Fig 1. OA induced tau hyperphosphorylation and oligomerization at different time points in mouse neuroblastoma N2a cells. (a). Immunoblots of N2a cells extracted protein (20 μg) using total and phospho-tau antibodies: DA9 (a.a. 102–140), CP13 (pSer202), and PHF-1 (pSer396/pSer404). ⍺II-Spectrin antibody was used to assess neuronal apoptotic pathway activation through monitoring intact spectrin (240 kDa), SBDP150/145 (calpain activation), and SBDP120 (caspase-3 activation). Different tau species are pointed with colored arrows. Blue arrows present monomeric p-tau (48 kDa), and oligomeric p-tau (110 kDa, 170 kDa, and 240 kDa). Red arrows on PHF-1 points on two minor bands of oligomeric p-tau (220 kDa and 260 kDa). Black arrows show non-phospho tau band (46 kDa). SNJ-1945 (abbreviated as S; a calpain inhibitor, 100 μM) and Z-DCB (abbreviated as Z; a caspase-3 inhibitor, 60 μM) were added for all experimental conditions for 1h before the treatment with OA (100 nM) for 6h or 24h, to prevent apoptosis-mediated proteolysis of tau and ⍺II-Spectrin. A reverse-time course followed OA treatment, and all cells were collected at the same time and conditions. (b). Immunoblots quantification. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as ± SEM for n = 3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant.

Fig 2. Screening of protein kinase inhibitors on OA-induced Tau hyperphosphorylation and oligomerization in N2a cells. (a). Immunoblots of N2a cells extracted protein (20 μg) using phospho-tau antibodies (CP13, PHF-1), total tau (DA9), and αII-Spectrin. αII-Spectrin was probed to assess neuronal cell injury monitored with SBDP145/150 and SBDP120. Kinase inhibitors effect on OA-induced tau bands (100 nM) was monitored by evaluating the levels of monomeric (48 kDa) and oligomeric p-tau immunoreactivity (110 kDa, 170 kDa, and 240 kDa; blue arrows), total tau, and non-phospho tau (46 kDa; black arrows). Phosphorylated tau break-down products are shown with PHF-1 immunoblot. For all experimental conditions, S (a calpain inhibitor) and Z (a caspase-3 inhibitor) were added for 1h to before the addition of OA for 24h followed by 6h incubation with the kinase inhibitors. The concentrations used for each protein kinase inhibitor are mentioned in materials and methods, cell treatment section. β-actin was probed as a loading control. All experimental conditions were collected and analyzed at the same time. (b). Immunoblots quantification. All data are normalized to β-actin and are expressed as a percentage of control. Data are presented as ± SEM for n = 3. Statistical analysis was performed with one-way ANOVA. For multiple comparisons, one-way ANOVA followed by Bonferroni’s post-hoc test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: non-significant.

Fig 3. Dose-response of TBB on OA-induced tau hyperphosphorylation and oligomerization in N2a cells. N2a cells were pre-treated with OA for 24h followed by treatment with different concentrations of TBB for 6h, as indicated in the Figure. (a) Immunoblots of cell extracted proteins using phospho-tau antibodies, including CP13 (pSer202), and total tau DA9 (a.a. 102–140). Blue arrows represent monomeric and oligomeric p-tau (48 kDa, 110 kDa, and 170 kDa). αII-Spectrin antibody used to monitor SBDPs with the increasing concentrations of TBB. The β-actin antibody was used as a loading control. All conditions included SNJ-1945 (calpain inhibitor) and Z-DCB (caspase inhibitor). (b) TBB dose-response treatment line chart. TBB concentration (in micromolar) is shown on the X-axis, and the inhibition percentage is presented on the Y-axis. The control sample values were designated as the standard response. The X-axis concentration values are logarithm-transformed to fit a straight line. The half-maximal inhibitory concentration (IC50) was used to measure the effectiveness of TBB in inhibiting OA-induced tau hyperphosphorylation and oligomerization. GraphPad Prism was used to calculate the IC50 (for DA9 and CP13 antibodies) and are presented on the figure. The statistical analysis was performed with one-way ANOVA, followed by Bonferroni's post-hoc test. *p<0.05, **p<0.01, ***p<0.001. Data are presented as ± SEM for n = 3.