Extended Data Fig. 3: Endogenous septin localization via immunofluorescence.
From: Blebs promote cell survival by assembling oncogenic signalling hubs
(A) MV3 cells adhered to fibronectin-coated glass slides showing either the mouse SEPT6-GFP probe or anti-SEPT2 immunofluorescence localization. While the well-established septin localization to actin stress fibers (https://doi.org/10.1016/S1534-5807(02)00366-0) is apparent in both samples, the anti-SEPT2 signal is irregular, punctate, and possesses low signal-to-noise ratio (SNR) compared to SEPT6-GFP. Scale bars are 10 µm. (B) Anti-SEPT2 signal in a rounded blebbing MV3 cell embedded in soft bovine collagen, with maximum intensity projections (top) and single z-slices of 0.16 micron thickness (below). Phalloidin signal from the same cell shown on right to visualize the cell surface. Anti-SEPT2 signal possesses low SNR, as seen in adhered cells. Despite this, the signal is enriched at the cell surface in blebby regions of the cell just as seen in cells expressing the SEPT6-GFP probe (see Extended Data Fig. 2a). (C) Cell surface distribution of anti-SEPT2 signal for the cell shown in Extended Data Fig. 3b. (D) Local anti-SEPT2 or SEPT6-GFP intensity and intracellular mean curvature as a function of distance from bleb edges. Mean intensity is comprised only of the brightest 50% of the cell surface to account for punctate and discontinuous immunofluorescent signal. (E) Fraction of cortical voxels (within 0.96 µm of surface) with anti-SEPT2 intensity higher than cytoplasmic mean intensity in MV3 cells with and without expression of SEPT6-GFP. Dashed lines separate quartiles and dots represent individual cells.
