Fig 1: Decoding the amyloidogenicity of c-MYC in vitro.(A) In vitro fibrillation assays using the human c-MYC peptide library (mean ± SEM, n=3 independent experiments, Two-way ANOVA). (B) ELISA quantitation of A11+–AOs generated by synthetic c-MYC peptides. Results of three independent experiments are presented as a heatmap. FC: fold change. (C) and (D) In vitro fibrillation assays using the P2 and P12 peptides that are subjected to alanine scan. Results are presented as heatmaps using the averages of three independent experiments.
Fig 2: c-MYC is intrinsically amyloidogenic.(A) In vitro fibrillation assays using recombinant c-MYC proteins (mean ± SEM, n=3 independent experiments, Two-way ANOVA). (B) ELISA quantitation of A11+–AOs generated by recombinant c-MYC proteins in vitro (mean ± SD, n=3 independent experiments, One-way ANOVA). (C) TEM visualization of protofibrils formed by recombinant c-MYC proteins in vitro (representative images of three independent experiments). Scale bars: 100 nm. (D) In vitro fibrillation assays using recombinant c-MYC /MAX dimers (mean ± SEM, n=3 independent experiments, Two-way ANOVA). (E) TEM visualization of recombinant c-MYC/MAX dimers in vitro (representative images of three independent experiments). Scale bars: 100 nm. (F) Quantitation of endogenous c-MYC AOs in HeLa cells treated with 10058-F4 by In-Cell PLA ELISA (mean ± SD, n=3 independent experiments, One-way ANOVA).
Fig 3: The amyloidogenesis of c-MYC contributes to its intrinsic tumor suppressor activity.(A) Measurements of the transcriptional activities of HA-MYCWT and HA-MYCΔC in HeLa cells using the dual MYC reporter system (mean ± SD, n=3 independent experiments, One-way ANOVA). (B) Quantitation of apoptosis induced by transient expression of HA-MYCWT or HA-MYCΔC in serum-starved NIH3T3 cells by flow cytometry using anti-cleaved caspase 3 Abs (mean ± SD, n=3 independent experiments, One-way ANOVA). (C) Detection of HA-MYC expression in serum-starved NIH3T3 cells by immunoblotting (representative images of four independent experiments). (D) Quantitation of A11+–HA-MYCΔC and its deletion mutants in NIH3T3 cells by In-Cell PLA ELISA using rabbit A11 Abs and mouse anti-HA Abs. The results are normalized by HA-MYC expression levels (mean ± SD, n=4 independent experiments, One-way ANOVA). (E) Quantitation of apoptosis induced by transient expression of HA-MYCΔC and its deletion mutants in serum-starved NIH3T3 cells by flow cytometry using anti-cleaved caspase 3 Abs. The results are normalized by HA-MYC expression levels (mean ± SD, n=4 independent experiments, One-way ANOVA).
Fig 4: c-MYC becomes detergent-insoluble and displays amyloid-like properties under proteotoxic stress and pathological conditions.(A) Detection of detergent-soluble and - insoluble c-MYC proteins by immunoblotting in HeLa cells pre-treated with 30 μM CR, followed by HS at 43°C for 30 min (representative images of three independent experiments). (B) and (C) Immunoprecipitation of either exogenously expressed HA-c-MYC or endogenous c-MYC proteins in HeLa cells by A11 antibodies (representative images of three independent experiments). (D) Detection of endogenous c-MYC proteins in HeLa cells by PLA using both the goat anti-c-MYC Ab and the rabbit anti-AO (A11) Ab. Nuclei are counterstained with Hoechst 33342. Scale bar: 10 μm. (E)-(G) Detection of endogenous c-MYC proteins in human prostate adenocarcinoma, hepatocellular carcinoma, and Alzheimer’s disease brain tissues by brightfield PLA using both the goat anti-c-MYC Ab and the rabbit anti-AO (A11) Ab. Scale bar: 100 μm for low magnification and 10 μm for high magnification. (H): Quantitation of endogenous c-MYC recognized by both the goat anti-c-MYC Ab and the rabbit anti-AO (A11) Ab through In-Cell PLA ELISA in HeLa cells treated with CR (mean ± SD, n=3 independent experiments, One-way ANOVA).
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