1316 15 7373 7 41785 document 7373 Images_timecourse.zip application/zip 92ab84f43cfc0aaabe7699401540885c MD5 310517787 2025-06-17 09:50:44 http://edata.bham.ac.uk/1316/1/Images_timecourse.zip 1316 1 2 application/zip archive public cc_by_4

Images_timecourse.zip

data 7374 7 41787 document 7374 Images_analysed.zip application/zip 54ee2caa213874de05502b422f64d482 MD5 3948304639 2025-06-17 09:55:50 http://edata.bham.ac.uk/1316/2/Images_analysed.zip 1316 2 1 application/zip archive public cc_by_4

Images_analysed.zip

data archive 660 disk0/00/00/13/16 2025-07-23 12:00:36 2025-07-23 13:11:34 2025-07-23 12:00:36 data_collection show O'Brien Shannon s.l.obrien@bham.ac.uk Calebiro Davide d.calebiro@bham.ac.uk 0000-0002-3811-1553 10col_mede HILO imaging, GPCR, G protein, intracellular signaling, intracrine activation This repository contains HILO microscopy images of either differentiated immortalized mouse brown preadipocytes or differentiated 3T3-L1 cells, as indicated in each image file name. Cells were transiently transfected with either YFP-tagged free fatty acid receptor 4 (FFA4) or Halo-tagged β2-adrenergic receptor (B2AR), labelled with JF646. These fluorescently tagged receptors were used to train a machine learning–based pixel classifier in Ilastik to categorize receptor pixels as either plasma membrane or intracellular. In some images, cells were also co-transfected with mini-G protein probes, which translocate from the cytoplasm to active receptors upon stimulation. MATLAB was used to quantify mini-G translocation to FFA4 or B2AR at both the plasma membrane and intracellular membranes. Each file name includes, in order: the corresponding figure, cell line, transfected overexpressed proteins, laser excitation wavelength, stimulant used, and cell number. 2025-07-23 published University of Birmingham https://doi.org/10.25500/edata.bham.00001316 Dataset research-data@contacts.bham.ac.uk Institute of Metabolism and Systems Research wt 212313/Z/18/Z yes yes yes TRUE Professor Calebiro Davide d.calebiro@bham.ac.uk Live-cell HILO imaging was performed on a custom total internal reflection fluorescence (TIRF) microscope (assembled by CAIRN Research). The system is based on an Eclipse Ti2 microscope (Nikon, Japan) equipped with four EMCCD cameras (iXon Ultra 897, Andor), 100x oil-immersion objective (SR HP APO TIRF NA 1.49, Nikon), an iLas2 TIRF illuminator (Gataca Systems), 405, 488, 561, and 637 nm diode lasers (Coherent, Obis), a quadruple beam splitter, quadruple band excitation and dichroic filters, 1.5x tube lens and hardware focus stabilization. Two of the four synchronized EMCCSs were used to acquire simultaneous image sequences at a rate of one image every 30 s. HILO images were acquired with MetaMorph 7.10.2.240. en 2018 2025