Brightline filter sets

Live-cell imaging was performed using a microscope stage top incubator INUG2 (Tokai Hit) equipped with an objective heater to maintain the cells at 37 °C with 5% CO₂. The incubator was installed on the stage of Olympus IX71 epi-fluorescence inverted microscope equipped with 150× 1.45NA TIRFM objective lens, iXon-EMCCD camera (Andor, Ireland), and BrightLine filter sets (Semrock, USA) for Alexa488 (485/524 nm excitation/emission), Rhodamine (543/593 nm excitation/emission) and CFP (434/479 nm excitation/emission). Image sequences were acquired using μManager open source software at 10 or 30 fps⁵⁴ . Images were further processed using ImageJ for cropping, background, and brightness/contrast adjustments⁵⁵ . To quantify the Rtnl1-driven constriction of the peripheral ER network in COS-7 cells still images extracted from the image sequences obtained at different times post-transfection were used. Two to three 30–40 μm² ROIs covering the peripheral ER (as in Fig. 3a) were used. All point histograms of the pixel fluorescence intensity were obtained for each ROI for the control COS-7 cells and cells expressing Rtnl1 and GFP-Rtnl1 (the corresponding probability densities are shown in Supplementary Fig. 2c).