EMBO/soda-nel huggingface dataset:
TLR4-dependent shaping of the wound site by MSCs accelerates wound healing (2020)
Figure 3

Figure 3. LPS primed MSCs increase expression of neutrophil regulatory genes in vivo.

(a) The upper scheme depicts the experimental design. Representative microphotographs show that injection of LPS primed MSCs into wounds increased the expression of CXCL6, IL-8 and IL-1β. Graphs display numbers of double positive cells for h-β2M + CXCL6, h-β2M + IL-8 and h-β2M + IL-1β, respectively. MSCs injected wounds served as controls. Statistical analysis was performed using unpaired t-test, values are represented as mean ± SEM, six biological replicates. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Es, eschar on wound margin; wm, wound margin; scale bars: 50µm.

(b) Results show that LPS primed MSCs injected into wounds provoke increased expression of MIP/KC in endogenous neutrophils. MIP/KC represents functional homologues of IL-8 in mice and are known as neutrophil chemoattractant. Similar results were found for IL-1β, which is a strong chemoattractant for neutrophil recruitment and bacterial clearance. In addition, LIX which shares 63% amino acid sequence identity with human GCP-2/CXCL6, a chemoattractant for neutrophils. The graphs display numbers of double positive cells for Ly6G MIP/KC, IL-1β and LIX, respectively. PBS and non-primed MSCs injected wounds served as controls. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates. Scale bars: 50µm.
TLR4-dependent shaping of the wound site by MSCs accelerates wound healing (2020)
Figure 4

Figure 4. TLR4 silenced MSCs fail to activate neutrophils and to enhance NET formation.

(a) Representative microphotographs of TLR4 silenced and non-silenced MSCs primed with LPS and co-cultured with neutrophils. Immunostaining was performed with an antibody detecting NETs (DNA-histone, red). Of note, TLR4 silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation (lower row, outer right panel) as compared to non-silenced, LPS primed MSCs co-cultured with PMA activated neutrophils (lower row, outer left panel). Neutrophils and DMSO served as a negative control (upper row, outer left panel), while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation (upper row, middle panel). Co-culture with MSCs suppressed enhanced NET formation (upper row, outer left panel) induced by PMA. Scale bars: 50μm.

(b) Quantitative assessment of NET bound elastase employing a specific ELISA in identical experimental setting as described in Figure 1. Similarly, to immunostaining, a significant reduction of NET bound elastase was observed in TLR4 silenced LPS primed MSCs when co-cultured with activated neutrophils as opposed to high NET bound elastase in non-silenced LPS primed MSCs co-cultured with activated neutrophils. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates.

(c) High resolution scanning electron microscope analysis depicting enhanced NET formation (red arrows) expulsed from neutrophils (blue arrows) in the presence of LPS primed MSC co-cultured with PMA activated neutrophils (middle row, outer left panel) as compared to reduced NETs in non-primed MSCs co-cultured with activated neutrophils (middle row, outer right panel). Of note, TLR4 silenced LPS primed MSCs failed to activate neutrophils and NETs (lower row, outer left panel). TLR4 silenced and non-primed MSCs display reduced NETs in co-cultures with activated neutrophils (lower row, outer right panel), suggesting that PMA activation shares TLR4 signaling components. PMA activated neutrophils alone served as positive control with highly enhanced NETs (upper row, outer right panel) and DMSO treated neutrophils as negative controls (upper row, outer left panel). Red stars indicate neutrophil derived granules. Scale bars: 0.1µm
TLR4-dependent shaping of the wound site by MSCs accelerates wound healing (2020)
Figure 5

Figure 5. LPS primed MSCs accelerate wound healing in TLR4 dependent manner.

(a) Representative clinical pictures of murine wounds at 0, 3, 5, 7 and 10 days after wounding. Enhanced wound healing in LPS primed MSCs group as opposed to all other groups.

(b) Statistical analysis of 20 wound areas per group at the indicated time points, expressed as percentage of the initial wound size (day 0), for PBS control, non-primed MSCs, LPS primed MSCs, non-primed scrambled siRNA-treated (Scr) MSCs, LPS primed Scr MSCs and LPS primed TLR4 silenced MSCs. Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments. Statistical analysis was performed using one way ANOVA.
SLC1A3 contributes to L-asparaginase resistance in solid tumors (2019)
Figure 1

Figure 1. A genome-wide CRISPR-Cas9 screen identifies SLC1A3 as a contributor to L-asparaginase (ASNase) resistance in PC3 cells.

IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase.

IncuCyte cell proliferation curves for ASNS knockout (sgASNS) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase.

Flow chart for a genome-wide CRISPR-Cas9 functional screen in PC3 cells.

Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen. Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate (FDR) <0.003.

IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase treatment. #3 and #4 represent 2 different sgRNAs targeting SLC1A3.

Radioactive labeled aspartate and glutamate uptake measurement in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells. #3 and #4 represent two different sgRNAs targeting SLC1A3. Radioactive labeled leucine uptake was used as a control. Data was normalized to the reads of control PC3 cells.

Endogenous levels of aspartate, asparagine, glutamate and glutamine in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells with or without ASNase for 3 days. Median peak intensity was used for the read normalization.

IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3#3) PC3 cells treated with ASNase and supplemented with either esterified-aspartate (6mM) or esterified-glutamate (6mM), and esterified-leucine (6mM) as a control.

Data information:

For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.
SLC1A3 contributes to L-asparaginase resistance in solid tumors (2019)
Figure 2

Figure 2. SLC1A3 expression is linked to ASNase resistance in different cancer cells.

RT-qPCR analysis was used to determine the relative SLC1A3 mRNA expression (to GAPDH) in different prostate and breast cancer cell lines, as indicated.

The same cell lines (as in panel A) were transduced with either control (sgNon-targeting) or sgSLC1A3. Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines. Leucine uptake level was used for normalization. The numbers above the control column denote the basal aspartate uptake capacity.

RT-qPCR was used to determine the relative mRNA levels (to GAPDH) of aspartate/glutamate transporter genes (SLC1A1, SLC1A2, SLC1A3, SLC1A6 and SLC1A7) in LNCaP, BT549 and SUM159PT cells.

The same batch of cancer cells (as in panel B) was subjected to IncuCyte cell proliferation assays in the absence or presence of ASNase at indicated concentrations. 'ns' indicates no significant difference.

MCF7 and DU145 cells were transduced with either lentiviral empty vector (control) or lentiviral vector containing a V5-tagged SLC1A3 coding sequence (V5-SLC1A3). Relative SLC1A3 mRNA levels (to GAPDH) were determined by RT-qPCR.

Immunofluorescence staining of the V5-tagged SLC1A3 in MCF7 and DU145 cells using anti-V5 antibody. Green staining indicates the plasma membrane localization of V5-SLC1A3 and blue DAPI staining marks the nuclei. Scale bar stands for 5 μm.

Relative aspartate uptake levels in control and V5-SLC1A3-expressed MCF7 and DU145 cells. Leucine uptake level was used for normalization.

Control and V5-SLC1A3 expressed MCF7 and DU145 cells were subjected to IncuCyte cell proliferation assays with or without ASNase at indicated concentrations.

DU145 cells were supplemented with cell-permeable aspartate (6mM, esterified) or glutamate (3mM, esterified) following ASNase treatment, with esterified leucine (6mM or 3mM) as control.

Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.
SLC1A3 contributes to L-asparaginase resistance in solid tumors (2019)
Figure 3

Figure 3. Combinational treatment of ASNase and SLC1A3 inhibition induced metabolic vulnerabilities and restrains cancer cell proliferation.

PC3, DU145 and V5-SLC1A3-DU145 cells were subjected to ASNase and TFB-TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay.

PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions. ASNase (0.3 U/ml), TFB-TBOA (5 μM).

DU145 and V5-SLC1A3-DU145 cells were treated under indicated conditions with ASNase (0.2 U/ml) or TFB-TBOA (20μM) or both, and subjected to IncuCyte analysis for apoptotic cell counts.

PC3 cells were treated under ASNase (0.3 U/ml), or TFB-TBOA (5 μM) conditions for 3 days and cell lysates were extracted and intracellular contents of aspartate, asparagine, glutamate and glutamine were determined by liquid-chromatography mass spectrometry (LC-MS).

From the same experiment as in panel D, key metabolites involved in urea cycle, pyrimidine synthesis, TCA cycle, oxidation, glycolysis and carnitines metabolism were determined. The NAD+/NADH ratio of the indicated conditions was calculated and normalized to control (mean± SEM). Dash line indicates indirect effect. TCA cycle: tricarboxylic acid cycle; OAA: oxaloacetic acid; UMP: uridine monophosphate; CMP: cytidine monophosphate; PEP: phosphoenolpyruvate; NADH: nicotinamide adenine dinucleotide (reduced form); NAD+: nicotinamide adenine dinucleotide (oxidized form); NADPH: nicotinamide adenine dinucleotide phosphate (reduced form); NADP+: nicotinamide adenine dinucleotide phosphate (oxidized form); FAD: flavin adenine dinucleotide; GSSG: glutathione disulfide; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA.

Data information: Median peak intensity was used for raw data normalization in (D and E). Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.
SLC1A3 contributes to L-asparaginase resistance in solid tumors (2019)
Figure 4

Figure 4. Gene expression changes pinpoint key pathways involved in SLC1A3-mediated ASNase resistance.

A-C. PC3 (A), DU145 (B) and DU145-V5-SLC1A3 (C) cells were treated with ASNase (0.3 U/ml in A; 0.2 U/ml in B and C), TFB-TBOA (5 μM) for 3 days as indicated and subjected to transcriptome analysis. Bioinformatics pathway or gene ontology (GO) biological process analysis was performed on the sets of genes that were upregulated or downregulated when PC3 cells were treated with ASNase and TFB-TBOA compared to mock. Transcriptome analysis was based on one biological replicate for each cell line and validated by real-time PCR experiments in Figures EV4A-C. Heatmap presents row scaled normalized read counts and the biological signaling pathway enrichment analysis was performed by ToppGene online program (Chen et al, 2009).
SLC1A3 contributes to L-asparaginase resistance in solid tumors (2019)
Figure 5

Figure 5. SLC1A3 expression promotes ASNase resistance and tumor progression in a mice model for breast cancer metastasis.

SUM159PT human breast cancer cells were orthotopically injected into the mammary glands of NSG mice. Once SUM159PT tumors reached 250mm3 volume, mice were treated with mock or ASNase (60U per day) for 5 consecutive days (n=3). Following treatment, mice were sacrificed, and blood, mammary glands and tumors were collected and subjected to mass spectrometry to determine the asparagine level. Essential amino acids were used for the raw data normalization. Data are presented as mean ± SD.

The mouse breast cancer cell lines 4T1 and 4T1-V5-SLC1A3 were orthotopically implanted into the mammary glands of pretreated NSG mice. Presented is the volume measurements of arising tumors at day 9. (n=13 mice for each group, except for 4T1+ASNase, n=12). Data are presented as mean ± SEM.

From the same experiment in panel B, tumors were surgically removed once reached a volume of ~500mm3 and collected and subjected to LC-MS to determine the levels of asparagine, aspartate, glutamine and glutamate. Leucine level was used as a control. Results are based on five tumor samples and presented as mean ± SEM.

The human breast cancer cell lines MDA-MB-231 and MDA-MB-231-V5-SLC1A3 cells were intravenously injected into pretreated NSG mice. Once mice showed breathing problems, they were sacrificed, and lung and liver were collected and blindly scored for metastasis lesions. The p-value was calculated by one-tailed unpaired t test in Prism7. Data are presented as mean ± SEM (n=8).

A schematic model depicting how SLC1A3-mediated aspartate and glutamate uptake promotes ASNase resistance.

Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.

Expanded View Figure legends
Soluble P-tau217 reflects amyloid and tau pathology and mediates the association of amyloid with tau (2021)
Figure 4

Figure 4. Plasma P-tau217, Aβ plaque density and tau tangle density by primary neuropathology.

(A-C) Individuals in the neuropathology cohort were divided in groups of no significant primary pathology (N=31), AD (N=36), PD (N=13), and others (N=8), and analyzed together with N=9 additional subjects with primary non-AD tauopathies (PSP/CBD). Data is shown for plasma P-tau217 (panel A), plaque density (panel B), and tangle density (panel C). Plasma P-tau217 was not elevated in PSP/CBD compared to subjects without significant primary pathology. AD, Alzheimer's disease; CBD, corticobasal degeneration; PSP, progressive supranuclear palsy; PD, Parkinson's disease. The central band of the boxes are medians and the boxes show inter-quartile ranges. The whiskers are defined as the smallest (largest) observation greater (less) than or equal to the first (third) quartile minus (plus) 1.5 times the inter-quartile range.
Soluble P-tau217 reflects amyloid and tau pathology and mediates the association of amyloid with tau (2021)
Figure 5

Figure 5. Associations between plasma P-tau217 with Aβ PET and tau PET.

Panel A shows R2 and AIC for different regression models, using either only Aβ PET ("A"), only tau PET ("T(I-VI)"), both Aβ and tau PET ("A+T"), or both Aβ and tau PET including their interaction term ("AxT"). All models included age and sex as covariates. The panel shows adjusted R2, together with AIC (above the bars) for each model. We compared R2 between the models using a bootstrap procedure (N=1000 iterations), verifying that the R2 for the "AxT" model was higher than for the "A+T" model (∆R2=0.026, 95% CI 0.010-0.058), the "T(I-VI)" model (∆R2=0.17, 95% CI 0.11-0.26) and the "A" model (∆R2=0.14, 95% CI 0.060-0.22).

Panel B shows associations between plasma P-tau217 and Aβ PET stratified by tertiles (T) of tau PET (T1: SUVR ≤ 1.05, T2: 1.05 < SUVR ≤ 1.11, T3: 1.11 < SUVR ≤ 3.03).

Panel C shows associations between plasma P-tau217 and tau PET stratified by tertiles (T) of Aβ PET (T1: SUVR ≤ 0.468, T2: 0.468 < SUVR ≤ 0.579, T3: 0.579 < SUVR ≤ 1.08).

(D-I) Panels D-I show associations with P-tau217 across groups of Aβ- and tau-PET positivity. Cut-points for Aβ-PET (> 0.533 SUVR) and tau-PET (> 1.36 SUVR in a ROI corresponding to Braak stages I-IV) were used to define the groups. Associations are shown for global cortical Aβ PET (panels D-F) and global cortical tau PET (corresponding to Braak Stages I-VI) (panels G-I) in individuals classified as negative for both Aβ and tau (N=264, panels D and G), positive for Aβ only (N=104, panels E and H), or positive for both Aβ and tau (N=58, panels F and I). The plots show R2 and P-values from linear regression models. No covariates were included in these models, in order to generate R2-values for the PET measures alone.

Data information: In panels B-C, tertiles were chosen to visualize the data, but the regression models used continuous PET uptake as predictors. In panel C, one outlier was beyond the range of the x-axis (Plasma P-tau217 = 17.8 ng/L, Tau PET = 3.03 SUVR, 3rd Aβ PET tertile). The solid lines represent mean effects from regression models. The shaded areas are 95% confidence intervals for the mean effects. Samples were analyzed in duplicates.
The dual role of c-src in cell-to-cell transmission of α-synuclein (2020)
Figure 1

Figure 1. α-Syn fibrils induce activation of c-src in a FcγRIIB-SHP-1/-2-dependent manner

A-B. SH-SY5Y cells and primary cortical neurons were treated with 1 μM α-syn fibrils (A) or monomers (B) for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to PBS, unpaired t-test.

C. Frontal cortices of A53T α-syn heterozygous transgenic mice and control mice at the age of 9 months (old) or 2 months (young) were lysed and Western blot was performed with the indicated antibodies (n=3). *** P < 0.001 compared to WT, unpaired t-test.

D-G. FcγRIIB KD SH-SY5Y cells (D), SHP-1 KD SH-SY5Y cells (E), SHP-2 KD SH-SY5Y cells (F), and SH-SY5Y cells with 20 μM NSC87877, 10 μM SSG and 1 μM PHPS-1 (G) were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.

Data information: Data are expressed as mean ± SEM.
The dual role of c-src in cell-to-cell transmission of α-synuclein (2020)
Figure 2

Figure 2. c-Src expressed in neurons mediates the uptake of α-syn

A. Schematic diagram showing the dual chamber assay.

B, D. SH-SY5Y cells (B) and primary cortical neurons (D) were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.

C, E. NT, c-src KD #1 and #2 SH-SY5Y cells (C) or primary cortical neurons downregulating c-src (E) were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells, one-way ANOVA (C), unpaired t-test (E).

F, G. SH-SY5Y cells (F) and FcγRIIB KD SH-SY5Y cells (G) were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.

Data information: Data are expressed as mean ± SEM. Blue indicates DAPI staining. Scale bar indicates 20 μm.
The dual role of c-src in cell-to-cell transmission of α-synuclein (2020)
Figure 3

Figure 3. c-Src mediates Lewy body-like inclusion body formation in SH-SY5Y cells

A. Schematic diagram showing the co-culture assay.

B. A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1, respectively, with 50 μM RA for 5 days.

C. NT or c-src KD #1 or #2 /A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured with 50 μM RA for 5 days. The samples were observed under confocal microscopy, and the number of cells containing double fluorescence labeled puncta were analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to PBS or NT cells, one-way ANOVA.

Data information: Data are expressed as mean ± SEM. Blue indicates DAPI staining. Scale bar indicates 20 μm.
The dual role of c-src in cell-to-cell transmission of α-synuclein (2020)
Figure 4

Figure 4. c-Src mediates α-syn release from SH-SY5Y cells by regulating autophagy

A. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h.

B. Differentiated NT, c-src KD #1 or c-src KD#2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured 12 h. Levels of α-syn in culture media were then measured using ELISA. Values were derived from three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to control, one-way ANOVA.

C. Schematic diagram showing the dual chamber assay.

D. SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, c-src KD #1 or c-src KD #2/ A53T α-syn-EGFP OE SH-SY5Y cells for 12 h. The cells in the lower chamber were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to NT cells, one-way ANOVA.

E. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 in the presence of DMSO or 50 μM bafilomycin A1 (BafA) for 12 h.

F, G. Differentiated NT and c-src KD/A53T α-syn-EGFP OE SH-SY5Y cells (F) and NT and c-src KD primary neurons (G) were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.

H. SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h. The cells were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). ** P < 0.01, one-way ANOVA.

Data information: Data are expressed as mean ± SEM. Scale bar indicated 20 μm. Blue indicates DAPI staining.
The dual role of c-src in cell-to-cell transmission of α-synuclein (2020)
Figure 5

Figure 5. c-Src is involved in lipid raft-dependent endocytosis

A, B. SH-SY5Y cells in the presence of 5 μM saracatinib, 10 μM SKI-1 (A) and NT, c-src KD #1 or c-src KD #2 SH-SY5Y cells (B) were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.

Data information: Data are expressed as mean ± SEM. Blue indicates DAPI staining. Scale bar indicates 20 μm.