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Published in final edited form as: Int J Hyperthermia. 2025 Aug 11;42(1):2544017. doi: 10.1080/02656736.2025.2544017

Figure 3. — Immunofluorescence assays confirm that multimodal treatment enhances apoptosis in HCT116 and BxPC-3 cell lines. HCT116 and BxPC-3 cells were treated with 5μM and 10μM ART, respectively, for 20h at 37°C, followed by treatment with or without 2 ng/mL rhTRAIL for an additional 4h. Some samples were exposed to hyperthermia at 42°C for 1 h, then incubated at 37°C for the remaining 3h. (A, C) Annexin V staining assay was immediately performed after multimodal treatment was finished by staining cells with Annexin V-FITC (green), propidium iodide (PI) (red), and hoechst (blue), and analyzed using an ECHO fluorescence microscope. Representative images are shown (scale bar: 100μm) (B, D) ImageJ program was used to analyze the relative fluorescence intensity of each of the samples, measuring the hoechst stain intensity, Annexin V stain intensity (green channel), and PI stain intensity (red channel). The bar plot represents the ratio of Annexin V stain fluorescence intensity to hoechst stain fluorescence intensity, as well as PI stain intensity to hoechst stain fluorescence intensity, with error bars representing the mean ± SD from triplicate experiments. (E) TUNEL staining assay was performed on treated HCT116 cells and fluorescent images were examined under an ECHO fluorescence microscope. Representative images are shown (scale bar: 100μm). (F) ImageJ program was used to analyze the relative fluorescence intensity of each of the samples, measuring the hoechst stain intensity and the TUNEL stain intensity. The bar plot represents the ratio of TUNEL stain fluorescence intensity to hoechst stain fluorescence, with error bars representing the mean ± SD from triplicate experiments.

Figure 3. — Immunofluorescence assays confirm that multimodal treatment enhances apoptosis in HCT116 and BxPC-3 cell lines. HCT116 and BxPC-3 cells were treated with 5μM and 10μM ART, respectively, for 20h at 37°C, followed by treatment with or without 2 ng/mL rhTRAIL for an additional 4h. Some samples were exposed to hyperthermia at 42°C for 1 h, then incubated at 37°C for the remaining 3h. (A, C) Annexin V staining assay was immediately performed after multimodal treatment was finished by staining cells with Annexin V-FITC (green), propidium iodide (PI) (red), and hoechst (blue), and analyzed using an ECHO fluorescence microscope. Representative images are shown (scale bar: 100μm) (B, D) ImageJ program was used to analyze the relative fluorescence intensity of each of the samples, measuring the hoechst stain intensity, Annexin V stain intensity (green channel), and PI stain intensity (red channel). The bar plot represents the ratio of Annexin V stain fluorescence intensity to hoechst stain fluorescence intensity, as well as PI stain intensity to hoechst stain fluorescence intensity, with error bars representing the mean ± SD from triplicate experiments. (E) TUNEL staining assay was performed on treated HCT116 cells and fluorescent images were examined under an ECHO fluorescence microscope. Representative images are shown (scale bar: 100μm). (F) ImageJ program was used to analyze the relative fluorescence intensity of each of the samples, measuring the hoechst stain intensity and the TUNEL stain intensity. The bar plot represents the ratio of TUNEL stain fluorescence intensity to hoechst stain fluorescence, with error bars representing the mean ± SD from triplicate experiments.