EMBO/soda-nel huggingface dataset:
Trehalose-6-phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana (2019)
Figure 3

Figure 3.

KIN10 mediates the T6P regulation of thermoresponsive hypocotyl growth

A, B Hypocotyl lengths of wild-type and KIN10-OX seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (A). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 15). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.

C Transient gene expression assays. The reporter vector containing 35S::Renilla luciferase and IAA19p::Firefly luciferase was co-transfected with 35S::PIF4 and 35S::KIN10 into Arabidopsis mesophyll protoplasts. The activity of Firefly luciferase under the control of IAA19 promoter was normalized to that of Renilla luciferase. Error bars indicate s.d. (n = 4). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05). T, 35S terminator.

D, E Hypocotyl lengths of seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (D). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 15). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.
Trehalose-6-phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana (2019)
Figure 4

Figure 4.

KIN10 directly interacts with and phosphorylates PIF4

A Box diagram of various fragments of PIF4 used in (B, C).

B, C Yeast two-hybrid assays showing the direct interaction between PIF4 and KIN10. Yeast clones were grown on the synthetic dropout medium with histidine (+HIS) or without histidine (-HIS) plus 25 mM of 3-amino-1,2,4-triazole (3-AT).

D Co-IP assays showing the interaction between PIF4 and KIN10 in vivo. Protein extracts from protoplasts expressing PIF4-Myc and KIN10-GFP (or GFP) were immunoprecipitated with anti-GFP antibody and analyzed by immunoblotting with anti-GFP or anti-Myc antibody.

E GRIK1-activated KIN10 phosphorylates PIF4. In vitro phosphorylation assays were performed at 30 °C for 30 min using 1 μg of purified recombinant proteins (KIN10, GRIK1, or PIF4). 32P-labeled proteins were visualized by autoradiography. CBB, coomassie brilliant blue-stained gel.

F T6P inhibits PIF4 phosphorylation by KIN10. This assay was performed with 1 μg of each protein at 30 °C for 30 min in the presence of 1 mM T6P or sucrose (Suc).
Trehalose-6-phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana (2019)
Figure 5

Figure 5.

KIN10 decreases the stability of PIF4 through proteasomal degradation

A The levels of PIF4-Myc are negatively correlated with the levels of KIN10-GFP. Total proteins were extracted from mesophyll protoplasts transfected with different amounts of KIN10-GFP and PIF4-Myc and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal loading.

B, C KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal protein loading. Representative western blots are shown in (B). Similar results were obtained from two other independent experiments (Appendix Fig S3). Quantification of relative PIF4-Myc levels is shown in (C). PIF4-Myc levels are presented as values relative to those at 0 h. In (C), different letters above each point indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 3). Error bars indicate s.d. (n = 3).

D, E Western blotting showing endogenous PIF4 protein levels in the seedlings grown in the same conditions as in (Fig 3A). Total protein extracted from the seedlings was analyzed by immunoblotting with anti-PIF4 antibody. Ponceau S staining shows equal protein loading.
Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia (2020)
Figure 1

Fig. 1 Overexpression of FLT3/ITD induced axis-duplication and ectopic expression of FST in zebrafish embryos.

A-D The morphology of uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected (150 ng per embryo) embryos on day 2 post fertilization (dpf).

E-H Whole mount in situ hybridization (WISH) of notochord-specific marker col9a2 in uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected embryos on 2 dpf.

I-J FLT3 signaling was detected by Western blotting in 293FT cells-transfected with FLT3/ITD mRNA (I) or in zebrafish embryos-injected with FLT3/ITD mRNA (J).

K The effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis-duplication phenotype-induced by FLT3/ITD mRNA injection in zebrafish.

L-N Quantification of fst expression by RT-qPCR (L), Western Blotting (M), and WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.

Data information: ov: otic vesicles; cm: cephalic mesoderm; nc: notochord. Scale bar = 500 μm. In (K and L), the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.
Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia (2020)
Figure 2

Fig. 2 FST was increased in FLT3/ITD-transgenic zebrafish and FLT3/ITD-mutated AML.

A-C WISH of fst in FLT3/WT- (A), FLT3/ITD- plasmid DNA injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35).

D-K Generation and characterization of FLT3/ITD-transgenic zebrafish. Diagrammatic representation (D and E) of the generation of Runx1-FLT3/ITD transgenic zebrafish (see Materials and Methods section). GFP expression was detected by florescent microscopy (F-H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1-FLT3/ITD transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD-positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1-FLT3/ITD transgenic zebrafish (F1) at two months old. Fish 4, 5 and 6 showed germline transmission of FLT3/ITD transgene (K).

L-N Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (M-N). (abbreviation for panel M: M, Myeloid cells; P, Progenitor cells; L, Lymphoid cells; E, Erythroid cells). Data are presented in box plot. The whiskers, boxes, and central lines in panel N represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).

O Expression of fst was detected by RT-qPCR in KM from WT sibling and Runx1-FLT3/ITD transgenic zebrafish at 18 months old. The RT-qPCR experiments were performed in triplicates and data were presented as mean ± SEM. **P<0.01.

P Detection of FST expression, p-ERK1/2, and p-CREB in mononuclear cells from normal peripheral blood stem cell (PBSC) and FLT3/ITD AML patients (diagnostic samples with leukemia blasts > 80%) by Western blotting. ^: non-specific staining of p-ATF1 protein due to the conserved motif.

Data information: Scale bar = 500 μm.
Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia (2020)
Figure 3

Fig. 3 FLT3/ITD upregulated FST through phosphorylation of CREB.

A In-silico analysis (DECipherment of DNA Elements, SABiosciences) and schematic model of transcription factor binding sites on human FST promoter. CBP: CREB-binding protein; CRE: cAMP response element; TSS: transcription start site.

B-C The direct binding of p-CREB to human FST promoter was detected by ChIP-PCR (B) and ChIP-qPCR (C). c-Fos was used as positive control of p-CREB target gene. Normal IgG was used as negative control of ChIP.

D Dual luciferase assay demonstrating the direct binding of pCREB on human FST promoter. pRL-CMV, Renilla luciferase vector; pGL-CRE- and pGL-CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE-) or wildtype (CRE+); p-GFPSpark, GFP expressing vector; p-CREBY134F, CREBY134F-GFP expressing vector.

E FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3-parental (P in short) and Ba/F3-FLT3/ITD (ITD in short) cells.

F-H Phosphor-flow analysis of p-CREB in Ba/F3-parental, Ba/F3-FLT3/ITD, and Ba/F3-FLT3/ITD cells-treated with FLT3 inhibitor Quizartinib (Qui in short). Isotype antibody were used as control to calculate the mean fluorescence intensity (MFI) ratio (F-G). The transcription and expression of Fst was detected by RT-qPCR after Quizartinib treatment (10 nM) in Ba/F3-FLT3/ITD cells for one day (H).

I-K The expression of FST and phosphorylation of CREB were detected by Western Blotting (I and K) and phospho-flow analysis (J) in MOLM-13 (I) and Ba/F3-FLT3/ITD (K) cells treated with Quizartinib and BRD7389 for one day, respectively.

L RSK and FST expression were detected by Western Blotting after p90RSK knockout by CRISPR/Cas9 in MOLM-13 cells.

M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba/F3-FLT3/ITD cells treated with CREB inhibitor 666-15 for one day. ^: non-specific staining of p-ATF1 protein due to the conserved motif.

N CREB and FST expression were detected by Western Blotting after CREB knockout by CRISPR/Cas9 in MOLM-13 cells.

O The growth of Ba/F3-parental (with IL-3), Ba/F3-FLT3/ITD (without IL-3) and Ba/F3-FLT3/ITD (with IL-3) cells was measured after three days treatment of CREB inhibitor 666-15 in vitro.

P The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.

Data information: In (C, D, G, H, J, and O), the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).
Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia (2020)
Figure 4

Fig. 4 FST promoted leukemia growth by activating ERK.

A FST expression in different AML cell lines was detected by Western Blotting.

B-C FST317 and FST344 overexpression resulted in significant increases of FST transcription by RT-qPCR and protein by Western Blot (B) and promoted ML-2 cell growth in vitro (C). Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. The RT-qPCR experiments were performed in triplicates (B).

D-E The clonogenicity of ML-2 overexpressing GFP, FST317, and FST344 in vitro for 14 days. The CFU experiments were performed in triplicates (E).

F-H The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 was quantified by bioluminescence imaging (F and G), and the survival of ML-2-engrafted NSG mice in vivo was recorded (H). Survival curve in panel H was analyzed by Log-rank test. *P<0.05, **P<0.01.

I-J RNA-seq and RT-qPCR validation of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells. RT-qPCR experiments were performed in triplicates (J).

K-L Overall survival analysis of patients from TCGA-AML based on the differential expression of IL2RA and CCL5.

M Activation of MAPK/ERK pathway in ML-2 by FST344 overexpression were validated by Western Blotting. Scale bar = 1 mm.

Data information: In (B, C, E, G, and J), data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.
Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia (2020)
Figure 5

Fig. 5 FST knockdown reduced AML cell growth in vitro and in vivo.

A FST knockdown in MOLM-13 by shRNA effectively reduced FST expression.

B-E The morphology (B), apoptosis (C), and clonogenicity of MOLM-13 (D and E) were measured after FST knockdown in vitro. Scale bar = 10 μm. The apoptosis assays (C) were performed in triplicates.

F-G. The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.

H The effect of FST knockdown on the survival of NSG mice-engrafted with MOLM-13. scr: scrambled sequence control (7 mice); sh: short hairpin RNA (8 mice for sh1 and sh2 respectively). The survival curve was analyzed by Log-rank test. **P<0.01.

Data information: In (C, E, and G), data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).
Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia (2020)
Figure 6

Fig. 6 FST targeting by CRISPR/Cas9 or antisense oligo significantly reduced leukemia cell growth in vitro and in vivo.

A-B The morphology and clonogenicity of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm.

C-D The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.

E The effect of FST knockout on the survival of NSG mice-engrafted with MOLM-13 cells. Cas 9: Cas 9 only (10 mice); sgRNA#3: Cas 9 + sgRNA#3 (10 mice); sgRNA#4: Cas 9 + sgRNA#4 (8 mice).

F The knockdown efficiency of different FST-specific antisense oligoes (ASOs) in MOLM-13 cells was detected by RT-qPCR after three days treatment in vitro. The knockdown and RT-qPCR experiments were performed in triplicates.

G MOLM-13 cell growth was measured after 3 days treatment of FST-ASO in vitro. The ASO treatment experiments were performed in triplicates.

H Intraperitoneal injection of FST-ASO (10mg/kg weekly, 6 mice) significantly prolonged the survival of MOLM-13-engrafted NSG mice. The random sequence was used for negative control (Neg-ASO, 6 mice).

Data information: In (B, D, F, and G), data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). In (E and H), survival curves were analyzed by Log-rank test. *P<0.05.
Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia (2020)
Figure 7

Fig. 7 Serum FST correlated with leukemia progression and therapeutic response in mouse and human FLT3/ITD AML.

A-D Flt3/ITD knock-in mouse were genotyped (A). The spleen weight (B and C, 4 mice each) and serum Fst level (D) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). In (C and D), data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).

E-H The engraftment of MOLM-13 in NSG mice was confirmed at week 1 post injection (E-F). Serum FST level was measured in MOLM-13-engrafted NSG mice at pre-injection and week 2 post injection (G, PBS group, 3 mice; MOLM-13 group, 4 mice). After FST knockdown, serum FST level was also measured in MOLM-13-engrafted NSG mice at week 2 post injection (H, 3 mice for each group).

I-K Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Human primary AML cells (FLT3/ITD-positive, leukemia blasts > 80%, 10 x 106, n = 7) were injected via tail vein into irradiated NSG mice at 6-8 weeks old (I). Human leukemic engraftments were confirmed by flow cytometry of human CD45 and mouse CD45.1 cells in recipient mouse BM aspiration (J). Serum FST from pre-injection and post-engraftment mouse were measured (K, one mouse for each primary AML sample).

L Correlation between serum FST levels and leukemia blast percentage from FLT3/ITD-mutated AML at diagnosis. Correlation analysis (Pearson correlation coefficient) was performed by Graphpad Prism 6.

M Serum FST decreased in CR and increased after relapse in 4 AML patients receiving Quizartinib monotherapy.

N Serum FST continued to rise during disease progression from a patient who did not respond to Quizartinib. Patients in M and N were recruited in the QUANTUM-R and patient accrual has been completed.

Data information: In (G and K), data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test). In (H, M and N), data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).
Ontogeny and function of the circadian clock in intestinal organoids (2021)
Figure 2

Fig. 2. kcHIEs and bHIEs show circadian rhythms with appropriate phase relationships between canonical clock genes.

(A-C) mRNA expression of Bmal1 (black), Rev-erbα (grey) and Per2 (red) in HIOs (A), kcHIEs (B) and bHIEs (C).

(D) Average expression of Bmal1 (black), Rev-erbα (grey) and Per2 (red) in HIO, kcHIE, and bHIE. (E) Amplitude of Bmal1 (black), Rev-erbα (grey) and Per2 (red). AU stands for arbitrary units.

Data information: The data in panels (A-C) are shown as mean (bold line) ± S.D. with replicates displayed as dashed lines. (D-E) Panels show mean ± S.D. of n=3 biological replicates. *p<0.05, **p<0.01, ***p<0.001 (Tukey's multiple comparison)
Ontogeny and function of the circadian clock in intestinal organoids (2021)
Figure 3

Fig. 3. Mouse enteroids show circadian phase-dependent necrotic cell death response to Clostridium difficile toxin B.

(A) Representative images of necrotic cell death (SYTOX orange - red fluorescence) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in control Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm.

(B) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f/f-EsrCRE enteroids without tamoxifen treatment.

(C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm.

(D) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids.

Data information: Data represented as mean ± S.D. of n=4 biological replicates (i.e. enteroids derived from different mice). All of the data were normalized to time and phase matched vehicle controls set to 1. **p<0.01 (Two-way ANOVA)
Ontogeny and function of the circadian clock in intestinal organoids (2021)
Figure 6

Fig. 6. Disruption of Rac1 abolishes circadian time-dependent necrotic cell death responses.

(A) Validation of anti-phasic Rac1 expression in mouse (mRac1, red) and human enteroids (hRac1, black) by qRT-PCR.

(B) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1-KD bHIEs.

(C-D) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f/f;CAGGCre-ERTM without tamoxifen (C) or Rac1 f/f;CAGGCre-ERTM with tamoxifen-treated (D) enteroids.

Data information: Data represented as mean ± S.D. of n=5 technical replicates. All of the data were normalized to time and phase matched vehicle controls set to 1. *p<0.05 (Two-way ANOVA)

Expanded View Figure Legends
Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease. (2005)
Figure 1.

Increased macroautophagy in PS1/APP mice and human brains. (A–D) EM images of cortical neuropil show an absence of AVs and normal neurite profile in 9-mo-old NTg mouse brains (A, arrowheads outline normal neurites) and a marked accumulation of AVs within enlarged or dystrophic neurites in PS1/APP mice (B, arrowheads outline dystrophic neurite profiles in C) and biopsied brain material from an AD patient (B, inset). At higher magnification (C), AVs include autophagosomes (arrows) and multilamellar bodies (arrowhead). In normal dendrites of PS1/APP mice, multiple AVs are frequently seen (D, arrows). (E and F) LC3 quantification analyzed from immunoblots of LC3-I and LC3-II (top) in prefrontal cortical homogenates from cases of nonaffected (Cont), early stage (preclinical) AD (AD-ES), and moderate AD (AD-MS; E), and from brains of 18–22-mo-old PS1/APP (PA) mice (n = 3; F) compared with nontransgenic (NTg) controls (n = 3; *, P < 0.01). Error bars represent SEM. (G–L) LC3 immunofluorescence in 9-mo-old PS1/APP mice can be seen mainly as puncta in dystrophic dendrites of the cortex (G, arrows) and along adjacent dendrites. LC3 (H, arrows) is strong in dystrophic neurites in the periphery (asterisks) of a thioflavin S–labeled plaque core (H, inset) but is less so in neurites closest (H, arrowheads) to the Aβ deposit. LC3 is diffuse and uniform in neurons of NTg mice (I and J) but is predominantly vesicular and distributed more to the dendrites (arrows) than the cell soma (arrowheads) in 9-mo-old PS1/APP cortex (K and L).
Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease. (2005)
Figure 2.

Identification of macroautophagy in the hippocampus of predepositing PS1/APP mice. Ultrastructural inspection of brain tissue from PS1/APP mice (A–D) shows that AVs (A and B, arrows) are five times more frequent in the dendrites of 8-wk-old PS1/APP than in those of age-matched NTg mice. The frequency of AVs per EM field (C) and mean number of AVs per EM field (D) within the hippocampal molecular layer (n = 3) are shown. LC3 immunoblot and analysis (E) and immunofluorescent labeling (F–K) of the hippocampal dendrites (brackets) in 8–9-wk-old PS1/APP and NTg mice show LC3-II elevation (P < 0.05) in 8-wk-old PS1/APP compared with NTg mice (E). (D) *, P < 0.001. (E) *, P < 0.05. Error bars represent SEM. LC3 immunoreactivity in pyramidal cell dendrites is increased in 9-mo-old (F–H) and 9-wk-old (I–K) PS1/APP mice and frequently exhibits a punctate labeling pattern, which is more evident at 9 mo than at 9 wk (H and K, arrows) and is uncommon in NTg mice (F and I). Bars (F, G, I, and J), 20 μm; (H and K), 10 μm.
Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease. (2005)
Figure 3.

Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1/APP mice. Cingulate cortex from 9-mo-old PS1/APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques (A). At higher magnification, anti-PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona (B). PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1/APP animals (C and D) and human brain (E and F) by IEM. Arrowheads identify tubulovesicular membrane labeling. PS1 (C–F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM). IEM followed by silver stain enhancement for PS1 was performed on a human brain that was diagnosed for AD (F).