DOI: 10.5281/zenodo.7116548
title: Autophagosomal/autolysosomal/lysosomal dynamics of FaDu and HGFb cells affected by autophagy modulators (dataset of dual labeling of autophagosomes and labeling lysosomes, confocal microscopy)

description: In this dataset, the impact of autophagy modulators on the auophagosomes and lysosomes in FaDu and HGF cells was investigated. Experimental details are described in the accompanying paper. This dataset contains total 702 CZI confocal image Z-stacks.

Relevant paper: HANELOVA, Klara, RAUDENSKA, Martina, KRATOCHVILOVA, Monika, NAVRATIL, Jiri, VICAR, Tomas, BUGAJOVA, Maria, GUMULEC, Jaromir, MASARIK, Michal and BALVAN, Jan. Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles. Cell Communication and Signaling. 24 May 2023. Vol. 21, no. 1, p. 120. DOI 10.1186/s12964-023-01126-z.





 



Model cell lines

 

The cell line FaDu (HTB-43TM), derived from a squamous cell carcinoma (SCC) of the hypopharynx, and the human gingival fibroblast cell line HGF (derived from the histologically normal gingival biopsy) were used in this study. The authenticated cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, Virginia, USA) within the last five years. 

Autophagy modulation

For autophagy modulation, FaDu cells were treated for 24 h with 5nM bafilomycin A1 (Sigma-Aldrich, B1793), 50 µM of hydroxychloroquine sulphate (Sigma-Aldrich, H0915), 100 µM of Cpd18 (Calbiochem), 50 nM of autophinib (Sigma, SML2632), 10 µM of EACC (MedChemExpress), 200 nM of rapamycin (Sigma-Aldrich, R0395), 3 nM of Torin-1 (MedChemExpress), or 30 nM of NVP-BEZ235 (MedChemExpress). To induce starvation, cells were cultured in DMEM F12 without glutamine and without FBS (Biosera). Modulation of autophagy did not reduce the viability  of FaDu cells.

Conditioned media preparation

see details in the accompanying paper

Fluorescence MicroscopyThe autophagosomal/autolysosomal/lysosomal dynamics of affected cells were observed using the combination of PremoTM Autophagy Tandem Sensor (P36239, Invitrogen) with the far-red emitting LysoTracker® Deep Red (L12492, Invitrogen) (Ex 647 nm/Em 668 nm). By combining acid-sensitive Emerald GFP (Ex 488 nm/Em 509 nm) with acid-insensitive TagRFP (Ex 555 nm/Em 584 nm) in the PremoTM kit, autophagosomes and autolysosomes labelling (yellow and red, respectively) is possible.Immediately after transduction with 12 µl PremoTM Autophagy Tandem Sensor/2ml cell suspension, cells were seeded at 5 × 105 into 35-mm glass-bottomed gelatin-coated dishes (Ibidi, μ-Dish 35 mm, high Glass Bottom) and cultured for 48 h to equilibrate expression levels. Subsequently, cells were exposed to the selected agents for 6, 12, 24 and 48 hours before imaging.  LysoTracker® Deep Red staining was performed 1 h before imaging. To monitor the uptake of isolated PS-EVs by fibroblasts, we stained EVs with PKH67 (Sigma, PKH67GL) and then removed the remaining dye using Exosome Spin Columns (MW 3000) (Thermo Scientific, #4484449). The stained EVs were then suspended in 400 ul of cultivation medium and added to HGFB cells growing for 24 h in Ibidi µ-Slide I Luer (Ibidi, 80176). Image acquisition was performed 24 hours after EVs addition. 1 µl of 1 µg/ml of Hoechst 33342 (Enzo) (Ex 350 nm/ Em 461 nm) nuclear stain was added 1 hour before imaging.To determine the viability of the cell population prior to isolation of EVs, cells were left in a culture dish with 1 ml of culture medium to which propidium iodide (Sigma-Aldrich) and Hoechst 33342 were added 45 minutes before capturing. To maximize the possibility of comparison between samples, all samples (from a single cell line) were captured on the same day in a single run using the same microscope settings. For each time and each treatment, 10–12 fields of view were captured from randomized sites of the culture dish. Epifluorescent microscopy images and confocal microscopy images were acquired using Laser scanning confocal microscope Zeiss LSM 880 with AiryscanFast module (Carl Zeiss Inc.) using a C-apochromat 40x/1.20 W and C-Apochromat 63 /1.20 W. LysoTracker® Deep Red was excited HeNe 633 nm solid-state laser and emitted light was detected at 638–759 nm. Emerald GFP was excited 488 nm ArgonRemote laser, and emitted light was detected at 493–576 nm. TagRFP was excited DPSS 561 nm laser, and emitted light was detected at 570–650 nm. Hoechst 33342 was excited with a 405 nm solid-state laser, and emitted light was detected at 410–508 nm. Fluorescence images were acquired by the transmitted light detector. Images were analyzed using ImageJ software and custom MATLAB software developed in our laboratory. The analysis process consists of the segmentation of cells from the background and extraction of the intensity of fluorescence channels (TagRFP, GFP, LysoTracker) inside cells. For segmentation, a thresholding-based method was used, where a manually selected threshold was applied. Segmentation was applied to the image created as the sum of all fluorescence channels to achieve segmentation independent of the intensity of individual channels. To achieve better segmentation without noisy pixels, fluorescence images were preprocessed with median filter (7x7) and Gaussian filter (standard deviation 1); additionally, binary segmentation was post-processed with morphological closing and removal of small binary connected components (<5000px). For intensity extraction, the mean value of segmented cell pixels was used for each field of view. Besides individual fluorescence channels, the mean colocalization of TagRFP and GFP was calculated with a pixel-wise multiplication of TagRFP and GFP channels.

File Naming

CZI images are organised in folders according to cell line, treatment time, and treatment. Names include magnification, cell line measured, treatment, and time of treatment,



40x_FADU_BAF_12h_BF_4.czi


The abbreviations used for treatments are as follows: BAF, bafilomycin A1; HCQ, hydroxychloroquine sulphate; Cpd18; APB, autophinib; EACC; RAPA, rapamycin; TOR1, Torin-1; BEZ, NVP-BEZ235; starv, starvation; 
description: This work was supported by the Grant Agency of the Czech Republic (GA21-06873S) and the Ministry of Health of the Czech Republic NU22-03-00202. We acknowledge the core facility CELLIM supported by the Czech-BioImaging large RI project (LM2018129 funded by MEYS CR) for their support with obtaining scientific data presented in this paper."