1. Volume 23, Issue 4, Pages 453-461 (April 2016)
Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin- Proteasome Degradation 
Ting-Ting Chu, Na Gao, Qian-Qian Li, Pu-Guang Chen, Xi-Fei Yang, Yong-Xiang Chen, Yu-Fen Zhao, Yan-Mei Li 
Cell Chemical Biology 
Volume 23, Issue 4, Pages (April 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 453-461DOI: (10. 1016/j. chembiol. 2016
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3. Figure 1
TH006 Induces the Greatest Reduction of EGFP Intensity, and Can Interact with Tau In Vitro as Well as Easily Penetrate Cells
(A) Flow cytometry results of Tau-EGFP overexpressed cells with treatment of synthetic molecules (100 μM) for 24 hr. The black horizontal line represents the fluorescence positive signal threshold.
(B) Fluorescence polarization assay shows the interaction between Tau and TH006 tested in vitro.
(C) Entry of CF-TH006 into wild-type N2a cells tested by flow cytometry. The black horizontal line represents the fluorescence positive signal threshold.
(D) Distribution of CF-TH006 in N2a cells tested by confocal microscopy. Blue, nucleus (top left); green, CF-TH006 (top right); red, Tau (bottom left) labeled with anti-Tau antibody (ab3931; Abcam), detected with Alexa Fluor 647-conjugated goat anti-mouse IgG (A0473; Beyotime). Bottom right: merged image. Arrows show the distributions of CF-TH006 in N2a cells. Scale bar, 10 μm.
(E and F) Mean fluorescence intensity of the flow plots of (A) and (C), respectively.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2
TH006 Can Induce Tau-EGFP Degradation through Enhancing the Poly-ubiquitination of Tau In Vivo
(A and B) Flow cytometry results of Tau-EGFP-overexpressed N2a cells treated with increasing amounts of TH006 for 24 hr (A) or with 200 μM TH006 for different times (B). The black horizontal line represents the fluorescence positive signal threshold.
(C and D) Mean fluorescence intensity of the flow plots of (A) and (B), respectively.
(E and F) Tau levels in Tau-overexpressed cells after treatment with (E) increasing amounts of TH006 for 24 hr or (F) 200 μM TH006 for the indicated times tested by western blotting assay. Ctrl in (E) denotes without CHX and TH006 treatment. Ctrl in (F) denotes with CHX treatment but not TH006 treatment.
(G) Tau levels in Tau-overexpressed N2a cells after treatment with TH006, CP1, and CP2 tested by western blotting assay.
(H) Tau reduced in Tau-overexpressed SH-SY5Y cells after treatment with TH006 (50 μM, 18 hr) tested by western blotting assay.
(I) Tau reduced in 3xTg-AD mouse primary neuron cells after treatment with TH006 (50 μM, 18 hr) tested by western blotting assay.
(J) Western blotting analysis shows the poly-ubiquitination levels of immunoprecipitated (IP) Tau from Tau-overexpressed cells after treatment with or without 200 μM TH006 for 24 hr. Top: the immunoblot was probed with anti-K48-linkage-specific poly-ubiquitin antibody (CST 4289S) to detect the poly-ubiquitination levels of Tau. Middle: Tau levels were tested in the same samples. Bottom: β-actin served as a loading control.
(K) Western blotting analysis shows the poly-ubiquitination levels of IP Tau from the same cell line after treatment with TH006 (200 μM) for 24 hr. MG132 (4 μM) and bafilomycin A1 (0.4 μM) were added, alone or in combination, for 6 hr before cell harvest.
(L) Immunoprecipitation-western blotting result shows that TH006 induces Tau poly-ubiquitination through VHL E3 ligase in vitro. Control peptide FLAG-CP2 cannot recruit VHL E3 ligase.
Immunoprecipitation-western blotting was performed with anti-FLAG antibody (F1804, mouse; Sigma), primary antibody anti-VHL E3 ligase (sc-5575, rabbit; Santa Cruz), anti-Tau antibody (sc-21796; Santa Cruz), anti-β-actin (A1978; Sigma-Aldrich), and GAPDH (sc-32233; Santa Cruz).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3
TH006 Can Rescue Mitochondrial Uneven Distribution Caused by Tau Overexpression
(A–C) Confocal microscopic images show the distribution of mitochondria in (A) wild-type N2a cells, (B) untreated Tau-overexpressing N2a cells, and (C) Tau-overexpressing N2a cells treated with 200 μM TH006 for 24 hr. The first column shows DAPI-stained nuclei (blue). The second column shows Tau immunofluorescence (green) detected with an anti-Tau (ab3931, Abcam) antibody, and stained with fluorescein isothiocyanate-coupled rabbit anti-mouse IgG antiserum (GF026129; GeneTech) in (A) or Tau-EGFP (no antibody required) in (B) and (C). The third column shows mitochondria (purple) labeled with Mito Tracker DeepRed (M22426; Invitrogen). The fourth column shows a merging of all three images in the row. Arrows show the distribution of mitochondria. Scale bars, 10 μm.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4
TH006 Can Reduce the Toxicity of Aβ to Cells and Reduce Tau Level in the Transgenic Mouse
(A) Viability of wild-type N2a cells treated with Aβ or Aβ in combination with TH006.
(B) Viability of Tau-overexpressing cells (Tau OE) with treatment of Aβ or Aβ in combination with TH006.
(C) Viability of primary hippocampus neuron cells treated with Aβ or Aβ in combination with TH006. The cell viabilities in (A), (B), and (C) was detected by a Cell Titer-Glo Luminescent Cell viability kit (G7570; Promega). Data are means ± SD (n = 5). *p < 0.05, **p < 0.01.
(D) Toxicity of TH006 to wild-type N2a cells was measured by the MTT assay. Data are means ± SD, n = 5.
(E) The Tau levels of brain cerebral cortex in transgenic mice (B6; 129-Psen1tm1Mpm Tg [APPSwe, Tau P301L] 1Lfa/Mmjax) were reduced after treatment with TH006 tested by western blotting assay. The control mouse was B6129SF2/J (n = 3).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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