EMBO/soda-nel huggingface dataset:
Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition (2019)
Figure 2

Fig 2. Inhibition of PKM2 by S-nitrosation facilitates the accumulation of reducing equivalents to attenuate oxidative stress.

A. Pyruvate kinase (PK) activity in FLAG immunoprecipitates from human endothelial cells expressing FLAG-tagged wild-type (WT), Y657F (YF) or Y657D (YD) eNOS; n=6 independent cell batches (1-way ANOVA and Tukey).

B. PK activity in eNOS immunoprecipitates from HEK293 cells expressing WT, YF, YD or S633D (SD) eNOS; n=5-9 independent experiments (1-way ANOVA and Bonferroni).

C. S-nitrosation of PKM2 in human endothelial cells expressing FLAG-tagged WT or YF eNOS and treated with H2O2 (30 µmol/L) for 15 min; n=8 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) was included to demonstrate specificity of the S-NO signal.

D. PKM2 S-nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG-tagged WT PKM2 or a C358S (CS) PKM2 mutant. SNO-FLAG-PKM2 was detected with an anti-FLAG antibody after biotin switch technique; n=4 independent experiments (2-way ANOVA & Bonferroni). DTT treatment was included to demonstrate specificity of the SNO signal.

E. PK activity in eNOS immunoprecipitates from HEK293 cells co-expressing YF or YD eNOS and either WT PKM2 or the CS PKM2 mutant; n=6 independent experiments (2-way ANOVA & Bonferroni).

F. PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate (PEP) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS. The data were fit with the Michaelis-Menten equation to determine Vmax and Km; n=4 independent experiments (2-way ANOVA & Bonferroni).

G. Quantification of pentose phosphate pathway (PPP) intermediates; gluconate-6-P (G6P), ribulose-5-P (Rl5P), ribose-5-P (R5P), sedoheptulose-7-P (S7P), fructose-6-P (F6P) and erythrose-4-P (E4P) in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).

H. Link between the pentose phosphate pathway, generation of NADPH and reduction of GSSG to GSH.

I. NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).

J. NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells treated with solvent (Sol) or the PKM2 inhibitor shikonin (SKN, 1 µmol/L) for 45 minutes; n=4 independent cell batches (Unpaired Student's t test).

Data information: Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition (2019)
Figure 3

Fig. 3. Enhanced NO bioavailability results in PKM2 S-nitrosation and inhibition in vivo.

A. Systolic blood pressure (SBP) in wild-type (WT) and YF-eNOS (YF) mice; n=9 animals per group (Unpaired Student's t test).

B&C. Phenylephrine (PE)-induced contraction (B) and acetylcholine (Ach)-induced relaxation (C) of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).

D. Acetylcholine (ACh)-induced vasodilatation of the buffer-perfused hindlimb in situ. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=12-13 animals per group (2-way ANOVA and Bonferroni).

E. PKM2 S-nitrosation in pulmonary endothelial cells from WT and YF mice; n=5-6 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent bands cropped from the same membranes.

F. PK activity in eNOS immunoprecipitates from WT and YF pulmonary endothelial cells; n=7-10 independent cell batches (Unpaired Student's t test).

G. PK activity in eNOS immunoprecipitates from hearts from WT and YF mice; n=6 mice per group (Unpaired Student's t test).

H. Representative images of eNOS-PKM2 interaction (PLA foci, red) in mouse pulmonary endothelial cells; the Golgi apparatus and endosomes were stained with Golph4 (green), the plasma membrane was stained with CD144 and nuclei were highlighted with DAPI (gray). Only rare PLA foci were found in samples incubated with control mouse and rabbit IgGs, demonstrating the specificity of the reaction; n=3 independent cell batches. Scale bars: 20 µm.

Data information: Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ns = not significant.
Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition (2019)
Figure 4

Fig. 4. Preserved NO bioavailability attenuates angiotensin II-induced endothelial dysfunction through preserved PKM2 S-nitrosation and redox homeostasis in vivo.

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days.

A. Phosphorylation of eNOS on Tyr656 in lung lysates from animals treated for 7 days, n=6-7 mice per group (Unpaired Student's t test).

B. Systolic blood pressure (SBP) in WT and YF mice treated for 28 days, n=10 mice per group (Unpaired Student's t test).

C. Acetylcholine (ACh)-induced relaxation of aortic rings from the same animals as in panel B, n=8 mice per group (2-way ANOVA and Bonferroni).

D. PKM2 S-nitrosation in lungs from WT and YF mice administered AII for 28 days, n=7-8 mice per group (Unpaired Student's t test). DTT treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent lanes from the same membranes.

E. NADPH/NADP+ and GSH/GSSG ratios in lungs from WT and YF mice treated with AII for 28 days; n=8-9 mice per group (Unpaired Student's t test).

F. 3-Nitrotyrosine (3-NT) levels in lungs from WT and YF mice treated with vehicle (Veh) or AII (AII) for 28 days; n=7-9 mice per group (2-way ANOVA and Bonferroni).

G. NADPH/NADP+ and GSH/GSSG ratios in pulmonary endothelial cells from WT and YF mice treated with AII for 30 min; n=8 cell batches (2-way ANOVA and Tukey).

H. H2O2 levels in pulmonary endothelial cells from WT and YF mice after treatment with AII (1 µmol/L) for 30 min; n=8 cell batches (2-way ANOVA and Tukey).

Data information: Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition (2019)
Figure 5

Fig. 5. Preserved NO bioavailability and PKM2 S-nitrosation protect against vascular oxidative stress in vivo.

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control.

A. Proximity ligation assays showing S-nitrosated PKM2 (yellow) in the RCA and LCA 2 days after ligation. Phalloidin staining (blue) was included to highlight the vessel wall; n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.

B. GSH (green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.

C. 3-nitrotyrosine (3NT, green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.

D. Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted LCA 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni).

E. Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted aorta 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni).

Data information: Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition (2019)
Figure 6

Fig. 6. PKM2 inhibition preserves endothelial cell GSH levels and protects against vascular oxidative stress in vivo.

ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control.

A. GSH (green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.

B. 3-Nitrotyrosine (3NT, green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.

Data information: Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition (2019)
Figure 7

Fig. 7. Preserved eNOS activity and PKM2 inhibition exert anti-inflammatory effects.

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet.

A. ICAM1 and VCAM1 (green) expression in the LCA 2 days after ligation. CD31 (red) was included to label endothelial cells and nuclei are highlighted with DAPI (gray). Comparable images were obtained in 4 additional animals in each group. Scale bars: 50 µm.

B-D. Percentage of neutrophils and monocytes infiltrated into the LCA 7 days after ligation and high cholesterol diet feeding; n=9-10 mice per group (Unpaired Student's t test).

E. Representative images and quantification of ICAM1 (green) and VCAM1 (red) staining in human endothelial cells pre-treated with the PKM2 inhibitor shikonin (SKN, 1 µmol/L) or solvent (Sol) for 45 minutes and then stimulated with interleukin-1β (IL-1, 20 ng/ml) or solvent (CTL) for 90 min. Nuclei are highlighted with DAPI (gray). n=6 independent cell batches (2-way ANOVA and Bonferroni). Scale bars: 20 µm.

F&G. Percentage of neutrophils and macrophages infiltrated into the LCA from ApoE-/- mice treated with Veh or SKN (1.2 mg/kg) 7 days after ligation and high cholesterol diet feeding; n=4-6 mice per group (Unpaired Student's t test).

Data information: Data are presented as mean ± SEM. *P<0.05, ***P<0.001.
Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition (2019)
Figure 8

Fig. 8. Preserved NO bioavailability delays atherosclerosis development.

A. Plaque burden in ligated carotid arteries from ApoE-/- (-/-) and ApoEYF (YF) mice fed a high cholesterol diet for 21 days; scale bars: 2 mm. Intimal and medial thickness was determined at four specific locations (proximal, middle 1, middle 2, distal) in cryo-sectioned arteries stained with Oil Red O; scale bars: 200 µm. n=7 mice per group (Unpaired Student's t test).

B&C. Oil Red O staining of plaques in aortae from mice fed a Western diet for four (B) or six months (C); scale bars: 3 mm. n=6 (B) or n=12 (C) mice per group (Unpaired Student's t test).

Data information: Data are presented as mean ± SEM. *P<0.05 , **P< 0.01.

Expanded View Figure Legends
The extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity during skin aging (2022)
Figure 2

Figure 2. Global gene expression analysis reveals molecular changes in slow- and fast-cycling epidermal stem cells during aging.

A. PCA map describes the transcriptomic clustering of LRCs (label-retaining cells) and nLRCs (non-label retaining cells) constructed from 3-fold differentially expressed genes among 2-month- (N=3) and 2-year-old (N=3) mice.

B. Table summarizes the number of signature genes in young LRCs vs. nLRCs and their respective changes in 2-year-old tail skin.

C-D. Gene ontology (GO) analysis obtained from ≥2-fold differentially regulated genes (P<0.05) in 2-year-old nLRCs compared to 2-month-old mice.

E. Heatmap shows basal and suprabasal signature genes of epidermal stem cells and HFSCs Ge et al., 2020() in 2-month-old vs. 2-year-old nLRCs. Scale bar reflects Z-score. IFE, interfollicular epidermis; HF, hair follicle; HFSC, hair follicle stem cells.

F. Schematic of fibulin 7 protein structure.

G. Fbln7 gene expression in 2-month- vs 2-year-old nLRCs. N=3 mice per group (t-test). *; P<0.05. Data shows mean ± S.D.

H-I. Fibulin 7 immunostaining in 2-month- vs. 2-year-old tail section (H) and its intensity quantification per basal epidermal stem cell/basement membrane (normalized to 2-month-old) (I). Dotted box areas were enlarged in the lower panels. White arrows indicate fibulin 7 basement membrane staining. Scale bar: 50 μm. Data shows mean ± S.D.**; P<0.01 (Mann-Whitney test). N=7 mice per age group (biological replicates).
The extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity during skin aging (2022)
Figure 3

Figure 3. Fbln7 knockout leads to aggravated fast-cycling stem cell loss and impaired injury repair.

A. Slc1a3CreER lineage tracing in the Fbln7 WT and KO mice. Low-dose tamoxifen was administered once at 2 months of age and samples were collected after 1-week, 3-month, and 1-year chases. Wholemount staining of tail epidermis with tdTomato, K10, and Hoechst. Scale bar: 200 μm. WT, wild-type; KO, knock-out.

B. The number of Slc1a3CreER+ clones per scale structure in 1-week and 3-month chases (left panel) and 1-year chase (right panel).

C. Quantitation of the area of Slc1a3CreER+ clones per total area of scale structure. Fbln7 WT mice for 1-week (N=5), 3-month (N=3), and 1-year chases (N=3). Fbln7 KO mice for 1-week (N=5), 3-month (N=4), and 1-year chases (N=6).

D-E. Ki67 immunostaining in 1-week-chase mice (D) and its quantitation (E). White arrows show Ki67 and tdTomato double positive cells, which were counted and summarized in (E). Scale bar: 20 μm. N=5 WT/het and 3 KO mice. Welch's t-test.

F. Representative pictures from tail wounds of 1-year-old Fbln7 WT and KO mice. Scale Bar: 4 mm.

G. Measurements of wound area over time in Fbln7 WT (N=5) and KO (N=7) mice. Two-way ANOVA, Tukey's multiple comparisons test.

H-J. Hematoxylin and eosin staining from tail sections of wounded Fbln7 WT vs. KO mice (H) and quantitation of the re-epithelialization length (I) and thickness at the healing front (J). Scale bar: 200 μm. N=3 WT and 5 KO mice.

Data information: All graphs show mean ± S.D. N reflects the number of biological replicates which are summarized from at least 2 independent experiments. *; P<0.05. **; P<0.01. (B, C, I, J) Mann-Whitney test. One sample from 1-year KO group was removed after an outlier test was performed in Graphpad Prism.
The extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity during skin aging (2022)
Figure 4

Figure 4. Loss of Fbln7 is associated with increased inflammatory response in aging epidermal stem cells and lineage misregulation.

A. Principal Component Analysis map constructed from 3-fold differentially expressed genes in epidermal stem cells from Fbln7 WT vs. KO dorsal skin in 2-month- and 1-year-old mice. Each dot represents one mouse (N=3 per group, except 1-year-old Fbln7 KO N=4).

B, C. Gene ontology (GO) analysis from ≥2-fold upregulated (B) or downregulated (C) genes in 1-year-old Fbln7 KO mice compared with WT.

D-E. Heatmap illustrating changes in inflammatory response genes (D) and basal or suprabasal signature genes of epidermal stem cells (IFE) and HFSCs Ge et al., 2020() (E) in 1-year-old Fbln7 WT vs. KO mice, constructed from ≥2-fold differentially expressed genes obtained from GO analysis in (B). Scale: Z-score.

F-G. K31 and K14 immunostaining in tail sections of 2-month- vs. 2-year-old C57BL/6J WT mice (F) and the quantification (G). Dotted box areas are enlarged in the lower panels. Scale bar: 50 μm. Quantification of K14 intensity in the suprabasal area was normalized to basal area per cell. N=4 (2-3-month-old) and N=5 (2-year-old) C57BL/6J mice (G).

H-J. K31 and K14 immunostaining in tail sections of 1-year-old Fbln7 WT vs. KO compared with 2-month-old Fbln7 KO mice (H) and the quantitation (I, J). Scale bar: 50 μm. N=5 for both WT and KO of 2-3-month-old mice (I). N=5 and N=7 for 1-year-old WT vs. KO, respectively (J). Data information (F-J): Two-way ANOVA, Tukey's multiple comparisons test. All graphs show mean ± S.D. N reflects the number of biological replicates which are summarized from at least 2 independent experiments. ****; P<0.0001, ns; not significant.
The extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity during skin aging (2022)
Figure 5

Figure 5. Fibulin 7 modulates Collagen IV and Fbln7 overexpression maintains the undifferentiated, slower proliferation state in primary keratinocytes.

A. Collagen IV immunostaining in tail sections of 1-year-old Fbln7 WT vs. KO. Dotted box regions are enlarged. Scale bar: 50 μm.

B. Quantification of Collagen IV basement membrane intensity per cell in Fbln7 KO mice normalized to WT mice. N=4 WT and N=6 KO mice. Graphs show mean ± S.D. Mann-Whitney test. **; P<0.01, ns; not significant.

C-E. Diagram of proteins binding to full-length fibulin 7 or fibulin 7 with coiled-coil deletion mutant (delta CC) (C) and workflow chart of screening experiments to investigate secreted fibulin 7-interacting proteins (D). List of the fibulin-7 binding proteins obtained from mass-spectrometry (E).

F. Solid-phase binding assay using recombinant fibulin 7 as liquid phase and purified collagen IV protein as solid phase. X-axis shows increasing doses of fibulin 7 (μg/well). Bovine serum albumin (BSA) was used at the same amounts as control liquid phase. Graph shows mean ± S.D. from 4 technical repeats in 2 independent experiments.

G. Brightfield images of mouse primary keratinocytes overexpressing GFP, full-length fibulin 7 (F7), or coiled-coil deletion mutant (F7 dCC) as shown in (C). Scale bar: 200 μm.

H­-K. Quantitative RT-PCR shows fold changes in gene expression of F7 or F7 dCC compared with GFP control. Keratinocytes were induced with CaCl2 for 1 day with or without Collagen IV coating prior to quantitative RT-PCR (J, K). N = at least 3 biological repeats. Statistical test: One-way ANOVA with Dunn's multiple comparison test (H); Welch's t-test. ***; P<0.001. **; P<0.01. *; P<0.05; ns, not significant (I). One-way ANOVA (Tukey's multiple comparisons test) was used for (J, K).

L, M. Cell proliferation assays comparing growth rates of GFP overexpression control with F7 and F7 dCC over 6 days in 15% FBS growth medium (L) or 3% reduced growth medium (M). FBS, fetal bovine serum. Abs, absorbance.

N. Cell proliferation assay comparing GFP or F7 overexpressing keratinocytes treated with 5 ng/ml IL-6 over 6 days. Statistical test two-way ANOVA (Tukey's multiple comparisons test). ****; P<0.0001; **; P<0.01. *; P<0.05; ns, not significant (L-N).

Data information: All graphs describe mean values ± S.D. from at least 3 biological repeats.

Expanded View figure legends
P-tau235: a novel biomarker for staging preclinical Alzheimer's disease (2021)
Figure 1

Figure 1. Exploratory and targeted IP-MS in brain. (A) Bar chart showing the relative abundance (signal intensity) of the phosphorylated tau species identified using an exploratory IP-MS approach using HT7 antibody on TBS-soluble control and Alzheimer's disease (AD) pools. Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP-MS: mono-phosphorylated p-tau231 and double phosphorylated p-tau(231+235). (B) Box-and-whisker plot showing quantification of mono-phosphorylated p-tau231 in control (n=10) and AD (n=10) TBS-soluble fractions using a targeted IP-MS method (P >0.05, non-significant, ns, Mann-Whitney U test). (C) Box-and-whisker plot showing the quantification of double phosphorylated p-tau(231+235) in control (n=10) and AD (n=10) TBS-soluble fractions using a targeted IP-MS method. Data information: Box-and-whisker plots show the median and the 25th and 75th percentiles. Group differences determined using Mann-Whitney U test (****P <0.0001). All samples were run in singlicates.
P-tau235: a novel biomarker for staging preclinical Alzheimer's disease (2021)
Figure 2

Figure 2. CSF p-tau235 performance in the discovery cohort. (A) Box-and-whiskers plot showing the levels of CSF p-tau235 in neurological controls (n=21) and biologically defined Alzheimer's disease (AD) cases (n=19). (B) ROC analysis indicating the diagnostic accuracy of CSF p-tau235 when discriminating AD from control.

Data information: Box-and-whisker plots show the median and the 25th and 75th percentiles. Group differences determined using Mann-Whitney U test (****P <0.0001). All samples were run in singlicates.
P-tau235: a novel biomarker for staging preclinical Alzheimer's disease (2021)
Figure 3

Figure 3. CSF p-tau235 assay across the Alzheimer's disease continuum (TRIAD cohort). (A) Box-and-whiskers plot showing CSF p-tau235 concentrations in the different groups: amyloid-negative cognitively unimpaired (CU-) participants (n=50), participants across the Alzheimer's disease (AD) continuum (amyloid-positive cognitively unimpaired (CU+, n=32); amyloid-positive mild cognitive impairment (MCI+, n=20); AD (n=20) and non-AD cases (n=19). CSF p-tau235 is highly specific for AD pathology. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL). (B) ROC analysis showing the high diagnostic accuracy of CSF p-tau235 discriminating AD from CU-, CU+, MCI+, and non-AD groups. (C) ROC analysis comparing AUC values of CSF p-tau235, p-tau231 and p-tau217 discriminating CU+ from CU-. CSF p-tau235 discriminating accuracy was statistically lower to that of CSF p-tau231, but not CSF p-tau217. (D) ROC analysis comparing AUC values of CSF p-tau235, p-tau231 and p-tau217 discriminating amyloid-negative cognitively impaired (CI-, non-AD) from amyloid positive cognitively impaired (CI+, MCI+ and AD). CSF p-tau235 performance was statistically similar to that of CSF p-tau217 and p-tau231. Data information: Box-and-whisker plots show the median and the 25th and 75th percentiles. P-values determined using one-way ANOVA adjusted by age and sex followed by Bonferroni-corrected post-hoc comparison (*P <0.05, ****P <0.0001). CSF p-tau235 cut-off was determined as the mean + 2 SD of the A-T- group in ALFA+ cohort (19.92 pg/mL). ROC analysis (B-D) indicating the diagnostic accuracy of the studied biomarkers as AUC values. DeLong test (C, D) was used to determine statistical differences between biomarker performances (P <0.05 is indicated in bold). All samples were run in singlicates.
P-tau235: a novel biomarker for staging preclinical Alzheimer's disease (2021)
Figure 4

Figure 4. CSF p-tau235 assay across in preclinical Alzheimer's disease (ALFA+ cohort). (A) Box-and-whiskers plot showing CSF p-tau235 concentrations in A-T- participants and across preclinical Alzheimer's disease (AD, dichotomised as A+T- and A+T+). CSF p-tau235 was increased already in A+T-, which represents the early cases within preclinical AD. A prominent increase was observed from A+T- to A+T+ cases, the latter representing late preclinical AD cases. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL). (B) ROC analysis comparing AUC values of CSF p-tau235, p-tau231 and p-tau217 discriminating A+T- from A-T-. Both CSF p-tau217 and p-tau231 a showed a statically higher accuracy discriminating A-T- from A+T- than CSF p-tau235 (C) ROC analysis comparing AUC values of CSF p-tau235, p-tau231 and p-tau217 discriminating A+T+ from A-T-. However, all three assays had statistically the same accuracy when distinguishing A-T- from A+T+. (D) ROC analysis comparing AUC values of CSF p-tau235, p-tau231 and p-tau217 discriminating A+T+ from A+T-. CSF p-tau235 demonstrated statistically superior diagnostic accuracy than CSF p-tau217 discriminating the two preclinical groups (A+T- and A+T+). In this scenario, no differences in diagnostic performance between CSF p-tau235 and p-tau231 were observed. Data information: Box-and-whisker plots show the median and the 25th and 75th percentiles. P-values were determined using one-way ANOVA adjusted by age and sex followed by Bonferroni-corrected post-hoc comparison (****P <0.0001). CSF p-tau235 cut-off was determined as the mean + 2 SD of the A-T- group in ALFA+ cohort (19.92 pg/mL). ROC analysis (B-D) indicating biomarker diagnostic accuracy as AUC values. DeLong test (B-D) was used to determine statistical differences between biomarker performances (P <0.05 is indicated in bold). All samples were run in singlicates.