Orca 2

Hek293T cells were imaged on a widefield Zeiss Axiovert 200 M microscope (Carl Zeiss MicroImaging, Jena, Germany) equipped with a 10x Plan Neofluar objective (NA 0.03 air) and a cooled charge-coupled device camera (Hamamatsu ORCA II). Cells were excited using a mecury lamp, at 500/20 nm with a 535/30 nm emission filter for YFP donor signal and a 620/60 nm emission filter for RFP sensitized emission. For OFP donor signals, 500/20 nm excitation was paired with a 572/28 nm emission filter, while the near-infrared sensitized emission was excited with 500/20 nm and detected by a 620/60 nm emission filter. Regular acceptor intensities were obtained by 577/20 excitation with a 620/60 nm emission filter. Cells were stimulated with a 1:1000 dilution of 10 mM paclitaxel stock dissolved in DMSO (Sigma-Aldrich) and imaged again 1 h after incubation. Background and flat-field corrections were applied to all images. Cells with RFP intensity counts above 200 were not analyzed in order to avoid artefacts in the FRET signal due to concentration. The RFP/YFP FRET ratio for each condition was obtained by calculating the slope of plotting Σ(donor × SE)/Σ(donor²) for n > 400. The error bars represent the 95% confidence intervals. Measurements were taken from distinct samples.

The cells were incubated with 0.016; 0.08; 0.4; 2; 10 or 50 μM EdU alone or together with 8 nM FdU for 8 hours. After fixation and permeabilization, the incorporated EdU was visualised by means of a click reaction using Alexa Fluor 488 azide (30 min, room temperature (RT), Life Sciences). The nuclear DNA was stained by DAPI (10 μM, 30 min, RT). The images were obtained by an Olympus IX81 microscope (objective: UPLFLN 10× NA 0.3) equipped with a Hamamatsu ORCA II camera with a resolution of 1344×1024 pixels using Cell∧R acquisition software (Olympus). The data were analysed using CellProfiller image analysis software, Microsoft Excel and the final graphs were made in GraphPad Prism 6 software. The measurements were performed for the three independent experiments. The graph was constructed using the standard four parameter logistic nonlinear regression according to the following equation: $y=bottom+\frac{top-bottom}{1+{10}^{\left(\right(\mathrm{l}\mathrm{o}\mathrm{g}EC50-x)\times hillslope}}$ , where x, y, top, bottom and hillslope represent the same parameters as mentioned above; EC50 is a half maximal effective concentration. The data are presented as mean values ± SEM. 10,000 cells were evaluated for every cell line.

Fluorescence imaging of live and fixed cell cultures was performed on an inverted fluorescence microscope (IX70, Olympus). Digital images were acquired using a cooled CCD camera (ORCA II, Hamamatsu) that was controlled using MetaMorph software (Molecular Devices). Time-lapse images of AChR vesicles were captured at an interval of 500 ms for 3 min or 3 s for 30 min under the control of an optical filter changer (Lambda 10-2; Sutter Instrument). Data were analyzed using ImageJ (National Institute of Health) and MetaMorph software (Molecular Devices). Data are shown as mean ± SEM and the statistical significance of differences between the control and experimental groups was assessed using Student’s t test.

In situ hybridization analysis was performed on 16-μm cryosections of eyes obtained from 2-month-old mice, as described elsewhere43 (link). An Adipor1-specific cDNA (base pairs 718–914 of NM_028320) was generated by PCR with primers that incorporate the T7 RNA polymerase promoter sequence into the PCR amplicon. This DNA template was used for in vitro transcription reaction with 80 μCi of αP33-UTP (NEN Life Science Products, Boston, MA). After hybridization at 60 °C for 16 h, sections were treated with RNase and washed in SSC buffer. Slides were dehydrated in a graded ethanol series and exposed to a 50% solution of autoradiographic emulsion type NTB2 (Eastman Kodak Company, Rochester, NY) for 3–6 days. Slides were developed using standard protocols44 , dehydrated and coverslips were applied (Permount; Fisher Scientific, Pittsburgh, PA). Digital images were acquired (ORCA II; Hamamatsu) using a cooled CCD camera mounted on a BX60 (Olympus) microscope equipped with dark-field optics.

Cells were grown to midlog in YM and stained live using Mitotracker Green and Rhodamine B hexyl ester (Y7530; Life Technologies) per the manufacturer’s instructions. Cells were imaged for differential interference contrast and fluorescence microscopy using an Eclipse 600-FN Nikon microscope with an Apochromat 100×/1.40 NA oil immersion objective, and a cooled charge-coupled device camera (ORCA-2; Hamamatsu Photonics). Images were processed with MetaMorph v7.0 software (Molecular Devices) and further processed using Image J.

To analyze the expression of selected surface markers, three-color flow cytometry was used. The cells were stained on ice for 30 min with fluorochrome-conjugated antibodies, then measured on a FACS Calibur flow cytometer (BD Biosciences Immunocytometry Systems, Franklin Lakes, NJ). The data were analyzed using Flowing Software (Cell Imaging Core, Turku Centre for Biotechnology, Finland) and the results were expressed as means of positive cells (%) ± SD. For immunohistochemistry studies, cell cultures were fixed in 4% PFA. Cytoskeletal actin filaments were labelled by phalloidin-TRITC (Sigma-Aldrich, Budapest, Hungary) and the nuclei by Hoechst 33342 (Invitrogen, Oregon, USA). Samples were examined under an Olympus IX81 inverted microscope with MT20 station (Olympus, Münster, Germany) and Orca2 (Hamamatsu Photonics K.K., Japan) camera. Surface carbohydrate molecules were labelled with lectins (Vector Labs, Burlingame, CA) diluted in Hepes buffer (Sigma-Aldrich)and examined by Olympus FluoView 1000 confocal LSM (Olympus).

For immunohistochemistry studies, cell cultures were fixed in 4% PFA. To visualize endogenous LC3, fixed samples were stained with anti-LC3B antibody (Sigma-Aldrich, St. Louis, MO, USA), and a fluorochrome-conjugated secondary antibody was used to label the cells. Samples were examined under an Olympus IX83 inverted microscope with an Olympus ScanR high-content imaging screening platform (Olympus, Tokyo, Japan) equipped with an Orca2 (Hamamatsu Photonics K.K., Shizuoka, Japan) camera. The images were subjected to line-scan fluorescence-intensity analysis and 3D surface plotting performed with Image J (v.1.53m, 28 September 2021).