Comparison of PIP2 sensor dissociation from PM by retinal vs a known photosensitizer, rose bengal. (A) Images of HeLa cells expressing PIP2 sensor (mCherry-PH). (A) PIP2 sensor translocation was induced by rose bengal (50 µM), incubated with cells for 5 minutes, in the presence of blue (4.86 µW of 445 nm) and green (light (0.22 µW of 515 nm) respectively. Plot shows the cytosolic fluorescence of PIP2 sensor in HeLa cells upon exposing to light. (mean ± S.E.M., n = 6). (B) HeLa cells were incubated with CoCl2 (100 µM) for 24 h to expose cells to hypoxia. The control cells were kept in same conditions without CoCl2 treatment. Cells were incubated with ATR (50 µM) and 445 nm imaging for 10 minutes was performed. Cell in hypoxic condition did not exhibit detectable PIP2 sensor accumulation in cytosol while control cells showed a gradual PIP2 sensor accumulation from PM to cytosol. (C,D) Antioxidants were tested to examine if they prevent PIP2 sensor translocation induced by retinal and blue light. HeLa cells expressing PIP2 sensor were incubated with antioxidants, alpha-tocopherol (1 mM) and reduced-glutathione ethyl ester (500 µM) overnight. Prior to imaging experiments ATR (50 µM) was added and incubated for 5 minutes followed by exposure of blue light (4.86 µW of 445 nm) for 5 minutes. (C) Cells treated with reduced-glutathione ethyl ester exhibited PIP2 sensor translocation from PM to cytosol upon blue light exposure. Plot shows the dynamics of PIP2 sensor (mean ± S.E.M., n = 14 cells). Overview of the antioxidant mechanism exert by reduced glutathione in vivo (right). (D) Cells treated with alpha-tocopherol showed a reduced rate and extent of PIP2 sensor translocation from PM to cytosol upon blue light exposure. Plot shows the dynamics of PIP2 sensor translocation (mean ± S.E.M., n = 6 cells). Note the reduction of PIP2 sensor accumulation in IMs of cells. Right: Overview of the antioxidant mechanism exert by alpha-tocopherol in vivo. (E) Proposed mechanism for blue light excited retinal induced PIP2 distortion process. (F) TD-DFT calculations (CAM-B3LYP/6–31++G**) of retinal’s energy states and the Jablonsky diagram shows strong absorption band due to the π → π* transition where triplet excited states are energetically and symmetrically matched to allow for efficient intersystem crossing and energy transfer to O2 which allows for singlet oxygen and ROS generation. Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box and green light (GL) = green box). Scale = 5 µm.