Journal: Molecular Oncology

Article Title: Mutant p53 R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation

doi: 10.1002/1878-0261.70341

Figure Lengend Snippet: Mutant p53 R273H disrupts PDPK1 homodimerization. (A) A schematic illustration of FRET from EGFP‐PDPK1 to PDPK1‐sREACh under different conditions that affect PDPK1 homodimerization and alter the EGFP donor fluorescence lifetime. (B) Fluorescence lifetime images of NCI‐H1299 cells transfected with the indicated plasmids. Pseudo‐color represents the EGFP fluorescence lifetime; blue indicates longer lifetimes, and warmer colors (yellow) indicate shorter lifetimes due to FRET, reflecting increased PDPK1 homodimerization (representative of n = 3 independent experiments). Scale bar = 20 μm (applies to all the images in the panel). (C) Quantification of FRET‐FLIM analysis. Fluorescence lifetime images were acquired using a time‐correlated single‐photon counting board controlled by custom software. The fluorescence signals from all pixels within each cell expressing EGFP‐PDPK1 were summed to generate an EGFP fluorescence lifetime curve. The homodimerization fraction, defined as the proportion of EGFP‐PDPK1 molecules undergoing FRET with PDPK1‐sREACh, was then calculated by fitting the fluorescence lifetime curve with a double‐exponential function convolved with a Gaussian pulse‐response function, incorporating a mixture of non‐FRET and FRET fractions. A higher homodimerization fraction indicates increased PDPK1‐PDPK1 dimer formation. Gray circles represent individual cells (number of cells = 87, 126, 62, 105, 106 from left to right). Blue horizontal lines and gray error bars represent the mean homodimerization fraction ± SEM for each group (representative of n = 3 independent experiments). Statistical analysis was performed using a one‐way ANOVA with post hoc Tukey's test. ** P value < 0.01, **** P value < 0.0001.

Article Snippet: Membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween‐20 and incubated with antibodies [mouse anti‐FLAG (Sigma‐Aldrich, #F1804), mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126), rabbit anti‐p53 (Santa Cruz Biotechnology, #sc‐6243), rabbit anti‐PDPK1 (Cell Signaling Technology, #3062), rabbit anti‐AKT (Cell Signaling Technology, #9272), rabbit anti‐phospho‐AKT (T308) (Cell Signaling Technology, #13038), mouse anti‐Actin (Merck Millipore, #MAB1501), mouse anti‐GST (Santa Cruz Biotechnology, #sc‐138), and rabbit anti‐MYC (Proteintech, #16286‐1‐AP)].

Techniques: Mutagenesis, Fluorescence, Transfection, Analysis, Counting, Software, Expressing

Journal: Molecular Oncology

Article Title: Mutant p53 R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation

doi: 10.1002/1878-0261.70341

Figure Lengend Snippet: PRIMA‐1 Met disrupts p53 R273H ‐PDPK1 interaction. (A) U‐251MG cells were treated with the indicated doses of PRIMA‐1 Met for 24 h. Lysates were immunoprecipitated with anti‐p53 antibody and analyzed by western blot using the indicated antibodies (representative of n = 2 independent experiments). Additional validation is provided in Fig. . (B) Proximity ligation assay (PLA) in U‐251MG cells. Cells transfected with FLAG‐PDPK1 were fixed, permeabilized, and stained using anti‐p53 and anti‐FLAG antibodies for PLA. Confocal microscopy images show nuclei (DAPI, blue) and green fluorescence signals indicating positive PLA interactions (representative of n = 3 independent experiments). Scale bar = 50 μm; applies to all the images in the panel. (C) FRET‐FLIM analysis of NCI‐H1299 cells transfected with the indicated plasmids. Gray circles represent individual cells (number of cells = 171, 245, 115 from left to right). Blue horizontal lines and gray error bars represent the mean homodimerization fraction ± SEM for each group (representative of n = 3 independent experiments). Statistical analysis was performed using a one‐way ANOVA with post hoc Tukey's test. **** P value < 0.0001. (D) U‐251MG cells were treated for 24 h in DMEM containing 0.1% FBS with DMSO, TNFα, PRIMA‐1 Met , or a combination of TNFα and PRIMA‐1 Met . Lysates were analyzed by western blot using the indicated antibodies (representative of n = 2 independent experiments). (E) A schematic model illustrating the p53 R273H ‐PDPK1 interaction contributing to increased AKT phosphorylation. The p53 R273H mutant disrupts the inactive PDPK1 homodimer, resulting in the formation of active PDPK1 monomers that promote AKT phosphorylation. Treatment with PRIMA‐1 Met , which has been reported to restore p53 R273H to a wild‐type‐like conformation, disrupts the p53 R273H ‐PDPK1 interaction and inhibits this gain‐of‐function effect of p53 R273H .

Article Snippet: Membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween‐20 and incubated with antibodies [mouse anti‐FLAG (Sigma‐Aldrich, #F1804), mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126), rabbit anti‐p53 (Santa Cruz Biotechnology, #sc‐6243), rabbit anti‐PDPK1 (Cell Signaling Technology, #3062), rabbit anti‐AKT (Cell Signaling Technology, #9272), rabbit anti‐phospho‐AKT (T308) (Cell Signaling Technology, #13038), mouse anti‐Actin (Merck Millipore, #MAB1501), mouse anti‐GST (Santa Cruz Biotechnology, #sc‐138), and rabbit anti‐MYC (Proteintech, #16286‐1‐AP)].

Techniques: Immunoprecipitation, Western Blot, Biomarker Discovery, Proximity Ligation Assay, Transfection, Staining, Confocal, Microscopy, Fluorescence, Analysis, Phospho-proteomics, Mutagenesis

Journal: Molecular Oncology

Article Title: Mutant p53 R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation

doi: 10.1002/1878-0261.70341

Figure Lengend Snippet: Mutant p53 R273H binds to PDPK1. (A) A schematic workflow of the screening approach. Lysates from U‐251MG cells expressing p53 R273H ‐FLAG were immunoprecipitated with anti‐FLAG antibody, and the immunoprecipitated proteins were analyzed by mass spectrometry. (B) KEGG pathway analysis of the 151 p53 R273H ‐interacting proteins using DAVID (Database for Annotation, Visualization and Integrated Discovery). The top 10 enriched pathways are shown. (C and D) Co‐immunoprecipitation of exogenous p53 R273H and FLAG‐PDPK1. Lysates of NCI‐H1299 cells transfected with the indicated plasmids were immunoprecipitated with anti‐p53 (C) or anti‐FLAG (D) antibody and analyzed by western blot using the indicated antibodies (both panels are representative of n = 3 independent experiments). (E) GST pulldown assay. Purified 6 × His‐PDPK1 was incubated with GST or GST‐p53 R273H proteins immobilized on glutathione agarose beads and analyzed by western blot using the indicated antibodies (representative of n = 4 independent experiments). (F) Co‐immunoprecipitation of endogenous p53 R273H and PDPK1. Lysates of U‐251MG cells transduced with shControl‐ or sh TP53 ‐expressing retroviruses were immunoprecipitated with anti‐p53 antibody and analyzed by western blot using the indicated antibodies (representative of n = 5 independent experiments). (G) Proximity ligation assay (PLA) in U‐251MG cells. Cells transfected with FLAG‐PDPK1 were fixed, permeabilized, and stained using anti‐p53 and anti‐FLAG antibodies for PLA. Confocal microscopy images show FLAG‐PDPK1 (red), p53 R273H (gray), nuclei (DAPI, blue), and green fluorescence signals indicating positive PLA interactions (representative of n = 3 independent experiments). Scale bar = 50 μm (applies to all the images in the panel).

Article Snippet: Membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween‐20 and incubated with antibodies [mouse anti‐FLAG (Sigma‐Aldrich, #F1804), mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126), rabbit anti‐p53 (Santa Cruz Biotechnology, #sc‐6243), rabbit anti‐PDPK1 (Cell Signaling Technology, #3062), rabbit anti‐AKT (Cell Signaling Technology, #9272), rabbit anti‐phospho‐AKT (T308) (Cell Signaling Technology, #13038), mouse anti‐Actin (Merck Millipore, #MAB1501), mouse anti‐GST (Santa Cruz Biotechnology, #sc‐138), and rabbit anti‐MYC (Proteintech, #16286‐1‐AP)].

Techniques: Mutagenesis, Screening, Expressing, Immunoprecipitation, Mass Spectrometry, Analysis, Co-Immunoprecipitation Assay, Transfection, Western Blot, GST Pulldown Assay, Purification, Incubation, Transduction, Proximity Ligation Assay, Staining, Confocal, Microscopy, Fluorescence

Journal: Molecular Oncology

Article Title: Mutant p53 R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation

doi: 10.1002/1878-0261.70341

Figure Lengend Snippet: F270A mutation in p53 R273H disrupts PDPK1 binding. (A) Lysates of NCI‐H1299 cells transfected with the indicated plasmids were immunoprecipitated with anti‐FLAG antibody and analyzed by western blot using the indicated antibodies (representative of n = 2 independent experiments). Additional validation is provided in Fig. . (B) Molecular Dynamics (MD) simulations on the p53 DNA binding domain (DBD) structural changes following point mutations at specific amino acid positions. Amino acid positions 270 (in orange), 273 (in blue), and 109–114 (in magenta) are shown. (C) Lysates of NCI‐H1299 cells transfected with the indicated plasmids were immunoprecipitated with anti‐FLAG antibody and analyzed by western blot using the indicated antibodies (representative of n = 3 independent experiments).

Article Snippet: Membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween‐20 and incubated with antibodies [mouse anti‐FLAG (Sigma‐Aldrich, #F1804), mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126), rabbit anti‐p53 (Santa Cruz Biotechnology, #sc‐6243), rabbit anti‐PDPK1 (Cell Signaling Technology, #3062), rabbit anti‐AKT (Cell Signaling Technology, #9272), rabbit anti‐phospho‐AKT (T308) (Cell Signaling Technology, #13038), mouse anti‐Actin (Merck Millipore, #MAB1501), mouse anti‐GST (Santa Cruz Biotechnology, #sc‐138), and rabbit anti‐MYC (Proteintech, #16286‐1‐AP)].

Techniques: Mutagenesis, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Biomarker Discovery

Journal: Molecular Oncology

Article Title: Mutant p53 R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation

doi: 10.1002/1878-0261.70341

Figure Lengend Snippet: Mutant p53 R273H enhances PDPK1 activation. U‐251MG cells were transfected with siRNAs targeting TP53 or PDPK1 , either individually or in combination. Cells were lysed 48 h post‐transfection, and lysates were analyzed by western blot using the indicated antibodies (representative of n = 3 independent experiments).

Article Snippet: Membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween‐20 and incubated with antibodies [mouse anti‐FLAG (Sigma‐Aldrich, #F1804), mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126), rabbit anti‐p53 (Santa Cruz Biotechnology, #sc‐6243), rabbit anti‐PDPK1 (Cell Signaling Technology, #3062), rabbit anti‐AKT (Cell Signaling Technology, #9272), rabbit anti‐phospho‐AKT (T308) (Cell Signaling Technology, #13038), mouse anti‐Actin (Merck Millipore, #MAB1501), mouse anti‐GST (Santa Cruz Biotechnology, #sc‐138), and rabbit anti‐MYC (Proteintech, #16286‐1‐AP)].

Techniques: Mutagenesis, Activation Assay, Transfection, Western Blot

Journal: bioRxiv

Article Title: Single-cell analysis identifies TOP2α as a critical regulator of G2/M entry and differentiation-dependent productive HPV replication

doi: 10.64898/2026.09.23.753843

Figure Lengend Snippet: a , UMAP embeddings of CIN612 cells by condition and by cluster, with feature plots for CDK1 and TOP2A (D4 upper, UD lower). b , Feature plots for NUSAP1 and H2AX on the same embedding. c , Cell-cycle distribution determined by DNA content in undifferentiated (UD) and differentiated (D4) HFK and CIN612 cultures; percentages of cells in G1, S and G2/M are indicated. d , Immunoblot of FACS-sorted G1, S and G2/M fractions from UD and D4 HFK and CIN612 cultures probed for TOP2α, NUSAP1, pCDK1 (Thr14/Tyr15), γH2AX and GAPDH. e , Quantification of TOP2α levels from d , normalized to GAPDH (mean ± s.d., n = 3). f , Haematoxylin and eosin staining and TOP2α immunofluorescence of HPV-positive cancer biopsy tissue and matched normal epithelium on the same slide, with quantification of TOP2α enrichment (log scale, normalized to normal = 1). g , Immunoblot of CIN612 cells and HFKs expressing HPV31 E6 or E7 in UD and D4 states, probed for TOP2α, p53 and GAPDH, with quantification of TOP2α levels (mean ± s.d., n = 3). h , Co-immunofluorescence of UD and D4 HFK and CIN612 cells for DAPI, TOP2α and pCDK1 (Thr14/Tyr15), γH2AX or NUSAP1. i , Quantification of the CIN612 panels in h , showing the proportion of TOP2α-positive nuclei that are positive for pCDK1 (Thr14/Tyr15), γH2AX or NUSAP1 in UD and D4 cultures; cell numbers are indicated within each bar. Two-sided Fisher exact test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; ns, not significant.

Article Snippet: Lysates were resolved by SDS–PAGE, transferred to nitrocellulose and probed with antibodies against anti-rabbit TOP2α (1:500, CST), anti-rabbit TOP2β (1:500, CST), anti-rabbit FOXM1 (1:500, CST), phosphorylated FOXM1 (pT600FOXM1; 1:500, CST), anti-rabbit NUSAP1 (1:2000,, anti-rabbitpCDK1 (Thr14/Tyr15, 1:500, CST), Anti-rabbit pATR (1:500, CST), anti-rabbit pSer139-γH2AX (1:3000, CST), anti-mouse p53 (1:4000, Santa Cruz), anti-mouse cytokeratin 10 (1:5000, Santa Cruz) and anti-mouse GAPDH (1:6000, Santa Cruz) followed by respective secondary HRP-conjugated antibodies (1:3000, CST).

Techniques: Western Blot, Staining, Immunofluorescence, Tissue, Expressing