Journal: Heliyon
Article Title: USP14 is crucial for proteostasis regulation and α-synuclein degradation in human SH-SY5Y dopaminergic cells
doi: 10.1016/j.heliyon.2025.e42031
Figure Lengend Snippet: Oxidative stress with elongation of mitochondria in USP14 deficient cells. Control and USP14-deleted cells were cultured and analyzed as indicated below. (A) Live-cell imaging using MitoTracker DeepRed FM and a 100× objective of Nikon-Eclipse Ti-E inverted wide-field microscope equipped with an environmental chamber. MitoTracker DeepRed FM channel (644/665 nm) represented in grayscale. Bottom panels, higher magnification. Left-bottom panel, quantification of mitochondrial branch length in μM from 90 to 100 control and USP14 deleted cells. Scale bar: 100 μM. 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. p-value was calculated by Student's t-test. (D) Live-cell imaging using CellROX Green and a 20× objective of EVOS FL microscope. Control cells treated with 5 μM H 2 O 2 for 90 min served as a positive control for oxidative stress. Top, CellROX Green. Bottom, phase-contrast images. Scale bar: 200 μM. Typical experiment is shown and was repeated three times with similar results.
Article Snippet: The primary antibodies used included: USP14 (J6111-6D6, Sigma, Germany), α-synuclein (2642, CST, USA), pS129-α-synuclein (EP1536Y, Abcam, GB), 20S CP subunits cocktail (BML-PW8195, Enzo Lifesciences, USA), K48-linkage specific polyubiquitin (4289, CST), PSMD2 (sc-271775, SantaCruz, USA), PSMC2 (14395, CST), UCHL5 (sc-271002, SantaCruz, USA), TFEB (37785, CST, USA), GBA1 (sc-166407, SantaCruz, USA), GABARAP (13733, CST, USA), LC3B (3868, CST, USA), p62/SQSTM1 (P0067, Sigma, Germany), beta-Actin (A2066, Sigma, Germany), GAPDH (MAB374, Millipore, Germany), OXPHOS cocktail (MS604, Mitosciences, USA – Currently Abcam, GB).
Techniques: Control, Cell Culture, Live Cell Imaging, Microscopy, Imaging, Western Blot, Positive Control
