Fig 2: 7E protected corneal epithelial cells from hyperosmotic stress-induced cell death. HCE-2 cells were subjected to control, 40 mM + mIgG (4 µg/ml), 40 mM + 7E (4 µg/ml), 80 mM + mIgG, and 80 mM + 7E for 8 h. a, b The mRNA transcripts of BAX and BCL-2 were analyzed by real-time PCR with specific primers. GAPDH was used as an internal control. One-way ANOVA, **p < 0.01, ***p < 0.001, and ***p < 0.0001. Data are shown as the mean ± SEM. c CCK-8 assay was applied to analyze the cell viability of HCE-2 cells treated with different groups for 24 h or 48 h. One-way ANOVA, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are shown as the mean ± SEM. d Immunocytochemistry staining of BAX and BCL-2 of different groups was analyzed. The reaction was detected by the chromogen AEC (red), and the nuclei were counterstained with hematoxylin (blue). Original magnification: ×200. The experiments in a–d were repeated three times independently with similar results, and the data of one representative experiment was shown. CCK-8 cell counting kit-8

Fig 3: CMA3 suppresses CCA growth and induces apoptosis in the xenograft mouse model. KKU-213 cells were subcutaneously injected into both flanks of Balb/c RJ mice. CMA3 was administered at 0.5 mg/kg into the tail vein once a week for 3 times. Control mice were treated with DMSO. Mice were euthanized on day 22 of the experiment. (A) Tumor volumes were measured every 3 days and are represented as the mean ± SD. ***P<0.001. (B) The numbers of Ki-67-positive nuclei per pancytokeratin-positive areas were compared between the control and CMA3-treated groups (n=4/group, 3 images/tumor, total=12 images/group). Data are presented as the mean ± SD. ***P<0.001. (C) Representative Ki-67, pancytokeratin and a-SMA immunohistochemistry staining of the control and CMA3-treated tumors are shown. Scale bar=100 µm. (D) Cytokeratin 19, caspase-9 and Bax expression in representative areas from the control and CMA3-treated groups. Scale bar=50 µm. SD, standard deviation; CMA3, Chromomycin A3; CCA, cholangiocarcinoma; SMA, smooth muscle actin.

Fig 4: Protective effects of CRA-CM against fragmentation of nuclei in oxidatively stressed SH-SY5Y cells. (A) Hoechst 33258 (5 µg/mL) staining was performed in SH-SY5Y cells. Apoptotic cells were identified by the condensation and fragmentation of their nuclei (red arrow). The negative control Caco-2 cells were treated with PBS. The expression levels of apoptosis-related genes and proteins (Bcl-2 and Bax) in oxidatively stressed SH-SY5Y cells were measured by qPCR (B) and western blotting and cropped blots are displayed (C), respectively. Western blot analysis was quantified using ß-actin as an internal control (D). The full-length blots are shown in Supplementary Fig. S4. The data are representative of three independent experiments performed in triplicate.

Fig 5: Combined treatment of Andro and TRAIL induces caspase-dependent apoptosis in 786-0 cells. (A) Cell apoptosis was determined by Annexin V-FITC after DMSO, TRAIL (50 ng/ml), and/or Andro (5 µM) treatment for 24 h. (B) Indicated protein levels in 786-0 cells treated with TRAIL (50 ng/ml) and/or Andro (5 µM) for 24 h as detected by immunoblotting. (C) Cells were treated with DMSO, TRAIL (50 ng/ml), and Andro (5 µM), pan-caspase inhibitor Z-VAD (0.05 µM), and cell-necrosis inhibitor necrostatin-1 (Nec-1) (0.05 µM). Then cell viability was determined by MTS assay (one-way ANOVA, Tukey). (D) Images (magnification, ×400) show the apoptotic cells following treatment under different conditions. Data are shown as mean ± SD; n.s. (not significant), P>0.05, ***P<0.001, n=3). Andro, andrographolide; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; PI, propidium iodide; PARP1, poly(ADP ribose) polymerase 1; Bax, Bcl-2 associated X, apoptosis regulator, DR, death receptor.