E2F3 and survivin as possible therapeutic targets against PBL. (A) Effect of H and S inhibitors, alone or in combination, in PBL-1 cells at IC₅₀ dose on E2F3 and survivin protein levels, as assessed by western blot. (B) The effect of the indicated inhibitors on viability and apoptosis was assessed by flow cytometry at 24 hours by annexin V/TO-PRO-3 staining in PBL-1 cells. Relative viability (%; left) and apoptosis (proportion; right) of treatments normalized to DMSO are shown. (C) Figure shows the distribution of cells in the cell cycle phases at 24 hours, which was evaluated by flow cytometry and TO-PRO-3 staining at different doses specified in the supplemental Materials and supplemental Table 2. (D) Effect of H, S, and H + S in cell proliferation measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in PBL-1 cells. (E) Scheme followed to evaluate the effect of the H and S inhibitors alone and in combination in the chicken CAM based on the engraftment of PBL-1 cells on day 9, followed by 2 administrations of H, S, or H + S combination on days 12 and 14, and the collection of samples on day 16. (F) Tumors are weighted on day 16 (n = 7-14 eggs per group). Images of tumors under different conditions (top); and the relative reduction of tumor growth with the treatments (bottom). (G) Hematoxilin-eosin (HE), MUM-1, and Ki67 immunohistochemical (IHC) staining from sections of formalin-fixed, paraffin-embedded tumors from CAM under different treatment conditions (n = 5 eggs per group). (H) Relative proliferation of tumoral cells under different treatment conditions measured by Ki67 IHC staining. (I) The presence of CD138⁺ cells in tumors formed without treatment or treated with H, S, or H+S assessed by CD138 (APC) staining and flow cytometry (n = 5 eggs per group). (J) The relative percentage of CD138⁺ cells depending on the treatment of PBL-1 CAM model. All in vitro experiments were performed in biological triplicates. ANOVA test was used for statistical analysis. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. C, control; DMSO, dimethyl sulfoxide; H, HLM006474; IL-6, interleukin-6; S, S12.