSigDB Hallmark gene sets identifies pathways significantly enriched among differentially expressed genes. Up-regulated pathways are shown in red, whereas down-regulated pathways are shown in gray. Enrichment direction reflects normalized enrichment scores derived from genes ranked by differential expression. (F) Whole-cell proteins were extracted from NSG mice xenograft tumors and 10 μg of protein was loaded on 10% SDS-PAGE gel. Protein levels of NF-κB p105 and VEGF in endpoint MDA-MB-468 (Control) and MDA-MB-468 (TMZ + TAL) snap-frozen tumors were determined by Western blot analysis. In the box-and-whisker plots, each dot represents one mouse, median values are represented by horizontal lines. + indicated the mean. *P < 0.05, **P < 0.01, ***P <0.001. Mann-Whitney test.

Article Snippet: Proteintech: Cat# 84534-5-RR MDMX/MDM4 Rabbit; Cat# 10442-1-AP P53 Rabbit PolyAb; Cat# 10355-1-AP P21 Rabbit PolyAb; Cat# SA00001-2 HRP-conjugated Goat Anti-Rabbit IgG(H+L), Cell Signaling: Cat# 9718S Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb; Cat# 89190S Poly/Mono-ADP Ribose (D9P7Z) Rabbit mAb; Cat# 4717S NF-κB1 p105 Antibody; Cat# 7074S Anti-rabbit IgG, HRP-linked Antibody; Cat# 3879S Snail (C15D3) Rabbit mAb; Cat# 2661S Phospho-Chk2 (Thr68) Antibody; Cat# 2662S Chk2 Antibody, Abcam: Cat# AB6326-1001 Anti-BrdU antibody; Cat# AB259265-1001 Anti-MDM2 antibody; Cat# AB191217-1001 Anti-PARP1 antibody; Cat# AB46154-1001 Anti-VEGFA antibody.

Techniques: Control, Immunohistochemistry, Software, SDS Page, Western Blot, Staining, Flow Cytometry, Fluorescence, FACS, RNA Sequencing, Derivative Assay, Quantitative Proteomics, Whisker Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mutant p53 Directs PARP to Regulate Replication Stress and Drive Breast Cancer Metastasis

doi: 10.64898/2026.03.26.713220

Figure Lengend Snippet: (A) Breast cancer PDX model WHIM25 (A) WHIM6 (B) cells were subcutaneous implanted in the flank of female NSG mice. Once tumor volumes reached approximately 150 mm 3 , mice were randomized to treatment via oral gavage either with vehicle (Control), or in combination of talazoparib 0.2mg/kg daily and temozolomide 6 mg/kg every 4 days (TMZ +TAL) treatment group. The CTCs from WHIM25 or WHIM6 Control or TMZ + TAL mice were stained with human specific PE-conjugated EGFR antibody, and the number of CTCs was determined by flow cytometry. The number of CTCs per milliliter was obtained by dividing the number of positive events by blood volume from individual animals. (C) Whole cell protein levels of PAR, PARP, p53, MDMX, MDM2 and γ-H2AX in endpoint Control and TMZ + TAL snap-frozen tumors were determined by Western blot analysis. Whole-cell proteins were extracted from NSG mice xenograft tumors and 10 μg of protein was loaded on 10% SDS-PAGE gel. (D) Soluble and Chromatin fractions were prepared from endpoint WHIM25 or WHIM6 Control and TMZ +TAL snap-frozen tumors. Protein levels of PARP, MDMX and p53 were determined by Western blot analysis. In the box-and-whisker plots, each dot represents one mouse, + indicated the mean. *P < 0.05, **P < 0.01, ***P <0.001, Mann-Whitney test.

Article Snippet: Proteintech: Cat# 84534-5-RR MDMX/MDM4 Rabbit; Cat# 10442-1-AP P53 Rabbit PolyAb; Cat# 10355-1-AP P21 Rabbit PolyAb; Cat# SA00001-2 HRP-conjugated Goat Anti-Rabbit IgG(H+L), Cell Signaling: Cat# 9718S Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb; Cat# 89190S Poly/Mono-ADP Ribose (D9P7Z) Rabbit mAb; Cat# 4717S NF-κB1 p105 Antibody; Cat# 7074S Anti-rabbit IgG, HRP-linked Antibody; Cat# 3879S Snail (C15D3) Rabbit mAb; Cat# 2661S Phospho-Chk2 (Thr68) Antibody; Cat# 2662S Chk2 Antibody, Abcam: Cat# AB6326-1001 Anti-BrdU antibody; Cat# AB259265-1001 Anti-MDM2 antibody; Cat# AB191217-1001 Anti-PARP1 antibody; Cat# AB46154-1001 Anti-VEGFA antibody.

Techniques: Control, Staining, Flow Cytometry, Western Blot, SDS Page, Whisker Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mutant p53 Directs PARP to Regulate Replication Stress and Drive Breast Cancer Metastasis

doi: 10.64898/2026.03.26.713220

Figure Lengend Snippet: (A) Top: Schematic of DNA fiber combing assay with CldU/IdU pulse-labeling protocol and treatment with PARGi, followed by S1 nuclease treatment. Bottom: Representative DNA fiber images of MDA-MB-468 cells with mtp53 R273H, and mtp53 (R273HΔ347-393 with vehicle (DMSO) or PARGi with or without S1 nuclease treatment. (B) Quantification of IdU tracts after 10 μM PARGi treatment with or without S1 nuclease. Each dot represents one fiber. Around 130-220 fibers were analyzed for each condition. Mean values are represented by horizontal lines. ns., not significant *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001, two-way ANOVA test followed by Tukey’s post-hoc test. Representative of two independent experiments. (C) Whole cell protein levels of PAR, mtp53, γ-H2AX, RPA32, Phos-Chk2, Chk2 and MDMX were determined by Western blot analysis. Representative of two independent experiments. (D) Model for mutant p53-PARP axis enables replication stress tolerance and drive cancer metastasis, created in BioRender.

Article Snippet: Proteintech: Cat# 84534-5-RR MDMX/MDM4 Rabbit; Cat# 10442-1-AP P53 Rabbit PolyAb; Cat# 10355-1-AP P21 Rabbit PolyAb; Cat# SA00001-2 HRP-conjugated Goat Anti-Rabbit IgG(H+L), Cell Signaling: Cat# 9718S Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb; Cat# 89190S Poly/Mono-ADP Ribose (D9P7Z) Rabbit mAb; Cat# 4717S NF-κB1 p105 Antibody; Cat# 7074S Anti-rabbit IgG, HRP-linked Antibody; Cat# 3879S Snail (C15D3) Rabbit mAb; Cat# 2661S Phospho-Chk2 (Thr68) Antibody; Cat# 2662S Chk2 Antibody, Abcam: Cat# AB6326-1001 Anti-BrdU antibody; Cat# AB259265-1001 Anti-MDM2 antibody; Cat# AB191217-1001 Anti-PARP1 antibody; Cat# AB46154-1001 Anti-VEGFA antibody.

Techniques: Labeling, Western Blot, Mutagenesis