Journal: Advanced Science

Article Title: Mitochondrial Localization of Cx26 Promotes Colorectal Cancer Malignancy and Exposes a Fatal Vulnerability to Drug Targeting

doi: 10.1002/advs.78117

Figure Lengend Snippet: PIMT promotes mitochondrial translocation of Cx26 in highly malignant CRC cells. (A) HEK293T cells were transfected with Cx26‐HA and PIMT‐Flag, and then immunoprecipitated with IgG as a control or anti‐Flag antibody or anti‐HA antibody, followed by western blot for the indicated proteins. (B) Analyze the correlation between the mRNA levels of PIMT and Cx26 in colorectal cancer cell lines through the TCGA database. (C) Immunoblot of PIMT and Tubulin in SW480 and SW620 cells. Tubulin was used as a loading control. *** p < 0.001 vs . SW480; by unpaired student's t ‐test (mean ± SEM). (D) Lysates of SW480 and SW620 cells were co‐immunoprecipitated with anti‐Cx26 or anti‐PIMT antibodies, and then PIMT and Cx26 protein levels were detected by immunoblot. IgG was used as a loading control. (E) Immunofluorescence of Cx26 (green) and PIMT (red) in SW480 and SW620 cells. Nuclei were stained with DAPI (blue). Scale bar: 10 µm. (F) SW480 cells were treated with TGF‐β for 0, 24, and 48 h; cell lysates were collected for co‐immunoprecipitation with anti‐Cx26 antibody. IgG was used as a loading control. (G) Western blot of the protein level of Cx26 in the cytoplasm and plasma membrane in SW620 cells transfected with shRNA‐NC or shRNA‐PIMT. Na, K‐ATPase and Tubulin were measured as loading control of membrane and cytoplasm, respectively. (H) Immunofluorescence of Cx26 (green) in SW620 cells transfected with or without shRNA‐PIMT. Mitochondria were stained with MitoTracker Red (red). Nuclei were stained with DAPI (blue). Scale bar, 50 µm. (I) Immunoblot of Cx26, PIMT, COX4, and Tubulin in the cytoplasmic and mitochondria‐enriched fractions of SW620 cells transfected with or without shRNA‐PIMT. Tubulin and COX4 were used as markers and loading controls for the cytoplasmic and mitochondria‐enriched fractions, respectively. *** p < 0.001 vs . SW620 cells transfected without shRNA‐PIMT; by unpaired student's t ‐test (mean ± SEM).

Article Snippet: Anti‐Cx26 antibody, anti‐Calnexin antibody, and anti‐Flag antibody were purchased from Proteintech Group (CHI, USA).

Techniques: Translocation Assay, Transfection, Immunoprecipitation, Control, Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Clinical Proteomics, Membrane, shRNA

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: Briefly, cells were fixed with 4% paraformaldehyde, permeabilized, blocked, and incubated with mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126) and rabbit anti‐FLAG (Proteintech, #80010‐1‐RR; probing for FLAG‐tagged PDPK1) primary antibodies.

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: Briefly, cells were fixed with 4% paraformaldehyde, permeabilized, blocked, and incubated with mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126) and rabbit anti‐FLAG (Proteintech, #80010‐1‐RR; probing for FLAG‐tagged PDPK1) primary antibodies.

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: Briefly, cells were fixed with 4% paraformaldehyde, permeabilized, blocked, and incubated with mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126) and rabbit anti‐FLAG (Proteintech, #80010‐1‐RR; probing for FLAG‐tagged PDPK1) primary antibodies.

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