Figures & data
Table 1 Tested TCM extracts (number)
Figure 1 Misfolded aggregation by ΔK280 tauRD in HEK293T cells.
Notes: (A) Chromatograms of DNA sequencing of wild type and ΔK280 tauRD. (B) Expression plasmid with the tauRD-DsRed downstream of CMV/TetO2 promoter. The tauRD contained a four-repeat domain (Gln244–Glu372) involved in microtubule binding and paired helical filament aggregation. (C) Expression and aggregation of wild type and ΔK280 tauRD-DsRed in HEK293T cells. Tau aggregation was monitored by thioflavine S staining (green).
Abbreviations: CMV, cytomegalovirus; HEK, human embryonic kidney; ΔK280, deletion of the lysine residue at codon 280; tauRD, tau repeat domain.
Abbreviations: CMV, cytomegalovirus; HEK, human embryonic kidney; ΔK280, deletion of the lysine residue at codon 280; tauRD, tau repeat domain.
Figure 2 Tet-On ΔK280 tauRD-DsRed 293 cell model for AD.
Notes: (A) Western blot analysis of ΔK280 tauRD-DsRed (tau antibody staining) and β-Actin (loading control) after a 2-day induction with doxycycline (+Dox) or without doxycycline (−Dox). (B) Real-time PCR analysis (n=3) of ΔK280 tauRD-DsRed and HPRT1 (internal control) mRNA levels after a 2-day induction with doxycycline (+Dox) or without doxycycline (−Dox). (C) Fluorescent images of ΔK280 tauRD-DsRed cells with or without Congo red (10 μM) treatment (blue, nuclei; red, expressed ΔK280 tauRD-DsRed protein). (D) DsRed fluorescence analysis (n=3) of ΔK280 tauRD-DsRed cells treated with Congo red (10 μM) or untreated (0 μM). To normalize, the relative DsRed fluorescence of untreated cells was set as 100%.
Abbreviations: AD, Alzheimer’s disease; Dox, doxycycline; PCR, polymerase chain reaction; HPRT1, hypoxanthine phosphoribosyl transferase 1; H33342, Hoechst 33342; tauRD, tau repeat domain.
Abbreviations: AD, Alzheimer’s disease; Dox, doxycycline; PCR, polymerase chain reaction; HPRT1, hypoxanthine phosphoribosyl transferase 1; H33342, Hoechst 33342; tauRD, tau repeat domain.
Figure 3 High-content screening of TCM extracts.
Notes: (A) Experimental flow chart. Cells were plated into 96-well plates, grown for 24 hours, and treated with different concentrations of Congo red or TCM extracts for 8 hours. Then, doxycycline (1 μg/mL) was added for 3 days and DsRed fluorescence was assessed by the HCA system. (B) DsRed fluorescence assay (n=3) with Congo red (1–20 μM) or TCM extracts (NH001–NH042; 100–500 μg/mL) treatment. To normalize, the relative DsRed fluorescence of untreated cells (Untr.) was set as 100%. *P<0.05, treated vs untreated cells. (C) The cells were pretreated with Congo red (20 μM) or TCM extracts (NH008, NH014, NH016, NH021, NH027, NH034, and NH037; 500 μg/mL) for 8 hours and ΔK280 tauRD-DsRed expression induced for 3 days for ROS measurement (n=3). P-values compared induced vs uninduced cells or TCM/Congo-red-treated vs untreated cells.
Abbreviations: Dox, doxycycline; HCA, high-content analysis; ROS, reactive oxygen species; SD, standard deviation; TCM, traditional Chinese medicine.
Abbreviations: Dox, doxycycline; HCA, high-content analysis; ROS, reactive oxygen species; SD, standard deviation; TCM, traditional Chinese medicine.
Figure 4 Cytotoxicity of the selected TCM extracts.
Notes: HEK293 and SH-SY5Y cells were treated with TCM extracts (NH008, NH014, NH016, NH021, NH027, NH034, and NH037; 5–30 mg/mL). Cell proliferation was measured the next day using MTT viability assay (n=3). To normalize, the relative viability in untreated cells was set as 100%. The IC50 of each TCM extract is shown under the columns. Red lines, 50% viability.
Abbreviations: HEK, human embryonic kidney; IC50, half maximal inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TCM, traditional Chinese medicine.
Abbreviations: HEK, human embryonic kidney; IC50, half maximal inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TCM, traditional Chinese medicine.
Figure 5 Neuroprotective effects of the selected TCM extracts in SH-SY5Y ΔK280 tauRD-DsRed cells.
Notes: (A) Fluorescence microscopy images of differentiated SH-SY5Y cells with ΔK280 tauRD-DsRed expression without or with NH021/NH034/Congo red treatment for 7 days. (B) Treatment with TCM extracts (NH008, NH014, NH016, NH021, NH027, NH034, and NH037; 500 μg/mL), Congo red (20 μM), and neurite outgrowth assay (n=3). To normalize, the relative neurite outgrowth of uninduced cells was set as 100%. P-values compared induced vs uninduced cells or TCM/Congo-red-treated vs untreated cells (only those with improved neurite outgrowth are shown).
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Dox, doxycycline; SD, standard deviation; tauRD, tau repeat domain; TCM, traditional Chinese medicine; TUBB3, tubulin, beta 3 class III.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Dox, doxycycline; SD, standard deviation; tauRD, tau repeat domain; TCM, traditional Chinese medicine; TUBB3, tubulin, beta 3 class III.
Table 2 Genes identified by the unfolded protein response plus PCR array
Figure 6 Alterations of UPR-related gene expression in Tet-On ΔK280 tauRD-DsRed SH-SY5Y cells.
Notes: (A) Heat map illustrating RNA expression changes in ΔK280 tauRD-DsRed uninduced (−Dox, −NH021), induced (+Dox, −NH021), and NH021-treated (+Dox, +NH021) cells. Red shade, high expression level; green shade, low expression level. (B) Relative ERN2, ERP44, DNAJC3, and SERP1 protein levels were analyzed by immunoblotting with specific antibodies. ERN2, ERP44, DNAJC3, and SERP1 levels were normalized to GAPDH internal control (n=3). The relative ERN2, ERP44, DNAJC3, and SERP1 protein levels are shown below the representative Western blot images. To normalize, the expression level in uninduced (−Dox, −NH021) cells was set as 100%.
Abbreviations: Dox, doxycycline; DNAJC3, DnaJ (Hsp40) homolog, subfamily C, member 3; ERN2, endoplasmic reticulum to nucleus signaling 2; ERP44, endoplasmic reticulum protein 44; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SERP1, stress-associated endoplasmic reticulum protein 1; tauRD, tau repeat domain; UPR, unfolded protein response.
Abbreviations: Dox, doxycycline; DNAJC3, DnaJ (Hsp40) homolog, subfamily C, member 3; ERN2, endoplasmic reticulum to nucleus signaling 2; ERP44, endoplasmic reticulum protein 44; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SERP1, stress-associated endoplasmic reticulum protein 1; tauRD, tau repeat domain; UPR, unfolded protein response.
