EMBO/soda-nel huggingface dataset:
NPM1 ablation induces HSC aging and inflammation to develop MDS exacerbated by p53 loss (2022)
Figure 2

Figure 2. Npm1 loss causes an HSC aging phenotype.

(A) Npm1 expression levels (relative quantitation, RQ) in BM-MNCs of young (3-6 months old) and old (12-18 months old) wild type (WT) mice (n = 3 mice per group).

(B) Representative flow cytometry analysis of phenotypic HSCs [c-Kit+Sca-1+Lin- (KSL) CD135-CD48-CD150+] in the bone marrow of 4-month-old Ctrl (Npm1F/FVav1Cre-), 4-months old Npm1 cKO (Npm1F/FVav1Cre+) and 2-year Old WT mice (left). Mean percentages of the indicated fraction are shown (right, n = 4-9 mice per group).

(C) Representative distribution of Tubulin and CDC42 in Ctrl, 4-months old Npm1 cKO and 2-years old WT HSCs determined by IF. Scale bars, 5 μm.

(D) Quantification of percentage of cytoskeletal polarized HSCs (n = 79 cells in Ctrl, n = 120 cells in cKO, n = 82 cells in Old, from 4 Ctrl and Old mice and 3 cKO mice).

(E) In vitro colony replating ability of HSCs from Ctrl, 4-months old Npm1 cKO and 2-years old WT mice determined by limiting-dilution assay. Overall test for differences in stem cell frequencies between any of the groups calculated by Chisq test, * p<0.05, ***p<0.001, **** p <0.0001. The slope of the line is the log-active cell fraction. The dashed lines give the 95% confidence interval.

(F ) HSCs of Ctrl, 4-months old Npm1 cKO and 2-years old WT mice were sorted and stained for TOM20. Representative images (top) and quantification of mitochondrial volume and number (bottom) in single cells (n = 58 cells in Ctrl, n = 70 cells in cKO and n = 23 cells in Old, from 3 mice per group), identified by volume rendering of TOM20. Scale bars, 2 μm.

(G) Representative flow cytometry histogram (left) and quantification of TMRM median intensity (right, n = 3 mice per group) in HSCs from the indicated genotyped mice.

(H) Representative flow cytometry histogram (left) and quantification of mitoSox median intensity (right, n = 4 mice per group) in HSC fraction.

(I) NPM1 expression levels (relative quantitation, RQ) in MDS-L cells transfected with siNPM1 or control (siCTRL) (n = 3 mice per group).

(J) Representative images (top) and quantification of mitochondrial volume and number (bottom), identified by volume rendering of TOM20, in MDS-L cells transfected with siNPM1 or control (siCTRL) (n = 58 cells per group, from 3 transfected MDS-L cultures). Scale bars, 2 μm.

(K) Representative flow cytometry histogram (left) and quantification (right, n = 3 transfected MDS-L cultures) of TMRM median intensity in MDS-L cells transfected with siNPM1 or control.

(L) Representative flow cytometry histogram (left) and quantification (right, n = 3 transfected MDS-L cultures) of mitoSox median intensity in MDS-L cells transfected with siNPM1 or control.

Data information: All data are shown as mean ± SD. Panels A, and G-L, Unpaired two-tailed t-test: panels B,D and F, ANOVA test, compare to Ctrl, * p<0.05, ** p<0.01, *** p< 0.001, **** p< 0.0001.
NPM1 ablation induces HSC aging and inflammation to develop MDS exacerbated by p53 loss (2022)
Figure 3

Figure 3. Inflammation and myeloid expansion in Npm1 cKO mice.

(A) Levels of Nlrp3 expression (relative quantitation, RQ) in Ctrl (Npm1F/FVav1Cre-) and cKO (Npm1F/FVav1Cre+) mice (4 months old, n = 4 mice per group).

(B) Quantification of IL-1β, Il-6, IFNγ, and IL-27 inflammatory cytokines in serum from Ctrl and cKO mice (4 months old, n = 8-10 mice per group).

(C) Representative flor plot (top) and quantification (bottom) of myeloid fractions (CD11b+Gr1+) and myeloid fraction subpopulation related to malignancy (CD11b+Gr1mid) in the bone marrow of Ctrl and cKO mice (n= 5-8 mice per group).

(D) Representative flow plot (top) and quantification (bottom) of myeloid progenitors: common myeloid progenitor (CMP), Lin-Sca1-c-Kit+CD16/32lowCD34+; granulocyte-macrophage progenitor (GMP), Lin-Sca1-c-kit+CD16/32highCD34+; megakaryocyte-erythroid progenitor (MEP), Lin-Sca-1-c-Kit+CD16/32low/negCD34low/neg in the bone marrow of Ctrl and cKO mice (n = 4 mice per group).

(E) In vitro colony-forming capacity of progenitor cells. Sorted KSL cells were cultured in semisolid medium. Counting and classification of colonies were performed in independent littermate pairs (n = 3 mice per group). GEMM, Colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte; GM, Colony-forming unit-granulocyte and macrophage; M, Colony-forming unit-macrophage; E, Burst-forming unit-erythroid.

(F) Representative peripheral blood (PB) smears. Insets show the differentiated granulocytes (a, b) in control mice, and anisocytosis (c), dysplastic myeloid cells (d-g) in Npm1 cKO mice (Npm1F/FVav1Cre+). Npm1 cKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead; schistocyte, blue-outlined arrowhead; tear drop, Howell-Jolly bodies, black arrowhead; black-outlined arrowhead), dysplastic monocytes (black arrow), and dysplastic neutrophils (d-f). Scale bars, 10 μm.

(G) Representative pictures (right) and weights (left, n = 6-8 mice per group) of spleens of 4-month-old Ctrl and Npm1 cKO mice.

(H) c-Kit positivity in myeloid fraction in the spleen (n = 5 mice per group).

Data information: All data are shown as mean ± SD. Unpaired two-tailed t-test, ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001.
NPM1 ablation induces HSC aging and inflammation to develop MDS exacerbated by p53 loss (2022)
Figure 4

Figure 4. Npm1 p53 double KO mice develop a myeloid dysplastic syndrome-like disease.

(A) Survival curves of Ctrl (Npm1F/FVav1Cre-), cKO (Npm1F/FVav1Cre+), p53 KO (Npm1F/Fp53-/-Vav1Cre-) and DKO (Npm1F/Fp53-/-Vav1Cre+) mice. Overall survival of the indicated mice was examined by plotting Kaplan-Meier survival curves. Log-rank test was used to generate p values, *** p<0.001.

(B) Amount of total bone marrow mononuclear cells (BM-MNCs) in Ctrl, 4-months old Npm1 cKO mice and moribund Npm1 cKO (cKO†) (n = 3-5 mice per group).

(C) Npm1 and p53 protein levels in KSL cells of Ctrl (Npm1F/FVav1Cre-), and cKO (Npm1F/FVav1Cre+) mice.

(D) p21 expression levels in BM-MNCs of Ctrl and Npm1 cKO mice (4 months old, n = 4 mice per group).

(E) White blood cell (WBC) count (left) and myeloid fraction (CD11b+Gr-1+, right) in PB (n = 3-8 mice per group).

(F) Representative PB smears of DKO mice. Insets show anisocytosis (a), poikilocytosis (b) and dysplastic myeloid cell (c). DKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead), Howell-Jolly bodies, (black arrowhead in insert a), dysplastic monocytes (black arrow, and i-v), dysplastic neutrophils (vi), a pseudo-Pelger-Huet anomaly (ii), and dysplastic platelets (giant platelet, iv and vii). Scale bars, 10μm.

(G) Pictures of spleens (top) and spleen weight quantification (bottom, n = 3-5).

(H) Percentages of myeloid fraction (CD11b+Gr1+), c-Kit positivity, and B220 positivity in spleen samples (n = 3-5).

(I) Representative H&E staining of sections of the spleen of 2-month-old DKO mice with myeloid disorders. Arrowheads (right) indicate the infiltrating myeloid blasts. Scale bars, 250 μm (left) and 10μm (right), respectively.

(J) Myeloid subtype (CD11b+Gr1mid), and c-Kit positivity inside myeloid fraction in BM (n = 3-5).

(K) c-Kit+Sca1-Lin- (KL Sca1-) and granulocyte-macrophage progenitor (GMP) in the BM (n = 3-4).

Data information: All data are shown as mean ± SD. Panel D, unpaired two-tailed t-test: Panels B,E,G,H,J,K, ANOVA test, compare to Ctrl, ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p <0.0001.
Tau acetylates and stabilizes β-catenin thereby promoting cell survival (2019)
Figure 2

Fig.2 Tau directly acetylates β-catenin in concentration-, pH-, time- and the acetyltransferase activity domain-dependent manner

(A) Tau directly acetylates β-cat in test-tube. Both Tau40 and β-cat were purified from E. coli using Ni-NTA resin (n = 3 from three independent experiments).

(B and C) Mutation of Tau40 at acetyltransferase activity region abolishes its acetylation activity on β-cat. eGFP-Tau40, eGFP-Tau-2CA and eGFP-Tau-k18(-) were purified from HEK293 cells (using anti-GFP beads) and GST-β-cat was from E. coli (B), or both Tau40 and β-cat were from E. coli (C) (n = 3 from three independent experiments).

(D and E) Mutation of β-cat K49 abolishes its acetylation by Tau. Both myc-Tau40 and eGFP-β-cat were purified from HEK293 cells (using anti-GFP or anti-Myc beads) (D), or both from E. coli (E) (n = 3 from three independent experiments).

(F-J) Kinetics of β-cat acetylation by Tau. Tau acetylates β-cat in concentration- (F), time- (G), pH- (H), and its acetyltransferase activity domain-dependent manner. The affinity (Km) of Tau to β-cat was measured by the initial rate of Tau as a function of the β-cat concentrations (I, J). Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).
Tau acetylates and stabilizes β-catenin thereby promoting cell survival (2019)
Figure 3

Fig. 3 β-catenin K49-acetylation inhibits its ubiquitylation-associated proteolysis and decreases its phosphorylation

(A and B) Expressing Tau inhibited β-cat ubiquitination in HEK293 cells, measured by immunoprecipitation using antibody against β-cat and Western blotting using anti- ubiquitination (anti-Ub) and anti-β-cat, respectively (n = 3 from three independent experiments).

(C and D) Overexpressing Tau inhibited β-cat ubiquitination in hippocampi of Tau transgenic mice, measured by immunoprecipitation using anti-Ub and Western blotting using anti-β-cat, respectively (n = 3 from three independent experiments).

(E and F) Expressing Tau-k18(-) attenuated Tau-induced β-cat elevation. HEK293 cells were transfected with Vec, or Tau-40, or Tau-k18(-) for 24 h, and then were treated with Chx for 12 h or 24 h, followed by Western blotting. β-cat protein level was normalized to β-actin (n=4 biological replicates each group).

(G and H) K49 acetylation of β-cat inhibited its ubiquitination. HEK293 cells were co-transfected with HA-ubiquitin and GFP-β-cat or pseudo-acetylated K49-β-catenin (K49Q), and then β-cat was immunoprecipitated by anti-GFP and blotted by anti-Ub and anti-β-cat (n=3 from three independent experiments).

(I and J) K49 acetylation of β-cat inhibited its degradation. HEK293 cells were transfected with β-cat WT or K49Q for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group).

(K and L) β-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group).

(M and N) K49 acetylation of β-cat inhibited its phosphorylation at N-terminal domain. HEK293 cells were transfected with GFP-vec, or eGFP-β-cat (WT) or K49Q for 48 h, then treated with wortmannin (WO, 1 μM) plus GF109203X (GFX, 1 μM) to activate GSK-3β for 1 h followed by Western blotting (n=3 biological replicates each group).

Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec (B), or vs Tau- (D), or vs Tau (F), or vs β-cat WT (H, J), or vs Tau+β-cat WT (L), or vs β-cat WT plus WO/GFX (N). Data in (B, D and H) were analyzed by Student's t test. Data in (F, J and L) were analyzed by Two-way repeated-measures ANOVA, and data in (N) was analyzed by one-way ANOVA.
Tau acetylates and stabilizes β-catenin thereby promoting cell survival (2019)
Figure 4

Fig. 4 β-catenin K49-acetylation mediates the anti-apoptotic effect of Tau

(A and B) Expressing Tau-k18(-) abolished its effect on β-cat K49-acetylation and c-cas-3 (cleaved caspase-3). N2a cells were transfected with Vec, or Tau40, or Tau-k18(-) for 48 h, and then treated with 0.5 μM staurosporine (STP, an apoptotic inducer) for 4 h, followed by Western blotting (n=3 biological replicates each group).

(C-F) Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with eGFP-Vec, or eGFP-Tau40, or eGFP-Tau-k18(-), or Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by co-staining with c-cas-3 (red) (D, E); N2a cells, transfected with myc-Vec, or myc-Tau40, or myc-Tau-k18(-), or myc-Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by flow cytometry using Annexin-FITC (C), or CCK8 assay (F). The apoptotic rate (E) was presented as the ratio of yellow (c-casp-3 and GFP co-stained) to green (the total GFP-expressing cells) staining. In panel D (middle row), the Tau expression (white arrowheads) was largely not co-localized with c-casp-3 (yellow arrowheads) in the same visual field (n=4~8 biological replicates each group; scale bar, 10 μm).

(G) Non-acetylation modifiable mutation of β-cat at K49 (K49R), not K19 (K19R), abolished the anti-apoptotic effect of Tau. N2a cells were co-transfected with Tau40 and β-cat WT, or K49R, or K19R for 48 h, and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group).

(H) Expressing β-cat K49Q most significantly increased cell viability. N2a cells were transfected with β-cat WT or K49R or K49Q or the empty Vec for 48 h, and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group).

(I) Overexpressing Ser199-phosphorylation mimic Tau (TauS199E) further augmented cell viability. N2a cells were transfected with empty vec, Tau40 or TauS199E for 48h and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group).

Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs vec (B), or vs Tau plus STP (C, E and F), or vs Vec plus STP (G, H and I); #, P<0.05, ##, P<0.01, ###, P<0.001 vs Vec plus STP (B), or vs β-cat + Vec plus STP (G), or vs β-cat plus STP (H), or vs Tau plus STP (I); $$$, P<0.001 vs Tau plus STP (B) or vs β-cat + Tau plus STP (G), or vs β-cat K49Q plus STP (H) ; &&&, P<0.001 vs β-cat K49R+ Tau plus STP (G). ♧♧♧, P<0.001 vs vec (F, G and H). Data were analyzed by one-way ANOVA.
Tau acetylates and stabilizes β-catenin thereby promoting cell survival (2019)
Figure 5

Fig. 5 β-catenin K49-acetylation promotes its nuclear translocation and increases the expression of survival signaling

(A) Co-expressing myc-Tau and eGFP-β-catenin increased nuclear translocation of β-catenin, while co-expressing myc-Tau and eGFP-β-cat K49R abolished the nuclear translocation of β-catenin in HEK293 cells. The nuclei were stained using DAPI (n=3 from three independent experiments, scale bar, 5 μm).

(B-D) Expressing β-cat K49Q increased its nuclear translocation. HEK293 cells were transfected with eGFP-β-cat WT or K49Q for 48 h, and then β-cat protein level in different fractions was measured by Western blotting (B, C) and immunofluorescent labeling using a laser scanning confocal microscope (D). The nuclei (blue) was stained by DAPI (n=3 from three independent experiments, scale bar, 2 μm).

(E) Expressing β-cat K49Q further increased its transcription activity measured by dual Topflash/Fopflash reporter luciferase assay after transient transfection of β-cat WT, or K49Q, or Vec in HEK293 cells for 48 h, and the luciferase activity was normalized by β-cat WT or K49Q protein level (n=7 biological replicates each group).

(F) Expressing Tau-k18(-) abolished Tau-induced upregulation of mRNA levels of survival genes (bcl2 and survivin) and dkk1 (downstream of Wnt signaling) in N2a cells measured by Q-PCR (n=6~9 biological replicates each group).

(G) Expressing Tau-k18(-) or Tau-2CA abolished Tau-induced Wnt/β-catenin transcriptional activation measured by dual reporter luciferase activity assay in HEK293 cells (n=7 biological replicates each group).

(H) Expressing phosphor-mimic TauS199E (TauS199E) further increased the transcription activity of Wnt/TCF/LEF1 by Tau in HEK293 cells (n=7 biological replicates each group).

(I-L) The increased mRNA levels of bcl2, survivin and dkk1 in the AD brains compared with the age-/sex-matched controls detected by Q-PCR (n=7 each group), and the positive correlations with the increased K49-acetylated β-catenin (see Fig. 1I, J) analyzed by Pearson analysis.

Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs β-cat WT (C), or vs Vec (E and H), or vs Tau (F and G), or vs Ctrl (I); ##, P<0.01, ###, P<0.001 vs β-cat WT (E), or vs Tau (H). Data were analyzed by Student's t test (C and I), one-way ANOVA (E, F, G and H).
UNC13D inhibits STING signaling by attenuating its oligomerization on the endoplasmic reticulum (2022)
Figure 1

Figure 1. UNC13D negatively regulates cytosolic DNA-triggered innate immune signaling.

A - L HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (A and B), UNC13D stable knock-down (KD) (C, E, G, I and K) or knock out (KO) ( D, F, H, J and L) HeLa cells, and their control cells were infected with VACV (A, C and D), HSV (E and F), or SeV (K and L), or they were transfected with plasmid DNA (pDNA) (1 μg/mL) (B, G-J) for the indicated time, before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -p-P65, -p-IκB-α, -actin or -tubulin antibody.

M - Q UNC13D stable knock-down (KD) HeLa cells and their control cells were transfected with plasmid DNA (1 μg/mL) (M-P) or infected with SeV (Q) for the indicated time. UNC13D, IFN-β and IL6 mRNA levels were measured by qPCR (M-O). The secretion of IFN-β was measured by bioassay (P and Q). n = 3 technical replicates.

R, S UNC13D knock-out (KO) HeLa cells and their control cells were infected with GFP-HSV for 18 h or 24 h. GFP expression was visualized (left) and the green fluorescence intensity was determined by ImageJ. Scale bar: 100 μm. n = 3 biological replicates.

Data information: Statistically significant differences were determined using ANOVA. Data are shown as the mean ± s.d. ns, not significant, p > 0.05; * p <0.05; ***p < 0.001.
UNC13D inhibits STING signaling by attenuating its oligomerization on the endoplasmic reticulum (2022)
Figure 2

Figure 2. UNC13D inhibits STING signaling

A HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (IB) with anti-HA and -Flag antibody.

B - E HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (B), UNC13D stable knock-down (KD) (D) or knock-out (KO) (C and E) HeLa cells, and their control cells were stimulated with cGAMP (100 nM) for 30 min (B and C) or for the indicated time (D and E) before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -GAPDH, -actin or -tubulin antibody.

F- H HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h (F and G). UNC13D knock-out (KO) and control HeLa cells were stimulated with cGAMP (100 nM) for 30 min (H). Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies (F and G), IgG and anti-STING antibodies (H), followed by immunoblotting (IB) with anti-HA, -Flag antibody or the indicated antibodies.

I, J UNC13D knock-out (KO) HeLa cells and HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. Confocal microscopy was used to determine the localization of endogenous STING (red), ERGIC marker ERGIC-53 (green) and nuclei (blue). Scale bars: 15 μm (I). The co-localization of STING and ERGIC was analyzed using ImageJ (J). n = 5 biological replicates.

K, L UNC13D stable knock-down (KD) HeLa cells and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. The aggregation of endogenous STING (red) was examined via confocal microscopy (K). Scale bar: 25 μm. The red fluorescence intensity was determined by ImageJ (L). n = 3 biological replicates.

Data information: Statistically significant differences were determined using ANOVA. Data are shown as the mean ± s.d. ns, not significant, p > 0.05; * p <0.05; ***p < 0.001.
UNC13D inhibits STING signaling by attenuating its oligomerization on the endoplasmic reticulum (2022)
Figure 5

Figure 5. STING inhibitor H-151 mitigates the overexpression of proinflammatory factors induced by UNC13D dysfunction

A qPCR analysis of IFN-β, IL6, TNFα and CXCL10 mRNA levels in UNC13D stable knock-down (KD) HeLa cells without stimulation. n = 3 technical replicates.

B, C UNC13D knock-out HeLa cells (UNC13D KO), UNC13D knock-out HeLa cells stably expressing FLAG-tagged wild-type UNC13D (UNC13D RE), UNC13D knock-out HeLa cells with STING knock-out (STING KO), UNC13D knock-out HeLa cells with cGAS stable knock-down (cGAS KD), and UNC13D knock-out HeLa cells stably expressing FHL3 mutation (FHL3 mut) were transfected with plasmid DNA (C) for 12 h, or not transfected (B). IFN-β, IL6 and CXCL10 mRNA levels were measured by qPCR. n = 3 technical replicates.

D UNC13D knock-out (KO) HeLa cells, UNC13D knock-out HeLa cells stably expressing FLAG-tagged wild-type UNC13D (UNC13D RE) and FHL3 mutation (FHL3 mut), and control cells were stimulated with cGAMP (100 nM) (or left unstimulated) for 30 min. Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-UNC13D, -Flag, -p-IRF3, -STING or -tubulin antibody.

E, F UNC13D knock-out (KO) cells, UNC13D knock-out HeLa cells stably expressing UNC13D FHL3 mutants (FHL3 mut), and their control cells were pretreated with H-151 (2 μM) or untreated for 2 h, followed by transfection with (F) plasmid DNA for 12 h, or not transfected (E). IFN-β, CXCL10 and IL6 mRNA levels were measured by qPCR. n = 3 technical replicates.

Data information: Statistically significant differences were determined using Student's t-test (A) and ANOVA (B, C, E and F). Data are shown as the mean ± s.d. ns, not significant, p > 0.05; * p <0.05; ** p < 0.01; ***p < 0.001.