Fig 1: Interaction of tau proteins with Hsp90 in ATRA-treated HMO6 cells. a Hsp90 and tau protein co-immunoprecipitation. Cells were subjected to 1 µM ATRA for 24 h, followed by a 6-h exposure to 10 ng/ml tau protein. Subsequently, Hsp90 was immunoprecipitated from whole cell lysates using an anti-Hsp90 antibody and the association between Hsp90 and tau protein was analyzed via Western blot employing both anti-Hsp90 and anti-tau antibodies. IgG was used as a control antibody. Tau-treated group was used as the control. Densitometric analysis of the protein bands was performed using the ChemiDoc MP Imaging System (n = 3). b Co-localization study of Hsp90 and tau proteins. The cells were fixed in formaldehyde, permeabilized with Triton X-100, and incubated with anti-human tau antibody overnight at 4 °C. This was followed by a 2-h incubation with FITC-conjugated anti-human tau antibody (green) and subsequent incubation with Alexa 633-conjugated anti-Hsp90 antibody (red). The co-localization (yellow fluorescence) of Hsp90 and human tau protein was visualized using a fluorescence microscope at a magnification of ×200. *p < 0.05
Fig 2: Degradation of ATRA-enhanced intracellular tau proteins in HMO6 cells via lysosomal and proteasomal pathways. a Measurement of tau protein in culture medium post-treatment. Following 24 h of 1 µM ATRA treatment, cells were incubated with 10 ng/ml tau protein for varying periods. Subsequently, the culture medium was collected, and tau protein levels were determined using ELISA. The baseline (0 h tau levels) represents the tau protein concentration immediately after the addition of tau protein. b–d Analysis of intracellular tau protein levels in the context of protease inhibition. Cells were treated with or without ATRA, in the presence of either lysosomal inhibitor (leupeptin, 50 µM) or proteasome inhibitor (epoxomicin, 1 nM) for 24 h, followed by 10 ng/ml tau protein treatment for another 24 h. The cells were then harvested, and intracellular tau protein was detected via Western blot (b). Densitometric analysis of the tau protein bands was performed using the ImageJ software (c). In parallel experiments, cells were treated with lysosomal (Leupeptin) or proteasome (Epoxomicin) inhibitors combined with ATRA for 24 h, followed by 10 ng/ml tau protein for an additional 24 h. Intracellular tau protein levels were then quantified using flow cytometry (d). *p < 0.05 compared to 0 h Tau (a), *p < 0.05 compared to Tau only (c, d), #p < 0.05 and ##p < 0.01 compared to ATRA+Tau (c, d)
Fig 3: Tau internalization is mediated by cell surface Hsp90. a Schematic illustration of cell surface/internalized protein isolation by biotinylation assay. b After treatment with 1 µM ATRA or GeB + ATRA for 24 h, cells were labeled with 0.25 mg/mL Sulfo-NHS-SS-Biotin for 30 min at 4 °C followed by treatment with 10 ng/mL of human recombinant tau protein and incubated at 4 °C or 37 °C for 6 h. The cell surface or internalized biotinylated protein was isolated using the avidin column and subjected to the western blot analysis with the antibodies against Hsp90 and human tau. Tau-treated group was used as the control. Densitometric analysis of the protein bands was performed using the ChemiDoc MP Imaging System (n = 3). c After treatment time, the cells were fixed in formaldehyde, permeabilized with Triton X-100, and incubated with anti-human tau antibody overnight at 4 °C. This was followed by a 2-h incubation with FITC-conjugated anti-human tau antibody (green) and subsequent incubation with Alexa 633-conjugated anti-Hsp90 antibody (red). The co-localization (yellow fluorescence) of Hsp90 and human tau protein was visualized using a fluorescence microscope at a magnification of ×200. *p < 0.05 vs tau 37 °C group; #p < 0.05 vs tau 4 °C group
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