Figure 4. Functional and cellular assays support GACC-associated stabilization of the KRAS promoter G-quadruplex and reduced KRAS transcription. (A–C) DNA polymerase stop assay using a KRAS promoter G4-containing template incubated with GACC (25–100 µM) prior to primer extension. (A) Representative denaturing PAGE showing full-length and stalled extension. (B,C) Quantitation of band intensities expressed as % of vehicle control (full-length products and stalled products as indicated), with PDS as a positive control and mutant/non-G4 templates and ligand-only controls included as specificity controls (lanes labeled in the gel). Data are mean ± SEM from n = 3 independent experiments. (D,E) BG4 immunofluorescence analysis in Hep3B cells transfected with Cy5-labeled KRAS-G4 oligonucleotide and treated with vehicle, GACC (200 µM) or PDS (10 µM) for 24 h. (D) Representative images showing nuclear BG4 signal (and Cy5/DAPI channels as shown). The Analysis panels are MetaMorph-generated spot/colocalization maps derived from the corresponding images and share the same spatial calibration. Scale bar: 20 µm (applies to all panels in (D), including Analysis). (E) Quantification of nuclear BG4 intensity per cell (analysis performed using identical thresholds and ROIs across conditions). Data are mean ± SEM; hundreds of cells pooled from 3 independent experiments. (F) KRAS mRNA levels in PLC/PRF/5 cells treated with GACC and PDS at the indicated concentration for 24 h, measured by qPCR and normalized to β-actin, expressed relative to vehicle. Data are mean ± SEM; n ≥ 3 biological replicates. Statistical tests: one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.