Fig 1.
Tau expression in stable HT22 cell lines.
Cell lysates (20 μg protein) from cells expressing wildtype (T4) and phosphomimetic tau (EC and EM) were analyzed by Western blot using site-specific and phosphorylation-dependent anti-tau antibodies. Tau expression levels were comparable across all groups. The control lane (ctrl) confirms the absence of endogenous tau in HT22 cells.
Fig 2.
Oxidative stress-induced mitophagy in HT22 cells.
Mitophagy was assessed in cells stably expressing mt-mKeima and respective tau constructs under unstressed conditions (A), and after induction with 5 μM PQ for 6 h (B). Fluorescence was measured using custom filter sets with excitation at 440 nm and 550 nm, and emission at 620 nm. Representative images were split into 550 nm (red: mitolysosome) and 440 nm (green: mitochondria) channels using ImageJ software. PQ exposure increased mitophagy, evidenced by increased red mitolysosome puncta. The mitophagy index, calculated as the ratio of the area and intensity of the mitolysosome (550 nm) to that of mitochondria (440 nm), was derived from three independent experiments. At least 5–7 cells per experiment were analyzed (C). Data represent mean ± SEM and were statistically analyzed with two-way ANOVA followed by Tukey’s post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bar: is 20 μm.
Fig 3.
Immunoblots analysis of mitophagy-related protein expression.
Cell lysates from unstressed (ctrl) cells and cells treated with PQ for 6 h to induce mitophagy were assessed using the indicated antibodies. Immunoblots of PINK1 and parkin protein expression (A). Densitometry quantification of PINK1 and parkin show no significant changes across all tau groups (B, C). Immunoblots for the indicated mitophagy receptors (D). Densitometry quantification of the respective mitophagy receptors show that FKBP8 expression was significantly reduced in cells expressing EC and EM compared to T4 (E), while BNIP3 and FUNDC1 receptor levels decreased significantly in all groups (F, G). Immunoblot shows changes in FKBP8 and FUNCD1 expression overtime (0 h, 6 h, 12 h) in cells expressing T4 (H). Proteins were normalized to their respective beta-actin and Ponceau-S. Data represent mean ± SEM from 3–5 independent experiments and were statistically analyzed with two-tailed Student’s t-test. *p < 0.05; **p < 0.01.
Fig 4.
Interaction of FKBP8 with tau.
Five hundred micrograms of lysate from cells expressing wildtype and phosphomimetic tau were immunoprecipitated with rabbit anti-tau (DAKO tau) or nonspecific rabbit IgG antibodies. Western blot analysis of the eluted proteins with mouse anti-FKBP8 shows co-immunoprecipitation of FKBP8 with tau. The blot was stripped and re-probed with mouse anti-tau antibodies (Tau5 and 5A6).
Fig 5.
Interaction of FKBP8 with ER specific protein and FKBP8 expression in mitochondria fraction during mitophagy.
Dual staining for FKBP8 (red) and BiP/GrP78 (green) was performed to analyze their colocalization in unstressed cells (A) and cells induced for mitophagy with PQ for 6 h (B). Representative immunofluorescence merged images from confocal microscopy reveal pronounced colocalization of these proteins during mitophagy. Magnified regions of the merged images are shown in the blue box. Mander’s coefficient of colocalization (C) was calculated from three independent experiments using Imaris 10.0 program (Oxford Instruments). Crude mitochondrial and cytosolic fractions were isolated by differential centrifugation from cells expressing wildtype and phosphomimetic tau. Equal protein amounts were subjected to Western blot analysis with an anti-FKBP8 antibody. β-tubulin (cytoplasmic marker) and TOM20 (mitochondrial outer membrane marker) were used to verify fraction purity. Representative immunoblot (D), densitometry analysis (E), and percentage decrease estimation (F) show reduced mitochondrial FKBP8 expression across all tau groups. Quantification is expressed as the FKBP8/β-tubulin ratio and represents mean values ± SEM from four independent experiments. Statistical analysis was performed using two-tailed Student’s t-test. *p < 0.05; **p < 0.01. Scale bar: 25 μm.
Fig 6.
Mitophagy in FKBP8 knockdown cells.
FKBP8 was knocked down in stable cell lines, confirmed by Western blotting (A). Mitophagy was assessed after induction with 5 μM PQ for 6 h. Fluorescence was measured using custom filter sets with excitation at 440 nm and 550 nm, and emission at 620 nm. Representative images were split into 550 nm (red: mitolysosome) and 440 nm (green: mitochondria) channels using ImageJ software (B). Mitophagy index calculated from three independent experiments and compared to stable cells treated with PQ for 6 h show that FKBP8 knockdown did not significantly suppress oxidative stress-induced mitophagy in all tau groups (C). Data represent mean ± SEM and were statistically analyzed using Student’s t-test. *p < 0.05. Scale bar: 20 μm.