Fig. 1: Evaluation of the aggregate forming ability of p53 and Δ133p53β.

a Western blot analysis of the soluble protein fraction from H1299 cells after transfection with WT, structurally and contact mutated p53 and Δ133p53β. n = 3. b Western blot analysis of the insoluble protein fraction from H1299 cells after transfection with WT, structurally and contact mutated p53 and Δ133p53β. n = 3. c 3D reconstruction of confocal immunofluorescent Z-stack images of H1299 cells transfected with WT, structurally and contact mutated p53. Green: α-Flag (p53), red: α-OC (Aβ42, amyloid aggregates) antibody, blue: Hoechst. Scale bar 8 μm. n = 3. d 3D reconstruction of the confocal immunofluorescent Z-stack images of H1299 cells transfected with WT, structurally and contact mutated Δ133p53β. Green: α-Flag (Δ133p53β), red: α-OC (Aβ42, amyloid aggregates) antibody, blue: Hoechst. Scale bar 8 μm. n = 3. e Immunofluorescence analysis of p53 (Δ122p53) localisation in tissues obtained from Δ122p53 homozygote mice performed using the PAb421 antibody. Yellow: p53 (Δ122p53), Blue: Hoechst. In total ten individual tumours from the Δ122p53 animal model were analysed. Arrows in upper panel indicate prominent cytoplasmic aggregates while in lower panel arrow demonstrates aggregates in between diffuse cytoplasmic staining. Scale bar 50 μm. f Upper panel: RNAscope to identify metastatic triple-negative breast tumours expressing Δ133p53β. Ubiquitin C (UBC) as a positive and the bacterial gene DapB as a negative control. Lower panel: Immunofluorescence analysis using the KJC8 antibody to detect Δ133p53β aggregates in a metastatic triple-negative breast tumour. Boxed areas: digital magnifications of regions with characteristic staining. n = 5. Scale bar 20 μm. g Immunohistochemical analysis of Δ133p53β aggregate presence in different primary or metastatic tumours as well as in normal breast tissue. Arrows indicate prominent Δ133p53β aggregates. Scale bar 50 μm. n = 5 (for primary breast cancers and corresponding brain metastases), n = 15 for normal associated breast tissue and n = 3 for brain metastasis of colon cancer. h Solution studies of the MBP-Δ133p53β construct. Size exclusion chromatography (SEC) profile of recombinant MBP-Δ133p53β injected on a Superdex 200 16/60. The maximum of the peak corresponds to an elution volume at 56 mL, which is close to the dead volume for that column. The size of the protein is estimated at 440 kDa according to the calibration. SDS-PAGE of SEC samples collected at elution times of 47 ml (line 1) and 56 ml (line 2). The observed SDS-PAGE bands are in agreement with the expected molecular weight of MBP-Δ133p53β (67.4 KDa). i. Dynamic light scattering (DLS) profile of the maximum peak from the SEC (2). The profile displays a unique peak with a maximum corresponding to a particle with a diameter of 16.7 nm and an estimated molecular weight of 480 KDa. The peak is broad and most probably corresponds to the equilibrium of multiple species of variable sizes. n = 3. The arrows indicate co-localisation in Fig. 1c and d or aggregate Fig. 1e–g. Molecular weight is expressed in kDa. Source data are provided as a Source Data file.