Fig 2: OSBP translocates from Golgi to PM via interacting with ORP4L.a Electron micrographs of ORP4L KI and wild-type T-cells, HepG2 and 293 T cells. Scale bar, 100 nm. Black triangles present in dividual data points for n = 20 cells from one mouse or cell line. The central mark indicates the median, the bottom and top edges of the box indicate the interquartile range, and the whiskers represent the maximum and minimum point. One-way ANOVA test with a confidence interval of 95% was used to compute statistics. b Co-immunoprecipitation analysis of ORP4L binding to OSBP in ORP4L KI T-cells with or without anti-CD3 stimulation. c The localization of OSBP in ORP4L KI T-cells. Scale bar, 10 µm. d The localization of OSBP in ORP4L KI and wild-type T-cells with or without anti-CD3 stimulation. Scale bar, 10 µm. e Western blot analysis of OSBP protein levels in PM and Golgi of ORP4L KI or wild-type T-cells. f A schematic representation describing the BiFC technique. g Interactions between ORP4L/pVn-C1 and OSBP/pVc-C1 in ORP4L KI T-cells as determined by BiFC. Scar bar, 10 µm. h The localization of OSBP in ORP4L KI T-cells upon ORP4L knockdown. Scar bar, 10 µm. i Western blot analysis of OSBP protein levels in PM and Golgi of ORP4L KI T-cells upon ORP4L knockdown. j The localization of overexpressed wild-type OSBP or OSBP (?ORP4L) in ORP4L KI T-cells. Scar bar, 10 µm. k Western blot analysis of OSBP protein levels in PM and Golgi of ORP4L KI T-cells with or without VAPA knockdown. l Western blot analysis of overexpressed wild-type OSBP or OSBP(?FFAT) protein levels in the PM and Golgi of ORP4L KI T-cells. Confocal microscopy and blot images are representative of n = 3 biological replicates with similar results. The same experiments were repeated twice in T-cells from two mice. In (c, d, h and j), the ration of OSBP fluorescence density from n = 10 cells from one mouse between PM and Golgi are shown below. Source data are provided as a Source Data file.

Fig 3: Inducible nuclear F-actin in the 24 h differentiating Th cells(A) Immunofluorescence staining of naive, 24 h- and 48 h-differentiating Th1 cells using DAPI (blue) and Phalloidin conjugated to biotin followed by cy3-streptavidin (red). Colocalization rate of the merged channels of nuclear F-actin and chromatin (overlapping signal in white). Images of wider fields and of the Th2 staining are presented in Figure S1A.(B) Comparison of the percentage of the visible nuclear F-actin-harbored cells between naive (n = 230), 24 h (n = 185)- and 48 h (n = 176)- differentiating Th1 cells. Data are represented as mean ± SEM. p values were computed with proportion Z-tests and exact Fischer’s test *p < 0.05, **p < 0.01, ***p < 0.001.(C) Z stacks of representative images of naive and Th1 cells from (A) were combined into a computational 3D-structures using the IMARIS 9.5 software (chromatin in transparent blue to facilitate F-actin visualization, and F-actin in red). The images are presented at the XY direction. The Th2 computational images are shown in Figure S1B.(D) Computational 3D-structure of 24 h-differentiating Th1 cells are presented at the XZ direction.(E) The differentiating Th cells were stimulated directly on the slide with anti-CD3 and anti-CD28 abs to maintain the orientation toward the TCR. The staining procedure was performed directly on the slide. Computational 3D-structure at XZ direction presenting F-actin (red), CD3e (green), and chromatin (blue). Wider fields and zoom-in images are presented in Figures S1C. The experiments were performed in two independent biological replicates with similar results.(F) (left) Immunofluorescence staining of 24 h-differentiating Th1 cells applying the cytospin protocol, using Phalloidin conjugated to biotin followed by cy3-streptavidin (red) and Hoechst (blue). (Right) Colocalization rate of nuclear F-actin and chromatin (overlapping signal in white). Images of Wider fields are presented in Figures S1C and S1D. The experiments were performed in two independent biological replicates with similar results.(G–K) (G) Immunofluorescence staining of the 24 h-differentiating Th1 cells using Phalloidin (red) and anti-Vav1 Ab (green). DNA was stained with Hoechst (blue). (H) Magnification of the white square in (I). The Th2 staining and images of wider fields are presented in Figure S1H. (I) Colocalization rate of nuclear F-actin and Vav1 (overlapping signal in white). The white dashed line, which was determined by Hoechst staining, defines the nuclear periphery (ROI) for nuclear colocalization assessment. (J) Colocalization rate of nuclear Vav1 and chromatin (overlapping signal in white). (K) Computational 3D-structure of Vav1, F-actin and chromatin. The Th2 images are presented in Figure S1K. Colocalization of Vav1, F-actin and chromatin in naive and 48 h differentiating Th cells are presented in Figures S1I and S1J. The experiments were performed in three independent biological replicates with similar results. Secondary Ab only was used as Control (Figure S1N). Images were acquired by super resolution (SR) Hyvolution microscope.