G4 Immunofluorescence imaging and data quantification

For immunofluorescence studies, 93T449 cells were seeded at 4 × 10⁴ cells/well on glass 12-mm-diameter coverslips in 24-well plates and grown overnight at 37 °C. Following treatment with entinostat (EPS002, Sigma Aldrich) 2–4 µM or actinomycin D (A1410, Sigma Aldrich) 0.5–1 μM for 48 h, cells were fixed in 2% paraformaldehyde in PBS 1× for 20 min at room temperature. Staining with BG4 was performed as previously reported¹⁴ with minor modifications: cells were permeabilized with 0.1% Triton X-100 (Sigma Aldrich) in PBS 1×, treated with 50 μg/ml RNase A (EN0531, ThermoFisher), blocked using BlockAid ({"type":"entrez-nucleotide","attrs":{"text":"B10710","term_id":"2091830","term_text":"B10710"}}B10710, ThermoFisher), incubated with BG4 1:100 (stock 0.25 mg/ml, MABE917, Merck), M2 anti-FLAG 1:800 (F3165, Sigma Aldrich), and anti-mouse Alexa 488 1:500 (A21204, ThermoFisher) antibodies. Nuclei were stained with TOTO3 (1:2000, ThermoFisher) for 20 min at RT. Digital images were acquired using a Nikon A1R Laser Scanning confocal microscope equipped with NIS-Elements Advanced Research software (Nikon Instruments), with 60× objectives. For BG4 (green channel), images were visualized at 488 nm excitation wavelength and 500–550 nm emission wavelength; for cell nuclei (blue channel), 641 nm excitation wavelength and 663–738 nm emission range were applied.

Confocal imaging quantification was performed using ImageJ software. 2D nuclear dye staining was employed for nuclei identification and nuclear area determination. The integrated fluorescence intensity values for BG4 channel were background subtracted and normalized to the nuclear area.