Fig 2: CA9 inhibition under hypoxia induced both apoptosis and ferroptosis in MM cells. (A) SYTOX Green staining of ACC-Meso-1 cells was performed to evaluate the effects of CA9 inhibitors (S4 and U104, 75 μM), which induced regulated cell death after 48 h-incubation under hypoxia (scale bar = 50 μm). Apoptosis was determined by detection of FITC-Annexin V with FACS (B) and TUNEL assay (C). (D) Expression of cleaved caspase-3 in ACC-Meso-1 cells was accessed by western blotting after 48 h-treatment with S4 or U104 under hypoxia. (E) Effects of Fer-1 (3 μM), DFO (0.5 μM) and Z-VAD (50 μM) on S4 and U104 induced cell death (S4 50 μM or U104 60 μM) were evaluated after 72 h-treatment under hypoxia. (F) Effect of erastin (20 μM) on CA9 and iron metabolism-associated proteins and cell viability (G) in hypoxic ACC-Meso-1 cell (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs each control (CTRL) unless indicated by bar). Refer to text for details. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: CA9 overexpression in MM cells correlates with increased catalytic Fe(II) in response to hypoxia. (A) Differential overexpression of Ca(r)9 in asbestos-induced rat MM previously analyzed by expression array. Normal mesothelium (N = 2)/Met-5A cells (N = 2) in combination (N = 4) were used as a control; SM (N = 13) or EM (N = 9) represent sarcomatoid or epithelioid subtypes of malignant mesothelioma (MM) [6,49]. (B)Ca(r)9 overexpression in MM samples (shown in A plus biphasic subtype; N = 2; total N = 24) exhibited an inverse correlation with rat survival [6]. Pearson's correlation coefficient and corresponding P value is indicated. Quantitative RT-PCR (C) using GAPDH and western blotting (D) using β-actin, as corresponding internal standards, were performed to access CA9 mRNA and protein levels by using two human EM (ACC-Meso-1 and NCI–H2052) and one SM (NCI–H2373) cell lines in comparison to non-tumorous human mesothelial cell (MeT-5A) after 48 h-incubation in normoxia (N) or hypoxia (H; 1% O2). (E) Western blotting was performed to determine levels of iron metabolism-associated proteins in MM cells after 48 h-incubation in normoxia or hypoxia. (F) Differential levels of catalytic Fe(II) in normoxia or hypoxia of ACC-Meso-1 cells were determined by staining with SiRhoNox-1 (5 μM), followed by evaluation with a flow cytometer after 48 h-incubation. (G) Localization of catalytic Fe(II) in normoxic or hypoxic ACC-Meso-1 cells were analyzed by co-staining of SiRhoNox-1 (5 μM), LysoTracker (200 μM) and MitoTracker (75 μM) after 48 h-incubation and observation with a confocal microscope. Integrated intensity per cell (N = 7) was quantified by ZEN Imaging Software (ZEISS) (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs each control unless indicated by bar). Refer to text for details. SM, sarcomatoid mesothelioma; EM, epithelioid mesothelioma; N, normoxia; H, hypoxia; A.U., arbitrary unit.

Fig 4: Immunohistochemical analysis of tumor nodule. (A) Correlation between tumor masses growth rates and: CA9 in untreated and (C) cisplatin-treated mice, (B) MCM2 in untreated and (D) cisplatin-treated mice. (E) Correlation between tumor masses growth rates and apoptosis in untreated and (F) cisplatin-treated mice. (G) Correlation between tumor masses growth rates and Ki67 in untreated (H) and cisplatin-treated mice. (I) Correlation between tumor masses growth rates and GD2 in untreated and (J) cisplatin-treated mice. The graphs show in left y-axis the growth rate of the tumor masses, expressed as mm3/day, and in the right y-axis the positive area of staining calculated by ImageJ software. On the x-axis, the mice involved in the study.

Fig 5: CA9 inhibition antagonizes MM cell growth and migration under hypoxia. ACC-Meso-1 and MeT-5A cells were cultured under hypoxia and treated with two CA9 inhibitors (S4 and U104). MTT (A) or WST-1 (B) assay was performed to evaluate anti-proliferation effect resulted from CA9 inhibitors in ACC-Meso-1 cells. MeT-5A was used as a non-tumorous control to evaluate the cytotoxic effect caused by CA9 inhibitors. (C) Wound healing assay was performed to estimate migration ability of ACC-Meso-1 cells in response to CA9 inhibition. Cell migration was evaluated by measurement of wound closure prior to (0 h) and after incubation with CA9 inhibitors (24 h). DMSO (0.5% v/v) as a vehicle was used as a negative control (scale bar = 100 μm; means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs control [DMSO vehicle] unless indicated by bar). Refer to text for details.