EMBO/soda-nel huggingface dataset:
Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice (2021)
Figure 5

Fig. 5: scRNA-seq analysis reveals that a lack of ATMs leads to major cell perturbations in the eWAT CD45- fraction.

A. Schematic representation of the scRNA-seq analysis used on a 1:1 mixture of CD45- and CD45+ eWAT cells (excluding F4/80hi resident-macrophages and Ly6C+ monocytes) obtained from lean/obese (HFD 16 weeks) WT and CD169-DTR mice (7 days DT injected, n = 5-8 mice per group). B. tSNE projection of 18,798 pooled cells (5032 cells from ND WT mice, 3494 cells from ND CD169-DTR mice, 5096 cells from HFD (16 weeks) WT mice, and 5176 cells from HFD (16 weeks) CD169-DTR mice) isolated from eWAT of lean and obese WT/CD169-DTR mice. The projection shows 23 distinct clusters of CD45- and CD45+ cell populations identified via unsupervised clustering. Six specific CD45- cell clusters were identified, including adipocyte stem cells (ASC1 and ASC2), stromal cells, vascular endothelial cells (VEC1 and VEC2), and mesothelial-like cells (MLCs). Seventeen different CD45+ cell clusters were identified, including B and T cells, NK cells, and several myeloid subpopulations. NK: natural killer; MAC: macrophage; Mono: monocyte; DC: dendritic cell; NKT: natural killer T. C. Violin plots depicting the expression of representative marker genes for each CD45- cell cluster: ASC1 (Gsn, Pdgfrb, Eln) (red), ASC2 (Pi16, Pdgfra, Cebpd) (yellow), stromal cells (Col1a1, Col1a2, Myl9) (green), VEC1/VEC2 (Pecam-1, Cldn5, Aqp1) (purple and blue), and mesothelial-like cells (MLCs) (Upk3b, Msln, Acta2) (orange). D. Four tSNE plots of 5032, 3494, 5096, and 5176 cells obtained from lean/obese WT and CD169-DTR mice. Orange and blue shaded areas depict the CD45- and CD45+ cell clusters, respectively. E. Hierarchical heatmap showing the percentage (log2) of all 23 cell clusters identified in lean/obese (HFD 16 weeks) WT and CD169-DTR mice.
Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice (2021)
Figure 6

Fig. 6: CD169+ ATMs are closely associated with the eWAT vasculature.

A. Representative 3D fluorescence imaging of lean and obese (HFD 16 weeks) eWAT samples stained with anti-MHCII (green in lean samples), anti-CD11c (green in obese samples), anti-CD31 (blue), anti-CD169 (pink), and DAPI (white). The two images at the bottom show the images merged for the lean and obese conditions without DAPI. Scale bar: 100 μm (lean) and 70 μm (obese). B. Higher magnification of specific eWAT areas. Scale bar: 30 μm (lean) and 50 μm (obese). C-D (left panels) Distance of different ATM subsets to blood vessels. Distances (in μm) were calculated using the Imaris software, as described in the Materials and Methods section. The box and whisker plots show the median value and 10-90 percentiles of distance values. The right panels show the distribution of ATM subsets to blood vessels within a certain distance (≤1 μm, 1-10 μm, ≥10 μm). C: CD169+MHCIIlow and CD169+MHChi ATMs in lean eWAT. D: CD169+CD11c- and CD169+CD11c+ ATMs in obese (HFD 16 weeks) eWAT. Four mice from two independent experiments were analyzed (n = 4-5; mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, ****P < 0.0001, bars with no asterisks showed no significant difference. E. Model construction based on 3D reconstructed images showing the relative distance of CD11c+ ATMs from traced large blood vessels. Blood vessels larger than 10µm diameter in the image were marked out on the image by tracing (white filaments). CD11c+ ATMs are close (< 10µm) to the traced large blood vessels (red ellipsoids) or far (>10 µm) (grey ellipsoids) from the traced blood vessel. Blood vessels are shown in green. Scale bar: 100 μm. The graph to the right shows the percentage of CD11chi ATMs showing a proximity of ≤10 or >10 μm to blood vessels quantified from 3 biological replicates from 1 experiment, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.
Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice (2021)
Figure 7

Fig. 7: Vascular integrity is impaired in eWAT that lacks ATMs.

A. Representative 3D fluorescence imaging of obese (HFD 16 weeks) eWAT stained with BODIPY (green) and anti-CD31 (pink) obtained from WT and CD169-DTR mice treated with DT over 7 days. Scale bar, 100 μm. B. The lack of a CD31+ fraction after cell isolation from eWAT obtained from obese (HFD 16 weeks) CD169-DTR mice following ablation of ATMs for 7 consecutive days. The flow cytometry analysis (on the left) shows the staining profile of CD45 (x-axis) and CD31 (y-axis) and the chart (on the right) shows the percentage of CD31+ CD45- fractions in the eWAT of control and CD169-DTR mice (both n = 4), data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ****P < 0.0001. C. Violin and feature plots of single cells in the VEC1 and VEC2 clusters showing expression of typical marker genes for capillaries or arteries (Rgcc, Car4, Acq7) and veins (Vwf, Vcam1, Acta2) in WT obese (HFD 16 weeks) eWAT. D,E. Vascular permeability is increased in eWAT obtained from obese (HFD 16 weeks) CD169-DTR mice. D. Two representative images of eWAT dissected from Evans blue-injected obese (HFD 16 weeks) WT and CD169-DTR mice treated for 7 days with DT. Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm (the absorption values were normalized to the tissue weight in grams). E. A chart showing the normalized mean optical density ± SD (n = 5-7 mice per group). Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001. F-H. Mmp12 (H) and Vegfα (F) are highly expressed in MHCIIhi and CD11c+ ATMs purified from obese (HFD 16 weeks) eWAT. qPCR analysis showed the relative expression in all three ATM subsets (n=3), data was expressed as mean ± SD . Statistical significance between the three ATM subpopulations was determined using one-way ANOVA test. *P < 0.05, **P < 0.01. G. qPCR analysis showing the relative expression of Vegfα in obese (HFD 16 weeks) eWAT of WT (n=3) or CD169-DTR mice (n=5) treated for 7 days with DT, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ****P < 0.0001.
Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice (2021)
Figure 8

Fig. 8: Absence of ATMs exacerbates eWAT fibrosis during obesity.

A. Elastin staining showing more pronounced ECM accumulation in eWAT of CD169-DTR (HFD 16 weeks) when compared with ECM accumulation in the WT obese (HFD 16 weeks) control (DAPI, blue; elastin, red; CD31, green). The fourth image in each row shows the three channels merged. Scale bar: 50 μm. B. Picrosirius red-staining depicts excessive collagen deposits in obese (HFD 16 weeks) eWAT of CD169-DTR mice when compared with collagen deposits in WT controls. Scale bar: 250 μm. Upper panels: brightfield microscopy; lower panels: fluorescence microscopy. C. qPCR analysis showing the relative expression of ECM marker genes in obese (HFD 16 weeks) eWAT of WT (blue bars) and CD169-DTR (red bars) mice (DT treatment for 7 days). n = 4-6 mice per group, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. D-E. Common signalling pathways of single cells from clusters of ASC1 (D) and stromal cells (E) showed increased ECM remodeling pathways in HFD (16 weeks) CD169-DTR mice when compared to WT mice. F. Violin plots of single cells showing upregulated ECM gene signatures in ASC1 (top, Fn1, Mfap4, Bgn, and Timp1) and stromal cell clusters (bottom, Mfap2, Mfap4, Ltbp2, and Gadd45b) in eWAT of obese (HFD 16 weeks) CD169-DTR mice when compared with the expression of these genes in WT mice. G. Violin plots showing the pro-inflammatory cytokine profile (Il-6, Tnf-α, and Il-1β) of all 23 cell clusters obtained from obese (HFD 16 weeks) WT and CD169-DTR eWAT. Cluster 0: ASC1; Cluster 1: Stromal cells; Cluster 2: VEC1; Cluster 3: VEC2; Cluster 4: NK1; Cluster 5: MAC/Mono1; Cluster 6: ASC2; Cluster 7: B; Cluster 8: cDC1; Cluster 9: mono-derived cDC; Cluster 10: Th2/ILC2/Treg; Cluster 11: MAC/Mono3; Cluster 12: Th17; Cluster 13: VEC2; Cluster 14: CD8; Cluster 15: MLCs; Cluster 16: CD11b+ DC; Cluster 17: Neutrophils; Cluster 18: cDC2; Cluster 19: cDC; Cluster 20: NKT; Cluster 21: NK2; Cluster 22: Mast cells/Basophils. ASC: adipocyte stem cell; VEC: vascular endothelial cell; MLC: mesothelial-like cell; NK: natural killer; MAC: macrophage; Mono: monocyte; DC: dendritic cell; NKT: natural killer T cells. H. qPCR analysis showing the relative expression of Il-6, TnF-α, and Il-1β in obese (HFD 16 weeks) eWAT collected from WT, CD169-DTR, CCR2-/-, and CCR2-/- CD169-DTR mice (n=3), data was expressed as mean ± SD. Statistical significance between the four experimental groups was determined using a Bonferroni two-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Expanded View Figure legends
NDST3 Deacetylates α-tubulin and Suppresses V-ATPase Assembly and Lysosomal Acidification (2021)
Figure 1

Figure 1: CRISPR/Cas9 knockout library screening shows NDST3 as a regulator of bafilomycin toxicity.

(A) A schematic diagram of the CRISPR genetic screen.

(B) Generation of a stable NDST3 KO RPE1 cell line.

(C) Generation of NDST3 polyclonal antibodies targeting a region of NDST3 unique in the NDST family.

(D) Immunoblot analysis of NDST3 in stable NDST3 KO RPE1 cells.

(E) Bafilomycin A1 (Baf A1) sensitivity assay for NDST3 KO cells. WT and NDST3 KO RPE1 cell lines were treated with 25 nM Baf A1 for 72 h, and cell survival was measured using calcein AM staining. The cell survival was calculated as the fluorescence intensity (percentage) of the treated cells relative to the untreated cells (n = 4 independent cultures, ****P < 0.0001).

(F) Detection of Flag-NDST3 and Flag-EGFP overexpressed in NDST3 KO cells by immunoblotting with anti-Flag antibodies.

(G) Baf A1 sensitivity assay for NDST3 KO cells after the NDST3 restoration. The NDST3 KO cells with stable expression of Flag-NDST3 or Flag-EGFP (control) were treated with 25 nM Baf A1 for 72 h, and cell survival was measured using calcein AM staining. The cell survival was calculated as the fluorescence intensity of the treated cells relative to the untreated cells (n = 4 independent cultures, ****P < 0.0001).

Data information: Error bars represent ± standard deviation. Scale bar, 200 μm.
NDST3 Deacetylates α-tubulin and Suppresses V-ATPase Assembly and Lysosomal Acidification (2021)
Figure 3

Figure 3: Loss of NDST3 promotes the assembly of the V-ATPase V1-V0 holoenzyme.

(A) To examine the levels of the V-ATPase V1-V0 holoenzyme in WT and NDST3 KO RPE1 cells, cytosolic and membrane fractions were analyzed by immunoblotting using antibodies against the V-ATPase subunits V1A (ATP6V1A), V1C1 (ATP6V1C1), and V0D (ATP6V0D). LAMP1 and vinculin were used as loading controls for membrane proteins and cytosolic proteins, respectively.

(B) Quantification of the levels of ATP6V0D relative to LAMP1 in the membrane fraction from (A) (n = 3 independent experiments, P = 0.7786).

(C, D) Ratios of ATP6V1A (C) and ATP6V1C1 (D) to ATP6V0D in the membrane fraction from (A) to represent the assembly of the V-ATPase V1-V0 holoenzyme (n = 3 independent experiments, ***P =0.0005, ****P < 0.0001).

Data information: Error bars represent ± standard deviation.
NDST3 Deacetylates α-tubulin and Suppresses V-ATPase Assembly and Lysosomal Acidification (2021)
Figure 5

Figure 5: NDST3 deacetylates α-tubulin in the cells and in vitro.

(A) Immunoblotting analysis demonstrated a large accumulation of α-tubulin acetylated at Lys40 (Ac-α-tubulin) in NDST3 KO RPE1 cells, while the total α-tubulin relative to GAPDH remained unchanged (n = 3 independent cultures, ***P = 0.0008).

(B) Immunoblotting analysis showed decreased levels of Ac-α-tubulin, but unchanged levels of total α-tubulin relative to GAPDH, in RPE1 cells over-expressing Flag-NDST3 compared to those in Flag-EGFP control cells (n = 3 independent cultures, *P = 0.0399).

(C) In vitro enzymatic assay quantitating the effect of NDST3 on α-tubulin acetylation. Flag-NDST3 was expressed in HEK 293 cells and immunoprecipitated by anti-Flag beads. Flag-EGFP was used as a control. The immunoprecipitated protein was incubated with purified tubulin heterodimers in vitro, with or without 1 mM NAD+, 400 nM TSA, or 5 mM nicotinamide (NAM) for 2 h at room temperature. The reaction products were examined by immunoblotting against Ac-α-tubulin or total α-tubulin (n = 3 independent experiments, *P = 0.0185, **P < 0.01, ****P < 0.0001).

(D) The same in vitro enzymatic assay as in (C), except that the purified tubulin was polymerized in the presence of 20 μM Taxol and 1 mM GTP (n = 3 independent experiments, **P < 0.01, ***P = 0.0001).

Data information: Error bars represent ± standard deviation. ns means non-significant.
NDST3 Deacetylates α-tubulin and Suppresses V-ATPase Assembly and Lysosomal Acidification (2021)
Figure 6

Figure 6: NDST3 is down-regulated in C9orf72-linked ALS patient cells and tissues.

(A, B) NDST3 protein levels relative to β-tubulin in B lymphocytes derived from healthy controls and C9orf72-linked ALS patients, as examined by immunoblotting (n = 3 biologically independent samples, **P = 0.0026).

(C-F) The protein levels of NDST3 relative to GAPDH (D) (**P = 0.0013), Ac-α-tubulin relative to total α-tubulin (E) (*P = 0.0379), and total α-tubulin relative to GAPDH (F) (P = 0.5700), as examined by immunoblotting, in spinal cord tissues from healthy controls and C9orf72-linked ALS patients (n = 4 biologically independent samples in the control group and n = 5 biologically independent samples in the ALS patient group).

(G-I) Immunoblotting assays showing down-regulation of NDST3 in C9orf72 shRNA (shC9orf72)-treated RPE1 cells, as compared to control shRNA (shCTRL)-treated cells. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (H) (n = 4 independent experiments, ***P = 0.0004) and (I) (n = 4 independent experiments, ***P = 0.0001), respectively.

(J-L) Immunoblotting assays showing up-regulation of NDST3 in RPE1 cells over-expressing Flag-C9orf72 compared to those expressing Flag-EGFP as control. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (K) (n = 3 independent experiments, **P = 0.0011) and (L) (n = 3 independent experiments, **P = 0.0033), respectively.

(M, N) Baf A1 sensitivity assay for WT, NDST3 KO, and Noco-treated NDST3 KO RPE1 cells expressing proline-arginine poly-dipeptide (PR82). 24 h after the transfection of PR82, WT and NDST3 KO cells were treated with vehicle or 10 μg/mL Noco overnight. The cell survival measured using calcein AM staining at 48 h post-transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells (n = 3 independent cultures, *P = 0.0369, ***P = 0.0007). Scale bar, 200 μm.

(O, P) Immunoblotting analysis of PR82 levels in WT, NDST3 KO, and Noco-treated NDST3 KO RPE1 cells from (M, N) after the drug treatment. Quantification of the levels of PR82 relative to β-tubulin is shown in (P) (n = 4 independent experiments, *P = 0.0141, **P = 0.0045).

Data information: Error bars represent ± standard deviation.
Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner (2019)
Figure 2

Figure 2. Phosphate availability determines Arabidopsis responses to GMVs

[A] Plants grown with 0.05 MS and GMVs (G 0.05) showed hyper-induction of IPS1 and PS2 gene expression in both shoots and roots, compared to plants grown with 0.05 MS alone (C 0.05). Values are means ± SE of three biological replicates.

[B] Root acid phosphatase activity as detected by the blue color from the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) treatment. Image contrast enhanced for improved visualization of the blue color in roots

[C], [D] Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-accumulation of anthocyanin (C) and hyper-induction of IPS1 gene expression (D) triggered by GMVs.

[E] Pi supplementation to the 0.05 MS medium partially restored GMV-induced plant vigor, as indicated by increases in total leaf area per plant.

[F] A low Pi level (LP), which was equal to 1/20 of that in the 0.5 MS medium, in plant growth medium blocked GMV-induced plant vigor.

[G] The Arabidopsis phr1phl1 mutant showed substantially decreased growth-promotion, compared to the wild type plants.

Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.
Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner (2019)
Figure 3

Figure 3. GMV-triggered plant hypersensitivity to Pi deficiency is mediated by diacetyl

[A] Images of Arabidopsis plants exposed to four individual GMV components, including diacetyl (DA), 2,3-butanediol (BTDL), 2-methyl-1-propanol (MP) and acetoin (ATN). The compounds were applied at dosages that, when the compounds totally evaporate from the agar-containing solid droplets, would yield in volatile concentrations of 9.7 µg (DA), 32.5 µg (BTDL), 7.9 µg (MP) and 28.5 µg (ATN) per mL free space in the petri dish, which resembled the 1 : 3.3 : 0.8 : 2.9 ratio among the four GMV components in natural GMVs22.

[B], [C] anthocyanin hyper accumulation (B) and PSR gene hyper induction (C) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components.

[D], [E] Compared to the wild type plants, phr1phl1 showed substantially decreased anthocyanin hyper accumulation (D) and PSR gene hyper induction (E) triggered by DA.

Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.
Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner (2019)
Figure 4

Figure 4. DA exacerbates plant sensitivity to Pi deficiency via activation of immunity

[A] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency.

[B] DA elevates SA accumulation levels in Pi-deficient Arabidopsis. Means ± SE of three biological replicates.

[C] Images of plants grown with different treatments using DA (9.7 µg per mL free space) or SA (100 µM in plant growth medium).

[D], [E] Exogenous application of SA mimics DA-induced anthocyanin accumulation (D) and IPS1 gene induction (E) patterns in Pi-deficient plants.

[F], [G] Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition, as shown by plant images (F) and quantification of anthocyanin accumulation levels (G).

Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.
Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner (2019)
Figure 5

Figure 5. DA enhances symbiotic colonization to Pi-sufficient plants via modulates immunity

[A] Clustering of DA-regulated immunity-related genes in Arabidopsis with sufficient Pi, as revealed by RNAseq analysis.

[B] DA suppresses plant ROS burst induced by flg22 or elf18. ROS accumulation was represented in relative luminescence units (RLU). Data bar indicate mean ± SE (n=24) readings for 60 min. Three independent experiments were performed and similar results were observed. **p < 0.01, student t-test.

[C] DA induces gene expression of AZI1 in Arabidopsis. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group. Bar indicates mean ± SE (n=3), two biological replicates were analyzed with similar results.

[D] flg22-induced MPK3/MPK6 phosphorylation in Arabidopsis is not decreased by DA. Kinase assays were performed at 5 min after flg22 treatment. Three independent experiments were performed and similar results were observed.

[E] Plant resistance to the pathogen Pst DC3000 is not reduced by DA. The severity of pathogen infection was measured as colony forming units (CFUs) per mg FW plants at 0 and 3 days after inoculation. Data bar indicate mean ± SE (n=8), two independent experiments were performed. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group.

[F] Root colonization of GB03 in wild type Arabidopsis (Col-0) and the mutants/transgenic lines npr1, NahG, and rbohd. *** p < 0.001compared with Col-0 without DA treatment, n= 9 biological replicates, student t-test.

[G] Bacteria chemotaxis with different concentrations of DA. Data point indicates mean± SE (n=6). Two independent experiments were performed and similar results were observed. *** p < 0.001, student t-test.
Overexpression of Atg5 in mice activates autophagy and extends lifespan (2013)
f1

(a) Kaplan–Meier survival curves of combined male and female WT and Atg5 Tg mice no. 25 (P0.001; n=65 for WT mice and n=70 for Atg5 Tg mice). (b,c) Lifespan extension in male Atg5 Tg mice no. 25 (P0.001; n=32 for WT mice and n=36 for Atg5 Tg mice no. 25) (b) and female Atg5 Tg mice no. 25 (P0.001; n=33 for WT mice and n=34 for Atg5 Tg mice no. 25) (c). (d–f) Kaplan–Meier survival curves of combined male and female WT and Atg5 Tg mice no. 43 (d), no. 47 (e) and no. 471 (f) (P0.001; n=25 for WT mice and n=30 for Atg5 Tg mice). P-values were calculated using the log-rank test. (g) Expression levels of Atg5 and Atg12-Atg5 conjugate, the conversion of LC3 I into LC3 II, and p62 level in the hearts of Atg5 Tg mice. Whole-tissue extracts were prepared from 3-month-old Atg5 Tg mice and WT littermates, and analysed by western blotting using the indicated antibodies. β-Actin served as a control. (h) Densitometric analysis of the signals on the blots shown in (g). The bars represent the mean±s.d. (n=3).