Fig. 4
In vitro experiments with MnO2-C/M-HA. a The cytotoxicity of 4T1 cells of varying formulations with or without 660 nm (1.0 W cm−2, 15 min) for 24 h. b Fluorescence images of Calcein-AM and PI double stained 4T1 cells treated by different formulations. Scale bar: 100 μm. c Flow cytometric analysis of apoptosis of 4T1 cells receiving different treatments and evaluated by Annexin V and PI staining. d Quantifcation results of apoptotic cells. I, Control; II, MnO2-HA + laser; III, MnO2-Ce6-HA + laser; IV, MnO2-MTZ-HA + laser; V, MnO2-C/M-HA; VI, MnO2-C/M-HA + laser. e Fluorescence microscope images of ROS generation by DCFH-DA staining in cells. Scale bar: 100 μm. f Western blot analysis of CRT, HMGB1 proteins expression after 4T1 cells treated with different approaches. I, Conttrol; II, MTZ; III, MnO2-MTZ-HA; IV, MnO2-C/M-HA; V, MTZ + laser; VI, MnO2-MTZ-HA + laser; VII, MnO2-C/M-HA + laser. g Fluorescence microscopic images to show the CRT exposure on and HMGB1 release from 4T1 cells after various treatments. Scale bar: 50 μm. h Detection of the released HMGB1. All the data were displayed as mean ± SD (*p < 0.05, **p < 0.01 and ****p < 0.0001)