Fig 1: The colocalization of USP10 with pTau aggregates in AD brain regions. (a,b) Representative immunohistochemical findings of USP10 and pTau in temporal cortex of AD patients (n = 3) are shown. The colocalization of USP10 with pTau aggregates was analyzed by a line profile analysis. Arrows indicate the colocalization suggested by the assay. The relative staining levels of USP10 and pTau on the indicated line were presented (b). Lower magnification pictures were presented in Supplementary Fig. S6. (c) The current working model for pathogenic Tau aggregation through SGs and aggresomes.
Fig 2: G3BP1-KD increase the amount of α-synuclein. (a–d) HeLa cells were transfected with G3BP1-siRNA (G3BP1), USP10-siRNA (USP10), p62-siRNA (p62), their combination (G1/U10 or G1/p62) or a control (NT), and then cells were transfected with the α-synuclein plasmid. Whole-cell lysates were subjected to a Western blot analysis using anti-α-synuclein, anti-G3BP1, anti-USP10, anti-p62, anti-ubiquitin and anti-β-actin antibodies. The ratios of the α-synuclein band to the β-actin one were presented as the means and SD from three experiments in (b,d). *P < 0.05; **P < 0.01; ****P < 0.0001.
Fig 3: USP10 does not interact with Tau. (a) HT22 cells were transfected with Tau (Tau4R1N) plasmid along with HA-USP10 and/or FLAG-TIA1 plasmid using Lipofectamine 2000. Prepared cell lysates were immunoprecipitated with an anti-HA antibody, and the amounts of Tau, FLAG-TIA1 and HA-USP10 were analyzed by Western blotting using respective antibodies. (b–e) HeLa cells were transfected with HA-USP10 plasmid along either with Tau3R1N, Tau4R1N or Tau3R1N/K317M plasmid by lipofection using Lipofectamine 2000. At 48 h after transfection, whole cell lysates (b,c), NP-40-soluble fractions and NP-40-insoluble fractions (d,e) were prepared and characterized by Western blotting using anti-Tau (TAU-5), anti-phosphorylated Tau (pTau) (AT8), anti-HA, anti-TIA1, anti-α-tubulin and anti-β-actin antibodies.
Fig 4: USP10-KD reduced the TIA1-induced Tau/TIA1-SG formation. (a–c) USP10-KD HT22 cells and the control (NT) were transfected with GFP-TIA1 plasmid using lipofection with Lipofectamine 2000. At 48 h after transfection, cells were treated with 5 µM MG-132 for 4 h, and SG formation was evaluated by GFP fluorescence together with anti-Tau (TAU-5) and anti-USP10 staining using a fluorescent microscope. Arrowheads indicate the colocalization of TIA1, Tau and USP10. The population (%) of cells with TIA1/Tau-SG or TIA1/Tau/USP10-SGs is presented as the mean and SD. **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bar: 10 µm.
Fig 5: USP10-knockdown reduces Tau/TIA1-SG in HT22 cells. (a) Knockdown of USP10 (USP10-KD) in HT22 cells was performed using the lentivirus vector encoding USP10/shRNA (USP10-3, USP10-4) or the control (NT). The expression of USP10, Tau, TIA1 and β-actin in these cells was measured by Western blotting using respective antibodies. The asterisk indicates a nonspecific band. (b,c) USP10-KD HT22 cells and the control (NT) were treated with 5 µM MG-132 for 4 h, and SG formation was evaluated by anti-Tau (TAU-5) and anti-TIA1 antibodies as well as Hoechst for nuclear staining using a fluorescent microscope. Arrowheads indicate the colocalization of Tau with TIA1. The population (%) of cells with TIA1-SGs or TIA1/Tau-SGs is presented as the mean and SD from three independent experiments in (c). *P < 0.05; **P < 0.01. Scale bar: 10 µm.
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