Fig 1: Heme Activates c-MYC Expression in Part via Binding to DNA G4 Structure in Its Promoter(A and B) qPCR (A) and western blot (B) analysis of c-MYC in PC3-sh-control and PC3-sh-c-myc cells treated with heme (50 µM) for 2 h or 4 h. Bar charts represent mean ± SEM. ANOVA, *p < 0.05, ***p < 0.001.(C and D) qPCR analysis of MMP2 (C) and uPA (D) in PC3-sh-control and PC3-sh-c-myc cells treated with heme (50 µM) for 2 h or 4 h. Bar charts represent mean ± SEM. ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001.(E) Anchorage-independent growth in soft agar of PC3-sh-control and PC3-sh-c-myc treated with heme (50 µM) for 3 weeks. Scatter plot represents mean ± SEM. n = 3 independent experiments. ANOVA, *p < 0.05. n.s., not significant.(F) Anchorage-independent growth in soft agar of PC3 cells cultured in the presence or absence of GQC-05 (10 µM) or vehicle (DMSO) with or without heme (50 µM) for 3 weeks. Bar chart represents mean ± SEM. n = 3 experiments performed in triplicate. ANOVA, ***p < 0.001. (G) Western blot analysis of c-myc, VEGF, and lamin A/C in cell lysates from PC3 cells treated with heme (50 µM) or GQC-05 (10 µM) for 0.5–8 h. A representative blot of three independent experiments is shown. (H) Crystal violet staining of PC3 cells treated for 48 h with GQC-05 (10 µM). Bar chart represents mean ± SEM. n = 3 independent experiments. (I) CD spectra with 1:1 heme:Pu27 ratio demonstrating molecular ellipticity at 262 nm. The peak is maintained upon addition of heme, indicating a parallel G4 structure. (J) Melting temperature of the heme:Pu27 complexes evaluated at 262 nm by CD. Melting temperature for pu27 oligonucleotide was 72.36°C, for Pu27:heme, 1:1 was 78.27°C, and for Pu27:heme, 1:2 was 84.15°C. (K) EMSA was performed using the wild-type G4-Pu27 (p27) or the mutated G4-Pu27 (mp27) sequence found in the c-MYC promoter region in the presence of an antibody against G4 (BG4). Results are representative of three experiments.(L) EMSA was performed using p27 and mp27 G4 sequences incubated for 20 min with 50 µM heme before loading in the presence of the BG4 antibody. n = 3 independent experiments.(M) ChIP analysis with anti-BG4 antibody in PC3 cells treated with heme (50 µM) for 0.5, 1, 2, and 4 h. n = 3–4 independent experiments performed in duplicate. Bar chart represents mean ± SEM. ANOVA, **p < 0.01, ***p < 0.001.(N) ChIP analyses with anti-nucleolin, anti-NM23-H2, anti-CNBP, and anti-hnRNPK antibodies in PC3 cells treated with heme (50 µM) for 2 h. n = 2 independent experiments performed in duplicates. Bar chart represents mean ± SEM. ANOVA, **p < 0.01, ***p < 0.001.(O) Immunofluorescence staining for BG4 in PC3 cells treated with 50 µM heme for 24 h. Scale bar 20 µm. n = 3 independent experiments.