Silencing P4HA2 reduces colony size and proliferation of breast cancer cells in 3D culture. (A) P4HA2 knockdown efficiency in T4-2 cells was verified by Western blot. (B) Phase images of control shRNA (shctrl) and shP4HA2-expressing ZR-75-1 and T4-2 cells in 3D culture for 4 days. The right column shows immunofluorescence images of α6-integrin (green) and DAPI (blue) staining. P4HA2-silenced T4-2 cells formed polarized acinar structures (arrows pointing to basal surface staining of α6-integrin), whereas control T4-2 cells still maintained disorganized mass-like structures (arrows pointing to lateral staining of α6-integrin; scale bar, 50 μm). (C) Ratio of polarized colony in control and knockdown P4HA2 T4-2 cells in 3D culture. (D) Quantification of colony size of control and P4HA2-silenced ZR-75-1 and T4-2 cells in 3D culture by measuring the diameter of at least 50 colonies. Knockdown of P4HA2 decreased the colony size. (E) EdU-staining was used to analyze the proliferation of control and P4HA2-silenced ZR-75-1 and T4-2 cells in 3D culture. Knockdown P4HA2 inhibited the proliferation of ZR-75-1 and T4-2 cells (*p < 0.05; **p < 0.01).