Fig 2: Increase of HO-1 expression by TChal. a Antibody array was performed to analyze the expression of 84 proteins on the membrane. Control or TChal-treated cell lysates from BT-20 cells were subjected to antibody array. The red box indicates HO-1, which was highly increased compared to the control. b Western blot analysis for the validation of the dose-dependent HO-1 expression in cancer cell lines treated with TChal at the indicated dose. β-actin was used as the internal control. c BT-20 cells were treated with 30 μM of TChal at different time points and cell lysates analyzed by western blotting. Equal loadings were measured by β-actin expression. d MDA-MB-231 and TPC-1 cells were treated with TChal and the cell lysates were subjected to western blotting. Equal loadings were measured by β-actin expression. e realtime PCR from BT-20 cells treated with different concentration of TChal for 24 h. ***p < 0.001. f Western blotting analysis for HO-1 in BT-20 cells treated with TChal for 1 h followed by the treatment of DMSO or epoxomicin for 24 h. Equal loadings were measured by β-actin expression

Fig 3: ALA regulates the level of the antioxidant system via Nrf2/HO-1 signaling. (a) Representative is the result of ROS; (b) immunoblot for the expression level of HO-1 in mouse brain (n = 5 mice per group; the number of experiments = 3; statistical analysis of one-way ANOVA with Tukey’s posthoc test was used for comparisons among the different groups; (c) double immunofluorescence result showing the expression level of Nrf2 and NOS-2 protein molecules. Magnification 10×, scale bar 100 um, and high resolution. Significantly different from the vehicle-treated control group, #, significantly different from the Cd-treated mouse group. Significance: * p < 0.05; # p < 0.05.

Fig 4: Analysis of HO-1 expression after siRNA transfection. a RT-PCR analysis using BT-20 cells transfected with HO-1 siRNA. The siRNA for HO-1 was transfected and the total RNA was isolated. RT-PCR for HO-1 was performed as described in the Methods section. GAPDH was used as the internal control. b Western analysis of HO-1 on BT-20 cells after HO-1 siRNA transfection. Equal loadings were measured by β-actin expression. c The toxicity assay on HO-1 siRNA-transfected BT-20 cells. After HO-1 siRNA transfection, the cells were tested for cell growth inhibition. Y-axis represents the percentage (%) of cell growth inhibition compared TChal to DMSO treated cells for 24 h. The data are representative of three independent experiments. **p < 0.01

Fig 5: Overexpressing HMOX1 or modulating its activity affects p38 MAPK/c-jun activation and MMP-9 expressionA. 10 x 106 MEC-1 cells were nucleofected with CRISPR/dCas9 HMOX1 activation (pAct H) or control (pAct C) plasmids and incubated with or without 3 μM ATO. After 24 h, cell lysates were analyzed by Western blotting. Values represent the average of two different experiments. B. 3-5 x 106 MEC-1 cells were incubated for 1 h with or without 3 μM CoPP or 2 μM ZnPP. 5 μM ATO was added or not and cells further incubated for 24 h. Cell lysates and conditioned medium were analyzed by Western blotting and gelatin zymography, respectively. The results of one representative experiment and the normalized average values of the three experiments performed are shown. *P ≤ 0.05; **P ≤ 0.01.