Fig. 4

TS regulated the activity, expression of CAXII, E2F1,3/Caspase-3 axis and pHi, pHe and extracellular lactate. Dose-dependent inhibition curves of CAXII esterase activity under different intervention were displayed s in  A and B. For TS group, the IC50 was 20.95 ± 5.8 µM in MDA-MB-231 cells and 30.92 ± 6.98 µM in BT-549 cells (A), and that in U-104 group were 5.47 ± 1.25 µM and 6.62 ± 1.8 µM, respectively (B). As RT-qPCR results showed, in 2D cultured MDA-MB-231 cells, the expression level of CAXII was 0.21 ± 0.024 folds compared to its NC (P < 0.001) after the intervention of TS, and that was 0.316 ± 0.02 folds of NC (P < 0.001) in 3D TS group. Similar results were observed in rest groups (C). As for Caspase-3 activity assay, in MDA-MB-231 cells, the value of TS group 2D (0.50 ± 0.01) was significantly higher than that of apoptosis inhibited TS group 2D (0.20 ± 0.02, P < 0.001). In 3D system, the value of TS group 3D (0.47 ± 0.02) was also significantly higher than that of apoptosis inhibited TS group 3D (0.19 ± 0.01, P < 0.001). Similar results were observed in BT-549 cells (D and E). Furthermore, calculated CASP3-specific activity in TS group 2D (0.12 ± 0.01) was significantly higher than that in NC group 2D (0.03 ± 0.01, P < 0.001). Similar result was also detected 3D cultured MDA-MB-231 and BT-549 cells (F). For MDA-MB-231 cells, the intracellular pHi value of 0 µM TS group (7.43 ± 0.04) was significantly higher than that of 20, 40, 80 µM TS group, respectively (7.00 ± 0.06, 6.66 ± 0.1, 6.41 ± 0.08, P < 0.001). Similar results were observed in BT-549 cells in extracellular lactate level (G and I). Opposite trends were displayed in extracellular pHe for both cells(H), NC negative control