Figure legends
Figure 1 . IR-780 specifically targets tumor tissue and locates to cancer cell mitochondria. (A) Nude mice with T24 subcutaneous tumor xenografts and C57 BL/6 mice with MB49 subcutaneous transplanted tumors were subjected to a single-dose intraperitoneal administration of IR-780 at 1 mg/kg (n=5). The dissected organs at 24 h after injection were subjected to near-infrared fluorescence (NIRF) imaging. (B) The fluorescence intensity of dissected organs and tumors in (A). (C) Tumor and the normal tissue from patients with MIBC were subjected to NIRF imaging after incubation with IR-780 at 10 μM for 2 h (n=5). (D) The fluorescence intensity of (C). (D) Colocalization of IR-780 with a mitochondria-specific tracker (MitoTracker Green) in T24 and MB49 bladder cancer cell lines was imaged using a confocal microscope; the HK2 and SV-HUC-1 normal cell lines were used as controls. The nuclei were stained with Hoechst 33258. Scale bar = 25 μm. (*, p<0.05).
Figure 2 . The anti-tumor effect of IR-780 and influence on the mitochondrial activities of T24 cells. (A) T24, 5637, TCCSUP, and MB49 bladder cancer cell viability were measured using a CCK-8 kit after treatment with various doses of IR-780 for 48 h. (B) T24 cells were treated with IR-780 for 24 h and harvested for detection of apoptosis using flow cytometry. (C) Mitochondrial ROS levels, (D) ATP production, (E) mitochondrial membrane potential, and (F) Mitochondrial complex I activity were detected after treatment with IR-780 for 24 h. (G) Western blot detection of recombinant human DNUFS1 protein treated with IR-780 in vitro. (*, p<0.05).
Figure 3 . HBO promotes IR-780 antitumor efficacy in vitro. (A) Different bladder cancer cell viability was tested after treatment with IR-780 with or without HBO for 48 h. (B) Image of colony formation by T24 cells after treatment with 0 or 7.5 μM IR-780 with or without HBO for 6 h before seeding. (C) The colony formation efficiency of T24 cells at 14 days after seeding. (D) T24 cells were treated with 0 or 7.5 μM IR-780 with or without HBO for 24 h and harvested for detection of apoptosis using flow cytometry. (E) Analysis of variance statistics of the cytometry data derived from samples presented in (D). (F) The morphology of T24 cells after treatment with 0 or 7.5 μM IR-780 combined or not with HBO was observed by inverted microscopy. Scale bar = 25 μm. (G) Expression of apoptosis-related proteins in T24 cells. Cleaved PARP, caspase-3, c-caspase-9, and cytochrome c levels were tested by western blotting. (H) Viability of T24 cells treated with IR-780+HBO in the presence of Z-VAD (an apoptosis inhibitor). (*, p<0.05).
Figure 4 . The antitumor efficacy of IR-780+HBO in vivo. Treatment was carried out by intraperitoneal injection every two days for a total of 5 treatments. The IR-780+HBO and IR-780 groups were treated with 3 mg/kg IR-780. (A) The transplanted tumor size was monitored every 3 days using a sliding caliper. After excision from the mice, the transplanted tumor were photographed (B) and weighed (C). Scale bar = 1 cm. (D) The body weights of mice were measured every 3 days before excision. (E) Long-term observation of tumor volume after mice were treated with IR-780+HBO or DDP. Images of the mouse transplanted tumor at day 28 are shown in (F). Scale bar = 1 cm. (G) H&E staining in major organs of mice. Scale bar = 50 μm. (*, p<0.05).
Figure 5 . HBO promotes uptake of IR-780 in cancer cells by increasing the plasma membrane potential. T24 cells were treated with the indicated doses of IR-780 with or without HBO and imaged using a fluorescence microscope (A), and fluorescence intensity was measured using flow cytometry (B) immediately after the completion of HBO. Scale bar = 25 μm. (C) The fluorescence spectrum of 7.5 μM IR-780 in cell culture medium combined or not with HBO with 740 nm using an excitation wavelength from 750~900 nm to scan their emission spectra. (D) T24 cells were treated with 0 or 7.5 μM IR-780 with or without HBO for 24 h, and NDUFS1 was detected by western blotting. (E) T24 cells were treated with pre-IR-780 (IR-780 was removed after incubation for 30 minutes)+HBO and stained with DiBAC4 to detect the plasma membrane potential by flow cytometry. (F) Plasma membrane potential was tested after cell incubation different doses of K+ medium for 1 h. Cells were pre-incubated for 1 h with the indicated doses of K+ medium followed by 2 μM IR-780 for 10 min, and the fluorescence intensity of IR-780 was examined (G, H). Scale bar = 50 μm. After 2 μM IR-780 treatment for 30min, the cells were incubated with the indicated doses of K+ medium and treated with HBO, and the fluorescence intensity of IR-780 was examined (I). (J) T24 cells were treated with IR-780, pre-IR-780 +HBO, or IR-780+HBO, and cell viability was measured. (*, p<0.05).
Figure 6 . IR-780+HBO induces excessive mitochondrial ROS production. (A) Fluorescence image of the ROS probe DCFDA by T24 cells after treatment with 0 or 7.5 μM IR-780 with or without HBO. Scale bar = 100 μm. (B) DCFDA fluorescence intensity was measured using flow cytometry. (C) Fluorescence images of T24 cells labeled with DCFDA and mitoSOX after treatment with IR-780+HBO. Scale bar = 25 μm. Mitochondrial ROS production detected using mitoSOX (D). (E) IR-780+HBO-induced cellular mitochondrial ROS levels were positively correlated with cell death. (F) Mitochondrial ROS levels were detected after treatment with IR-780+HBO in the presence of DPI (an NADPH inhibitor), MitoQ (a mitochondrial ROS inhibitor) or NAC, and then cell viability was detected (G). (H) Western blot detection of apoptosis induction by IR-780+HBO in the presence or absence of MitoQ or NAC. T24 cells were treated with 7.5 μM IR-780 with or without the indicated concentrations of H2O2 for 24 h and then harvested for detection of apoptosis using flow cytometry (I). Mitochondrial ROS levels of T24 cells were measured after treatment with IR-780+HBO in the present of 21% O2, 50% O2, 100% O2 (J) or TTFA (K). (L) Detection of mitochondrial membrane potential induced by IR-780+HBO. Mitochondrial morphology in T24 cells after treatment with 0 or 7.5 μM IR-780 combined or not with HBO was observed by transmission electron microscopy (M). Scale bar =1 μm. (*, p<0.05).
Figure 7 . The effects of IR-780+HBO on DDP-resistant T24 cells. (A) Staining of IR-780 in T24 and T24/DDP cancer cells was imaged using a fluorescence microscope, and the fluorescence intensity was determined by flow cytometry (B). Scale bar =50 μm. (C) NIRF imaging of T24/DDP subcutaneous tumor xenografts using IR-780. (D) Viability of T24 cells and T24/DDP cells treated with DDP. (E) Cell viability of T24/DDP cells treated with IR-780, IR-780+HBO or DDP. (F) The volumes of T24/DDP cell tumors were observed after nude mice were treated with IR-780+HBO or DDP. Images of the xenografts after excision from the mice at day 19 are shown in (G). Scale bar = 1 cm. (H) In the IR-780+HBO treatment group, the T24/DDP tumor xenografts appeared to be ruptured. (*, p<0.05).
Figure 8 . Proposed model of mitochondrial apoptosis-inducing mechanism by IR-780+HBO. IR-780 preferentially accumulates in the mitochondria of tumor cells by targeting mitochondrial protein NDUSF1, causing many electrons to leak out; combination with HBO could induce cancer cell apoptosis by promoting cancer cell uptake of IR-780 and inducing excessive mitochondrial ROS production.