Fig 2: SEPT2 organizes the polarized distribution of Orai1 in invading cells. A) Quantification of entotic cells cultured for 4 h with DMSO and the SEPTIN inhibitor, FCF (50 µm). Data represent mean ± SEM of triplicate experiments (n > 300 in each experimental group). B) Immunofluorescence images show endogenous SEPT2 (red) and GFP-Orai1 (green) in MCF7 cells cultured for 2.5 h in suspension. The arrow indicates enriched SEPT2 at ID. Line graphs show relative SEPT2 (red) and GFP-Orai1 (green) intensities for the indicated line scans. X axis: 5 µm, Y axis: 1 (A.U.). C) Bar graph showing the distribution of SEPT2 at different engulfment stages. Early-stage (E, n = 7); middle-stage (M, n = 9); and late-stage (L, n = 13). D) Immuno-fluorescent images of suspended cells taken after 2.5 h of culturing show endogenous SEPT2 (green) and CellBrite (red, PM marker). Line graphs show relative SEPT2 (green) and PM marker (red) intensities for the indicated line scans. X axis: 5 µm, Y axis: 1 (A.U.). E) Fluorescence images of GFP-SEPT2-expressing MCF7 cells cultured for 2.5 h in DMSO and FCF (50 µm). Line graphs show the distribution of GFP-SEPT2 in DMSO (green) and FCF (black dotted). F) Immunofluorescence images showing endogenous SEPT2 (red) and endogenous Orai1 (green) in MCF7 cells cultured for 2.5 h in DMSO or FCF (50 µm). The arrow indicates enriched SEPT2 at ID. Line graphs show the distribution of SEPT2 (red) and Orai1 (green). X axis: 5 µm, Y axis: 5000 (A.U.). G,H) Bar graphs showing the distribution of SEPT2 (G, n = 10) and Orai1 (H, n = 7) at ID in late engulfment stage (from Figure S10, Supporting Information). I) Immunoblots of whole-cell lysates (left) or IP (right) from cell co-expressing Flag-Myc-Orai1 and GFP-SEPT2 in HEK293T cells with or without 50 µm of FCF. J) Immunoblots of whole-cell lysates (left) or IP (right) from cell co-expressing Flag-Myc-Orai1 and GFP-STIM1 in HEK293T cells with or without 50 µm of FCF and TG (1 µm). Scale bar = 5 µm. Significance was determined using unpaired two-tailed t-test. ***p < 0.001; **p < 0.01. ED: engulfing cell distal region; CC: cell–cell contact site; ID: invading cell distal region.