Fig. 5

LPL-roGFP-Orp1 is spatially oxidized at protrusions of cells. a Intensity profiles of TRX1, LPL, and F-actin in untreated (left) and 50 μM H₂O₂-treated (right) MV3 wt LPL eGFP cells. MV3 LPL eGFP cells (green) that firmly adhered to poly-D-lysine-coated coverslips overnight were kept untreated or were treated with 50 μM H₂O₂ for 30 min. The samples were then fixed and stained for TRX1 (blue) and F-actin (red). The samples were imaged using a confocal microscope (n = 5). Scale bar = 10 µm; ×100 magnification. b Quantification of TRX1 in filopodia and cell bodies. At least 50 cells were analyzed from three independent experiments. The data are presented as the mean ± SEM (n ≥ 3; ****p < 0.0001, ns nonsignificant). P-values were calculated by t-test. c Schematic diagram of the LPL-roGFP-Orp1 sensor. d Representative time-lapse images of MV3 LPL-roGFP-Orp1 cells. The upper panel shows F-actin (white), and the lower panel shows the oxidation state of the roGFP-Orp1 probe. The magnified area shows filopodial extensions. The color scale from dark blue to green and red indicates the oxidation state of the sensor. Ratio imaging was performed with a confocal microscope equipped with dual excitation features (n ≥ 3). Scale bar = 10 µm; ×100 magnification. e Heat map showing the ratio of oxidized (excitation (Ex) 405 nm) to reduced (Ex 488 nm) roGFP-Orp1 in LPL-roGFP-Orp1-expressing cells in the absence of H₂O₂ (time-lapse imaging, t = 0 h, three independent experiments, n = 103 cells). Each lane represents a single cell. The color scale from blue to green and magenta indicates the oxidation state of the sensor in different cells