PMCID string | Title string | Sentences string |
|---|---|---|
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Many of these phosphorylation sites are found to be hyperphosphorylated in the context of neurodegenerative disorders like Alzheimer’s disease (AD) . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This is considered to contribute to Tau aggregation into intracellular Tau filamentous deposits, a process associated with neuronal cell death and the emergence of AD . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Specific phosphorylation events are linked to distinct stages of the disease . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | For instance, the AT8 epitope, involving phosphorylation at S202 and T205, is frequently detected in pre-tangle neurons, marking early AD pathology . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Among emerging biomarkers, phosphorylation of Tau at T217 has garnered attention for its utility in early-stage tauopathy diagnosis. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | It was recently shown to outperform the measure of phosphorylation at T181 as a cerebrospinal fluid (CSF) and blood biomarker for AD . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Similarly, phosphorylation at S422 is considered a marker for early disease events in both AD and other tauopathies . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Microtubule dynamics are essential for spindle assembly, chromosome alignment, and segregation during mitosis. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Since Tau is expressed not only in the nervous system but also in other cell types—such as lung epithelial cells, pancreatic acinar cells, enteroendocrine cells, and cancer cells —understanding Tau phosphorylation during mitosis is of significant interest. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Previous studies have revealed notable similarities between Tau phosphorylation patterns observed during mitosis and in AD, particularly at phosphorylation epitopes AT8, PHF1, p-S214 and p-S422 . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Specifically, phosphorylation of Tau at PHF1 and AT8 epitopes during mitosis was initially identified in cell lines stably expressing Tau and in neuroblastoma cell lines synchronized with nocodazole treatment . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Mass spectrometry analysis of Tau peptides in artificially synchronized cells further suggested that S214 phosphorylation occurs during cell division . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Additionally, studies using Xenopus meiosis and neuroblastoma cell lines demonstrated Tau phosphorylation at S422 during mitosis . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | In this study, we aimed to characterize Tau phosphorylation sites specifically enriched during mitosis compared to interphase under conditions of transient Tau expression, without the use of microtubule-depolymerizing drugs. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | To this end, we quantified the relative levels of phosphorylation at 12 Tau phospho-epitopes during interphase and mitosis within the same biological system to establish a preliminary Tau phospho-signature during mitosis. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | To circumvent the limitations of continuous Tau overexpression, we utilized an SHSY-5Y cell line with inducible Tau expression. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Immunocytochemistry experiments, featuring co-labeling of dividing cells, were performed to avoid artificial mitotic arrest with microtubule polymerizing or depolymerizing agents. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Finally, we validated this mitotic Tau phospho-signature in vivo using dividing epithelial cells of Drosophila melanogaster overexpressing human Tau. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Our results revealed strong phosphorylation of Tau during mitosis at AT8, p-T217, and p-S422 epitopes both in vitro and in vivo. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | These findings offer new perspectives on the intersection of fundamental biology focusing on cell division and the molecular mechanisms underlying Tau-associated diseases. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Tau-inducible cell line was established from the neuroblastoma cell line SH-SY5Y by using the Tet-on T-rex system . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Cells were grown in complete culture medium consisting of Dulbecco’s modified Eagle’s medium/F-12 medium supplemented with 10% heat-inactivated FBS (Gibco, ThermoFisher Scientific, Waltham, MA, USA, #10270-106), 50 U/mL penicillin/streptomycin, and 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA, #G7513) and maintained in humidified atmosphere containing 5% CO2 at 37 °C. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Selection was maintained with 5 µg/mL blasticidin and 100 µg/mL Zeocin. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Induction of Tau expression (1N4R isoform) was performed for 24 h with 1 µg/mL tetracyclin 24 h after having plated the cells in 24-well plates for immunocytochemistry experiments . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | At this time point, increased Tau expression is observed with no significant disruption of mitosis detected. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | We used the ptc-Gal4 activator strains as in Bougé and Parmentier . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | The UAS-hTau strain was a gift from Mel Feany . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Third instar stage larvae were used independently of their sex. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Cells grown on coverslips were fixed with 4% paraformaldehyde for 5 min at room temperature or. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Cells were then permeabilized with PBS 1×, Triton 0.3% (PBS-T), at room temperature and then incubated with blocking buffer (PBS-T, 0.3% BSA) for 30 min at room temperature. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Cells were then incubated with primary antibodies diluted in blocking buffer for 1 h at room temperature, followed by PBS-T wash and incubation with secondary antibodies. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Third instar larval imaginal discs were dissected in PBS 1× and fixed for 20 min in 4% paraformaldehyde. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | After permeabilization with PBS 1×, Triton 0.3% (PBS-T) for 30 min at room temperature, discs were incubated overnight at 4 °C with primary antibodies diluted in PBS-T, 0.3% BSA. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Fluorescent secondary antibodies were used at the recommended dilution and incubated for 1 h at room temperature with 300 nM DAPI to counterstain nuclei, when needed. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Preparations were mounted using Dako fluorescent mounting medium (Agilent Technologies, Inc., Santa Clara, CA, USA, Cat. # |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Primary antibodies were: Pan-Tau antibodies: rabbit polyclonal anti-Tau (Dako, Agilent Technologies, Inc, Santa Clara, CA, USA, #A002401, 1:2000) and mouse anti-Tau monoclonal T46 (ThermoFisher Scientific, Waltham, MA, USA, Cat. # |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | 13-6400, 1:200), mouse monoclonal anti-GFP (Roche, Basel, Switzerland, Cat. # |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | 11814460001, 1:5000), sheep polyclonal anti-tubulin (ATN02, Cytoskeleton Inc., Denver, CO, USA, 1:400), rat monoclonal anti-tubulin (CBL270, Merck KGaA, Darmstadt, Germany, 1:300), mouse anti-PH3 (phospho-Ser10, clone 3H10, 1:300), rabbit anti-PH3 (phospho-Ser10 + Thr11, Cat. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | ab32107, Abcam, Cambridge, UK, 1:1000). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Anti phospho-Tau antibodies used in this study are listed in the table below. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Name of Antibody or Phospho-EpitopeReferenceProviderDilutionAT8MN1020Invitrogen (ThermoFisher Scientific) Waltham, MA, USA)1:200S202[EPR2402] ab108387Abcam, Cambridge, U.K.1:200T20544-738 GInvitrogen 1:200PHF1Gift from P. Davies 1:500S396[EPR2731] ab109390Abcam1:200S404[EPR2605] ab92676Abcam1:200AT100MN1060Invitrogen1:200T21244-740 GInvitrogen1:200S21444-742 GInvitrogen1:200T21744-744Invitrogen1:200S416 Cell Signaling Technologies, Danvers, MA, USA1:200D7U2P (#15013) S422[EPR2866] ab79415Abcam1:200 Secondary antibodies were Alexa-Fluor-488, Alexa-Fluor-633 (ThermoFischer Scientific, Waltham, MA, USA), Cy3 and Cy5 (Jackson ImmunoResearch, Westgrove, PA, USA), all diluted between 1:1000 and 1:500. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Images were acquired using a LSM780 and LSM980 laser confocal laser scanning microscope with 20× Plan Apo 0.8NA and 40× Plan Apo oil 1.3NA objectives at the imaging facility MRI (Biocampus, UM-CNRS-INSERM, Montpellier, France). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | For adequate measurements and comparison of immunoreactivity intensities, confocal laser settings were set up to avoid saturation of signal and were not modified when measuring different conditions of a specific staining. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | After acquisition, images were processed with Fiji . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Images displayed in the figures are maximal intensity z-projections of 4 to 8 z-stack images with minor brightness adjustments to ensure consistent rendering across confocal sessions. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Quantification of the level of Tau and phospho-Tau immunoreactivity was made as follows: dividing cells were selected based on the mitotic spindle visible with the tubulin staining independently of the other stainings, and a cytoplasmic area was selected around the mitotic spindle to measure total Tau and/or phospho-Tau staining (mean intensity within the selected area). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Interphasic cells were chosen nearby measured mitotic cells for quantification of interphasic Tau and phospho-Tau stainings. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | In addition to qualitative observations, two to three biological replicates were dedicated to quantification, with a minimum of three images per condition (±tetracycline). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Each condition included 12–24 quantified cells. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Figures display the results of a representative experiment, alongside a selected image area for illustration. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | The Principal Component Analysis (PCA) was conducted using eigenvalues greater than 1, without applying a rotation method. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Components 1 and 2 accounted for 53.98% and 39.47% of the variance, respectively. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | The analysis was performed using Jamovi software (2024, Version 2.6; www.jamovi.org retrieved on 3 March 2025), and graphical representation was generated using the snowCluster Jamovi module (Version 7.4.8; github.com/hyunsooseol/snowCluster retrieved on 24 September 2025). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | For in vivo analyses, at least three wing discs from Tau-overexpressing Drosophila were examined for each phospho-epitope. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Each experiment was replicated three times to ensure reproducibility. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | We utilized the SH-SY5Y neuroblastoma cell line transfected with Tau 1N4R transgene, regulated by the tetracycline-controlled T-Rex mammalian expression system . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This approach enabled the investigation of Tau phosphorylation under two conditions: (1) low basal expression levels, and (2) a 20-fold increase in Tau expression following 24-h induction with tetracycline (Figure 1 and Figure 2, Table 1). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | To examine phosphorylation under normal cell cycling conditions, we did not arrest the cell cycle. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Instead, we performed immunocytochemistry on fixed cells, co-labeling for microtubules to identify mitotic spindles. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Total Tau or phospho-Tau labeling intensity was quantified in the cytoplasm of interphase and mitotic cells under both basal and Tau overexpression conditions. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This allowed us to determine which phospho-epitopes exhibited the greatest increase in staining during mitosis relative to interphase under conditions of Tau overexpression (Table 1, Figure 3A). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Hence, the first selection criteria were the degree of increase in phospho-epitope immunoreactivity between interphase and mitosis in conditions of Tau overexpression, indicating mitosis-specific staining. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | As a control, total Tau staining revealed no significant change in staining between interphase and mitosis in conditions of Tau overexpression (Table 1). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | The second selection criteria were the magnitude of the increase in phospho-epitope immunoreactivity during mitosis between basal and Tau-overexpression conditions, reflecting specificity for Tau staining (Table 1, Figure 3A). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | We initially focused on the previously characterized phospho-epitopes AT8 and PHF1 along with their associated phosphorylation sites: S202 and T205 for AT8, and S396 and S404 for PHF1. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | AT8 epitope was previously shown to be strongly phosphorylated during mitosis, whereas the increase in phosphorylation at PHF1 site was only partial . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Our findings revealed a 60-fold increase in AT8 staining specifically during mitosis under conditions of Tau overexpression, alongside a 16-fold increase in AT8 staining in mitotic cells when comparing basal and Tau-overexpression conditions (Figure 1 and Table 1). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This indicates that Tau undergoes specific phosphorylation at this epitope during mitosis. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | In contrast, PHF1 displayed only 4-fold increase in immunoreactivity in mitotic cells compared to interphase cells under Tau overexpression conditions. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This was accompanied by a marked 20-fold increase in staining associated with Tau overexpression during interphase (Figure 1 and Table 1). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | These findings suggest that phosphorylation at the PHF-1 epitope predominantly occurs during interphase under conditions of Tau overexpression. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Altogether, our results are in accordance with previously reported data on the AT8 and PHF1 epitopes during mitosis. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Analysis of individual phosphorylation sites revealed that mitosis-specific phosphorylation is associated with T205 for AT8 and S396 for PHF1, showing a 27- and 3-fold increase in immunoreactivity during mitosis under Tau overexpression conditions, respectively. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | In contrast, S202 and S404 displayed a pattern of increased phosphorylation primarily during interphase in response to elevated Tau levels, with 19- and 30-fold increases, respectively (Figure 1 and Table 1). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | We then analyzed a series of additional phospho-epitopes, including AT100, p-S214 and p-S422, all previously reported to be phosphorylated during mitosis . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | We examined the phosphorylation sites T212 and T217, which are components of the AT100 epitope along with S214 , and the recently characterized S416 as an additional distal phosphorylation site . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Under conditions of Tau overexpression, our analysis revealed a modest increase in staining for AT100 and S214 during mitosis (4-fold and 7-fold, respectively); however, there was no clear increase in staining of mitotic cells between basal and Tau overexpression conditions (0.75-fold and 1.42-fold, respectively) (Figure 2, Table 1). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This suggests an absence of specific Tau phosphorylation at these epitopes. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | For the other phosphorylation sites within the AT100 epitope, both T212 and T217 showed increased phosphorylation during mitosis under conditions of Tau overexpression, with T217 showing a much stronger response (6.6-fold and 30-fold, respectively). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Additionally, the distal phospho-epitopes p-S416 and p-S422 demonstrated pronounced increases in staining during mitosis, with 13-fold and 28-fold increases in immunoreactivity under Tau overexpression conditions, respectively (Figure 2, Table 1). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Overall, Principal Component Analysis using the three variables from Table 1 identified the mitotic phospho-epitope cluster, characterized by high values for Component 2 (driven by the two mitosis-related variables) and low values for Component 1 (primarily influenced by interphase overexpression effects) (Figure 3B). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | This cluster contains AT8, p-T205 (part of the AT8 epitope), p-T217, p-S416, and p-S422 as Tau epitopes highly and specifically phosphorylated during mitosis (Figure 3A,B). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Having identified this signature under normal cell cycle conditions in a neuroblastoma cell line, we assessed its generality and robustness in vitro, by extending our analysis to (i) a distinct cell line: HeLa cells stably expressing the 0N4R Tau construct , (ii) an alternative technique: Western blot analysis, which required cell cycle synchronization. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Our results in HeLa cells confirmed the mitotic phosphorylation of these epitopes (Supplementary Figure S1), thereby validating the signature across in vitro experimental systems. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | To further evaluate the relevance of mitotic phosphorylation at the specific phospho-epitopes identified in cell culture, we analyzed their behavior in vivo under conditions of human Tau overexpression. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | For this purpose, we used a Drosophila transgenic system that overexpresses human 0N4R Tau within a determined area of the epithelial wing disc during larval development, as described in Bougé and Parmentier . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | The tissue undergoes growth during development, with all cells undergoing mitosis. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Mitotic cells were identified using the phospho-histone 3 (PH3) marker. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Consistent with previous findings, PHF1 and p-S396 epitopes were detected throughout the entire domain of Tau overexpression (Figure 4), encompassing both mitotic and non-mitotic cells. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Double immunostaining confirmed the presence of PHF1 in all Tau-overexpressing cells (Figure 5). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Similarly to previous findings, we could not detect any specific signal associated with epithelial Tau-expressing cells for p-S214 and AT100 epitopes . |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | When assessing the phospho-epitopes identified in cell-culture experiments, we observed that immunoreactivity for AT8, p-T217, and p-S422 was predominantly restricted to mitotic cells. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | In contrast, immunoreactivity for p-T205 and p-S416 was also present in many non-mitotic cells (Figure 4). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | Double staining with either PHF1 or total Tau further confirmed that AT8, p-T217, and p-S422 epitopes were restricted to a subset of Tau-expressing cells (Figure 5). |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | These results validate the mitotic specificity of AT8, p-T217, and p-S422 epitopes. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | The use of an inducible Tau expression system to study Tau phosphorylation during mitosis has proven highly beneficial in multiple aspects. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | First, by comparing Tau phosphorylation levels before and after a 24-h transient induction, we could identify specific phosphorylation sites that were prominently phosphorylated during interphase under Tau overexpression conditions. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | These included PHF1, S202, S404, and, to a lesser extent, S396. |
PMC12562719 | Phospho-Tau Signature During Mitosis: AT8, p-T217 and p-S422 as Key Phospho-Epitopes | These findings are internally consistent, as p-S396 and p-S404 together constitute the PHF1 epitope. |
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