Additional file 1 of SETD7-mediated monomethylation is enriched on soluble Tau in Alzheimer’s disease
Creators: name: Bichmann, Maria
name: Prat Oriol, Nuria
name: Ercan-Herbst, Ebru
name: Schöndorf, David C.
name: Gomez Ramos, Borja
name: Schwärzler, Vera
name: Neu, Marie
name: Schlüter, Annabelle
name: Wang, Xue
name: Jin, Liang
name: Hu, Chenqi
name: Tian, Yu
name: Ried, Janina S.
name: Haberkant, Per
name: Gasparini, Laura
name: Ehrnhoefer, Dagmar E.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Chemical Sciences not elsewhere classified
subject: Sociology
subject: FOS: Sociology
subject: Mental Health

description: Additional file 1: Supplementary Figure S1. Methyl-Tau antibodies specifically recognize methylated over non-modified Tau peptides. Non-modified recombinant Tau 2N4R, Tau 2N4R subjected to reductive methylation as well as methylated and non-methylated Tau-derived peptides were spotted onto nitrocellulose membranes and probed with the different methyl-Tau antibodies used in this study. Supplementary Figure S2. Three of five methyl-Tau antibodies demonstrate specificity for their methylation site in a cell lysate context. HEK293T cells were transiently transfected with wt 2N4R Tau or mutant Tau proteins (K130R, K132R, K343R, K353R or K438R). While the antibodies directed against meK130, meK132 and meK353 specifically recognize wt, but not the corresponding mutant Tau, antibodies against meK343 and meK438 show no such specificity. Supplementary Figure S3. Sarkosyl Extraction scheme. Supplementary Figure S4. Treatment with λ-phosphatase abolishes the HMW Tau band. Cytosolic and soluble nuclear fractions derived from 8- and 30-week old tg4510 mouse cortex were subjected to dephosphorylation with λ-phosphatase. Staining with Tau 12 demonstrates that the high molecular weight (HMW) Tau band apparent in 30-week old animals is abolished upon phosphatase treatment. Furthermore, the pT231 antibody staining, which strongly stains the HMW bands in untreated samples, is also removed by the treatment. Stainings with the meK130 and meK132 antibodies demonstrate that Tau methylation is not removed by phosphatase treatment. Supplementary Figure S5. Subcellular localization of PKMTs identified by MS proteomics in iPS-derived neurons. Supplementary Figure S6. SETD7 levels and subcellular localization in rTg4510 mouse brain. Cortical tissue from 16 week old WT and rTg4510 mice was subjected to subcellular fractionation and Western blotting for SETD7 as well as the fraction markers Hsp90 and HDAC2. In agreement with the data from hiPSC-derived neurons, SETD7 also localizes to the cytoplasmic fraction in mouse brain samples. No statistically significant difference in the abundance of SETD7 between WT and rTg4510 mice was detected upon quantification and normalization to Hsp90. Statistical significance was determined by unpaired two-tailed Student’s t-test, n.s.: not significant. Supplementary Figure S7. Differential gene expression of SETD7 in human postmortem brain tissue for multiple AD related phenotypes in two independent cohorts. (A) SETD7 gene expression in the Mount Sinai Brain Bank study (AMP-AD) for dementia status. SETD7 is significantly higher expressed in cases with dementia (CDR 3-5) compared to no dementia (CD 0-0.5) or MCI (CDR 1-2). (B) SETD7 gene expression in the Mayo study (AMP-AD) for Braak stages. SET7 is significantly higher expressed in brain tissue of patients with Braak V-VI compared to patients with low (0-II) or medium Braak stages (III-IV). (C) SETD7 gene expression in the Mayo study (AMP-AD) for AD status. SETD7 is significantly higher expressed in brain tissue of AD patients compared to controls. Please refer to the methods section for details of the analysis. Suppl. Fig. S8. The influence of PKMT inhibition on neuronal viability and methylated Tau levels. (A) – (C) SH-SY5Y cells were treated with different PKMT inhibitors as described in the methods section. No influence on meK130 and meK132 levels or viability was observed. Statistical significance was determined by one-way ANOVA. ANOVA meK130: p = 0.1910, ANOVA meK132: p = 0.3367, ANOVA viability: p = 0.3750. Suppl. Fig. S9. SETD7 inhibitor treatment does not alter Tau phosphorylation in hiPSC-derived neurons. 48h of treatment with 10 μM (R)-PFI-2 does not alter AT8 or AT100 signal, but significantly decreases meK132 Tau signal as measured by ELISA. Statistical significance was determined by unpaired two-tailed Student’s t-test, n.s.: not significant, **: p&lt;0.01. Suppl. Fig. S10. (R)-PFI-2 treatment does not change nuclear Tau levels in hiPSC neurons. hiPSC-derived neurons were treated for 48h with 10 μM (R)-PFI-2, and levels of nuclear, overall monomethylated Tau were determined by PLA with the meK and the Tau 12 antibodies and confocal microscopy (A). The levels of total, nuclear Tau were determined by subcellular fractionation and Western blotting (B). Scale bars: 100 μm. Statistical significance was determined by unpaired two-tailed Student’s t-test, n.s.: not significant. Suppl. Fig. S11. The influence of SETD7 knockdown and K/R mutations on total Tau levels. (A) shRNA treatment against SETD7 leads to a slight reduction in total Tau levels in SH-SY5Y cells. For a representative Western blot, see Fig. 6C. (B) Total Tau levels do not significantly differ in HEK293T cells transiently transfected with wt 2N4R Tau or the K130R and K132R mutant proteins. For a representative Western blot, see Fig. 6D. Statistical significance was determined by Mann-Whitney test in (A), and one-way ANOVA in (B). **: p&lt;0.01, ANOVA p=0.8096.