Apt–cL–triGemcitabine conjugate treatment induces apoptosis and death of targeted TNBC cells. Cultured tumor cells were treated as indicated for two days and stained with Annexin V (green fluorescence) to mark apoptotic cells, propidium iodide (PI) (red) to mark dead cells, and Hoechst dye (blue) for cell tracking purposes. (A) Fluorescent microscopic images of MDA-MB-231 cells (TNBC) treated with Apt–cL–triGemcitabine conjugate or free gemcitabine drug at equimolar amounts of gemcitabine, or no treatment as background baseline controls. (B) Fluorescent microscopic images of T47D (non-TNBC) cells with the same set of treatments. (C,D) Quantitative analysis of apoptosis and death rates in MDA-MB-231 cells post treatments as indicated. (E,F) Quantitative analysis of apoptosis and cell death rates of off-target T47D cells under the same treatment conditions. Scale bar = 200 µm. Significant differences between groups are indicated with asterisks. p ≤ 0.05 was considered significant. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001 (Student’s t-test).