Journal: bioRxiv
Article Title: USP14 regulates pS129 α-synuclein levels and oxidative stress in human SH-SY5Y dopaminergic cells
doi: 10.1101/2024.05.09.592905
Figure Lengend Snippet: (A) Live-cell imaging using Mitotracker DeepRed FM and a 100x objective of Nikon-Eclipse Ti-E inverted wide-field microscope equipped with an environmental chamber. Bottom panels, higher magnification. Left-bottom panel, quantification of mitochondrial branch length in µM from 90-100 control and USP14 deleted cells. Scale bar: 20µM. Typical experiment is shown and was repeated three times with similar results. (B) EM imaging was done as described in Methods. Note elongated mitochondria (MT) in USP14 deleted cells. Red stars * mark the presence of electron-dense lysosomes/ autophagosomes/ vesicles that were increased in the USP14-deleted cells compared with controls. N represents nuclear compartment. The experiment was repeated with similar results. (C) Left panel, immunoblot using an OXPHOS antibody cocktail. Right panel, quantification of the densitometry ratio of CI-CV subunits (CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MTCO1 and CV-ATP5A) normalized to β-actin. Values are means ± S.E.M. *p ≤ 0.05, ns= not significant. n=3. (D) Live-cell imaging using CellROX Green and a 20x objective of EVOS FL microscope to visualize reactive oxygen species (ROS). Note increases of ROS in USP14-deleted cells Control cells treated with 5µM H 2 O 2 for 90 min served as a positive control for oxidative stress. Top, CellROX Green. Bottom, phase-contrast images. Scale bar: 200µM. Typical experiment is shown and was repeated three times with similar results.
Article Snippet: The stained live-cells were imaged utilizing Nikon Eclipse Ti-E inverted widefield microscope with full environmental chamber, Hamamatsu Orca Flash 4.0 V2 B&W camera for fluorescence and Lumencor Spectra X light engine (Biomedicum Imaging Unit, Medicum, University of Helsinki).
Techniques: Live Cell Imaging, Microscopy, Imaging, Western Blot, Positive Control
