Skip to main content
. 2021 Mar 11;11(11):5279–5295. doi: 10.7150/thno.55680

Figure 1.

Figure 1

C004019 induces tau clearance via promoting its ubiquitination and proteasome-dependent proteolysis in vitro. (A) Working model of C004019. (B) The designed C004019 composed of a tau binder, a linker and a Vhl (E3 ligase) binder with a molecular mass of 1,035.29 Dalton. (C) Compared with the vehicle, C004019 (0.01-100 µM for 24 h) did not significantly affect cell viability measured by CCK-8 assay in HEK293-hTau cells. (D-I) C004019 induced a concentration-dependent clearance of total tau (Tau5) or p-tau at multiple-doses AD-related sites in HEK293-hTau cells (D-E) and SH-SY5Y cells (F-I) measured by Western blotting. (H-I) SH-SY5Y cells were simultaneously treated with C004019 (1 or 10 µM) and wortmainnin (WO, 1 µM) and GF-109203X (GFX, 1 µM) for 24 h to induce tau hyperphosphorylation, and then tau was measured by Western blotting. (J) C004019 induced tau reduction in HEK293-hTau cells measured by immunofluorescence staining (Scale bar, 10 µm). (K) Co-immunoprecipitation analysis showed interaction of tau with Vhl (E3 ligase) and the increased tau ubiquitination in HEK293-hTau cells treated with 20 µM C004019 for 6 h. (L) C004019 (20 µM for 24 h) did not affect mRNA level of tau in HEK293-hTau cells measured by q-PCR. (M-N) Inhibition of proteasome with MG132 (10 µM) abolished C004019-induced tau clearance measured by Western blotting. Data were expressed as mean ± SEM, (E) ***P < 0.001 vs. 0 µM. (I) *P < 0.05, ***P < 0.001 vs. 0 µM + GFX + WO. (N) **P < 0.01 vs. Vehicle. Data in (C, E, G, I, N) were analyzed by one-way ANOVA. Data in (L) was analyzed by Student's t-test.