Fig 1: IsoLGs induce misfolding of p53 protein in vivo(A) The schematic representation of animal experiment.(B) Representative images of p53 protein co-localization with amyloid fibrils detected using amyloid OC antibody in murine esophageal tissues. Mice received 2-HOBA dissolved in water (1 g/L) 2 weeks after surgery. Control mice received vehicle (water). Esophageal tissues were collected after drug treatments for 10 days and analyzed for misfolding of p53 protein. Co-localization of p53 and amyloid staining was observed in mice with reflux surgery. The staining was not found in mice treated with 2-HOBA. Inset: high magnification image. Scale bar, 10 μm.(C) Representative immunohistochemical staining of murine esophageal tissues with p53 (PAb 240) antibody. Insets show high magnification images. Graph shows the IHC scores (sham control versus reflux mice, ***p < 0.001; reflux mice versus reflux mice treated with 2-HOBA, ***p < 0.001; Tukey’s multiple comparison). Mice with sham surgery were used as a control. Scale bar, 10 μm.(D) Representative images of p53 protein misfolding in the human esophagus. The human esophageal tissues from healthy subjects, GERD and BE patients were analyzed using p53 (PAb 240) antibody. Misfolded p53 protein was observed in the esophagus of GERD and BE patients, but not in normal (no GERD) esophageal epithelium (scale bar, 5 μm). Graph shows the immunofluorescence scores (normal versus GERD, *p < 0.01; normal versus BE, *p < 0.01).(E) Graphical representation of p53 protein regulation by isoLGs. All results are expressed as mean ± SD.
Fig 2: IsoLGs form amyloid-like p53 aggregates(A) Dot blot analysis of CP-A cell extracts using anti-amyloid OC antibody. Protein aggregation levels were found to be higher in CP-A cells treated with ABS than in control untreated cells (***p < 0.001; n = 3; Tukey’s multiple comparison). 2-HOBA prevented amyloid aggregation (***p < 0.001; n = 3; Tukey’s multiple comparison).(B) The same as (A) but the formation of amyloid aggregates was analyzed in EPC-2 cells.(C) Representative images of co-localization of p53 protein with amyloid fibrils detected with anti-amyloid OC antibody in CP-A cells (n = 3) (scale bar, 5 μm). All results are expressed as mean ± SD.
Fig 3: p53 adduction leads to p53 protein aggregation(A) Representative images of p53 protein aggregates in CP-A cells (scale bar, 5 μm). Immunofluorescence staining was performed with p53(D0-1) antibody in cells treated with ABS alone or in combination with 2-HOBA. 2-HOBA prevented aggregation of p53 protein (n = 3).(B) Representative images of Duolink PLA in CP-A cells treated with ABS. PLA was performed using D0-1 and D11: E-tag primary antibodies (scale bar, 5 μm). The isoLG-p53 adducts were detected in large cellular aggregates in cells treated with ABS. The adduct-specific PLA signals were not observed in cells treated with 2-HOBA (n = 3).(C) p53 protein adduction was analyzed in the cytoplasmic and nuclear fractions of CP-A cells after ABS treatment for 8 h. Equal amount of total protein (10 μg) was loaded in each well. Bottom panel shows experimental inputs.
Fig 4: ABS treatment causes conformation change of the p53 protein molecule(A) Dot blot analysis of CP-A cell extracts using D01 antibody.(B) The same as (A) but p53 (PAb 240) antibody was used. Staining was higher in cells treated with ABS than in control or 2-HOBA-treated cells (***p < 0.001; n = 3; Tukey’s multiple comparison).(C) The same as (B) but EPC-2 cells were studied (ABS versus ABS+2-HOBA, ***p < 0.001; n = 3; Tukey’s multiple comparison).(D) Native blue PAGE demonstrates misfolding and aggregation of p53 protein in CP-A cells collected after treatment with ABS. The graph shows the densitometric measurement of p53 aggregation detected by p53 (PAb 240) antibody (control versus ABS, ***p < 0.001; ABS versus 2-HOBA, ***p < 0.001; n = 3; Tukey’s multiple comparison). Aggregated p53 proteins are indicated by a red box. Protein expression in control cells was arbitrarily set at 1.(E) Representative images show co-localization of p53 protein with ProteoStat-positive aggregates (scale bar, 5 μm). p53 was analyzed with p53(D0-1) and ProteoStat dye was used to identify protein aggregates in CP-A cells treated with ABS alone or in combination with 2-HOBA (n = 3).(F) The same as (E) but PAb 240 antibody was used for detection of misfolded p53 protein (scale bar, 5 μm).(G) The same as (F) but the p53 misfolding was analyzed in EPC-2 cells. All results are expressed as mean ± SD.
Fig 5: Acidic bile salts increase the formation of isoLG-p53 adducts(A) p53 protein was immunoprecipitated from CP-A cells and analyzed for adduction of p53 protein with D11 scFv antibody by western blotting. ABS treatment increases levels of isoLG-p53 protein adducts, while 2-HOBA counteracts this effect by preventing the isoLG adduction of p53 protein (n = 3; Tukey’s multiple comparison) at 8 h (control versus ABS, ***p < 0.001; ABS versus ABS+2-HOBA, ***p < 0.001) and 21 h (control versus ABS, ***p < 0.001; ABS versus ABS+2-HOBA, ***p < 0.001). Levels of isoLG: p53 adducts was normalized to total levels of p53 protein, which were analyzed with p53(D01) antibody. Levels of p53 protein adduction in control cells was arbitrarily set at 1.(B) The same as (A) but the p53 protein adduction was analyzed in EPC-2 cells at 8 h (control versus ABS, ***p < 0.001; ABS versus ABS+2-HOBA, ***p < 0.001) and 21 h time points (control versus ABS, ***p < 0.001; ABS versus ABS+2-HOBA, ***p < 0.001) after treatment.(C) CP-A cells were treated with 0.5 μM synthetic isoLGs and analyzed for the adduction of p53 protein (control versus isoLG, **p < 0.01; isoLG versus isoLG+2-HOBA, **p < 0.01; n = 3; Tukey’s multiple comparison).(D) Analyses of p53 protein acetylation in CP-A cells by western blotting with antibody recognizing acetylated p53 at Lys382 (control versus ABS, ***p < 0.001; ABS versus ABS+2-HOBA, ***p < 0.001; n = 3; Tukey’s multiple comparison).(E) The same as (D) but acetylation of p53 protein was analyzed in EPC-2 cells (control versus ABS, ***p < 0.001; ABS versus ABS+2-HOBA, ***p < 0.001; n = 3; Tukey’s multiple comparison).(F) Cell-cycle analysis in CP-A cells transfected with either p53 siRNA or scrambled siRNA and treated with ABS alone or in combination with 2-HOBA. Cell cycle was analyzed by flow cytometry and compared between groups (scr siRNA: ABS versus ABS+2-HOBA, **p < 0.01; p53 siRNA: ABS versus ABS+2-HOBA; NS, not significant; n = 3; Tukey’s multiple comparison). All results are expressed as mean ± SD.
Supplier Page from Abcam for p53 Human Immunocapture Kit