Fig. 4. (A) Intracellular delivery efficiency of the NB-R10 fusion protein with TBN-R10 additives. Top: schematic design of NB-R10 fusion protein. Bottom: CLSM images of OS-RC-2 cells incubated with 5 μM 12E-R10-Cy5 or 4C-R10-Cy5 in the presence or absence of 10 μM TNB-R10 for 1 h at 37 °C. Excitation: 581 nm; emission: 580–650 nm; scale bar: 20 μm. Individual cytosolic fluorescence intensity of the CLSM images in the presence of TNB-R10 was processed with Image J software. (B) Viabilities of OS-RC-2 cells incubated with 12E-R10/4C-R10 and 10 μM TNB-R10 in the presence or absence of Ferrostatin-1 (−Fer-1, ferroptosis inhibitor) for 12 h at 37 °C. (C) Viabilities of L-02 cells incubated with 4C-R10/12E-R10 and 10 μM TNB-R10 additives, or small molecular inhibitors RSL3 and ML162 at 37 °C for 72 h. The results were repeated three times and presented as mean ± SD (n = 3). (D) Lipid peroxidation induced by 12E-R10, 4C-R10 and RSL3 based on C¹¹ BODIPY^581/591 cell imaging. OS-RC-2 cells were first incubated with 5 μM 12E-R10/4C-R10 (in the presence of 10 μM TNB-R10) and RSL3 (1 μM) at 37 °C for 2 h, and then incubated with 1 μM C¹¹ BODIPY^581/591 for 30 min for imaging; scale bar: 20 μm. Cells were also pre-incubated with (+Fer-1) or without Fer-1 (−Fer-1). Individual cell fluorescence intensity in different channels of the CLSM images (processed with Image J software) was indicated on the right of the images.