EMBO/soda-nel huggingface dataset:
Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment (2019)
Figure 4

Figure 4 Diflunisal affects the HMGB1/CXCL12/CXCR4 axis but not the HMGB1/TLR4 axis.

A) DFL does not affect the cytokine-inducing activities of disulfide HMGB1 (ds-HMGB1). Mouse Bone Marrow Derived Macrophages were activated or not for 3 h with 3 µg/ml ds-HMGB1 (~100 nM) or 10 ng/ml LPS, in the presence of the indicated concentrations of DFL. The data points represent n=3 biological replicates, with avg ± sd, in one representative experiment (of 2 performed in different days). The levels of TNFα mRNA are not significantly different in the presence of increasing concentrations of DFL (one-way ANOVA plus post tests).

B) DFL does not inhibit chemotaxis towards 3S. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM fully reduced HMGB1 (fr-HMGB1) (green) or 3S (red), or no chemoattractant (black), were added in the lower chamber, together with 1 µg/ml of anti-CXCL12 monoclonal antibody or 30 nM DFL. Data points (n=4) with avg ± sd in one representative experiment (of two performed in different days). Statistics: one-way ANOVA (p<0.0001), followed by Dunnett's post-test. ***, p<0.001 relative to migration towards HMGB1, the migrations towards 3S are not significantly different among each other.

C) Quantification of CD45+ cells isolated with immunobeads from injured muscles 6 hours after damage. Before damage mice were treated either with Phosphate Buffered Saline vehicle, Glycyrrhizin or DFL. Two independent experiments. Each point represents a different mouse, avg ± sd are shown (statistics: Kruskal-Willis plus post-tests).

Expanded View Figure legends
Gut microbiota regulate lacteal integrity by inducing VEGF-C in intestinal villus macrophages (2019)
Figure 1

Figure 1. Depletion of gut microbiota causes morphological defects in lacteals.

(A) Diagram for depletion of gut microbiota in C57BL/6 adult mice by administration of ABX starting at 8 weeks for 4 weeks and their analyses at 12 weeks.

(B) Comparison of bacterial colony forming unit (CFU) in feces from vehicle- and ABX-treated mice (n = 5 mice/group). n.d., non-detectable.

(C) Representative image and measurements of lengths for CD31+/LYVE-1+ lacteal, CD31+/LYVE-1- capillary plexus and DAPI+ villus.

(D, E) Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of vehicle- or ABX-treated mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group).

(F, G) Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (F) and VEGFR3 expression (G), presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.

Data information: All scale bars, 100 μm. Data are represented as means ± SD. *P < 0.05; **P < 0.01 vs. vehicle-treated mice by two-tailed unpaired Student's t-test.
Gut microbiota regulate lacteal integrity by inducing VEGF-C in intestinal villus macrophages (2019)
Figure 2

Figure 2. Depletion of gut microbiota alters LEC junctions in lacteals and impedes dietary lipid absorption to systemic circulation.

(A, B) Images and comparison of VE-cadherin LEC junctions in lacteals of jejunum between vehicle- and ABX-treated mice. Each boxed portion is highly magnified for visualization of detailed distributions of VE-cadherin to distinguish button-like (red arrowheads), zipper-like (open arrowheads) and mixed LEC junctions in lacteals (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 50 μm.

(C, D) Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals. Asterisks indicate the site in lamina propria where fluorescence intensity (FI) was measured. Relative FI divided by mean FI of controls at 16 min (left) and normalized FI divided by FI at 16 min in each villus (right) after initial loading of BODIPY-FA were quantified. For normalization of FI, villi that showed peak intensity earlier or later than 16 min were excluded (n = 5-7 mice/group, 5-10 villi/mouse). Scale bars, 100 μm.

(E, F) Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Jejunum of vehicle- and ABX-treated mice were harvested 2 hours after oral lipid loading. Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. CM, chylomicron; IS, interstitium of lamina propria; LC, large caveola; Lu, lacteal lumen; Ve, vesicle containing lipoproteins. Scale bars, 1 μm.

(G) Diagram for ABX treatment and sampling of systemic blood at indicated time points after oral loading of corn oil into C57BL/6 adult mice.

(H) Comparisons of serum triglyceride (TG) and free fatty acids (FFA) at indicated time points in vehicle- and ABX- treated mice (n = 5-7 mice/group).

Data information: Data are represented as means ± SD. *P < 0.05; **P < 0.01 vs. vehicle-treated mice by two-way ANOVA with Bonferroni post-hoc analysis.
Gut microbiota regulate lacteal integrity by inducing VEGF-C in intestinal villus macrophages (2019)
Figure 3

Figure 3. Depletion of gut microbiota delays lacteal maturation during postnatal period.

(A, B) Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.

(C, D) Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.

(E, F) Images and comparisons of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in right (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.

Data information: Data are represented as means ± SD. *P < 0.05; **P < 0.01 vs. vehicle-treated mice by two-tailed unpaired Student's t-test (B, D) and by two-way ANOVA with Bonferroni post-hoc analysis (F).
Gut microbiota regulate lacteal integrity by inducing VEGF-C in intestinal villus macrophages (2019)
Figure 4

Figure 4. Microbiota colonization restores lacteal defect in GF mice.

(A, B) Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of 8-week old SPF, GF or CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.

(C, D) Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (C) and VEGFR3 expression (D, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from SPF, GF and CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.

(E, F) Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum among SPF, GF and CONV mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.

Data information: Data are represented as means ± SD. **P < 0.01 vs. SPF mice; ## P < 0.01 vs. GF mice by one-way ANOVA with Tukey's multiple comparison test (B-D) and by two-way ANOVA with Bonferroni post-hoc analysis (F).
Gut microbiota regulate lacteal integrity by inducing VEGF-C in intestinal villus macrophages (2019)
Figure 5

Figure 5. Depletion of gut microbiota reduces the expression of VEGF-C, which is essential for lacteal maintenance in intestinal villi.

(A) Diagram for LEC-specific ablation of VEGFR3 in adult mice (VR3iΔLEC).

(B) Images and comparison of VEGFR3 expression (presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum between WT and VR3iΔLEC mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.

(C, D) Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and VR3iΔLEC mice. Note that, in addition to the alterations in the lacteal lengths in VR3iΔLEC mice, diameters of the lacteal also shrank by VEGFR3 ablation. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.

(E, F) Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from WT and VR3iΔLEC mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.

(G, H) Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum from WT and VR3iΔLEC mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.

(I) Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group).

(J, K) Comparison of protein (J) and mRNA (K) levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).

Data information: Data are represented as means ± SD. *P < 0.05; **P < 0.01 vs. WT by two-tailed unpaired Student's t-test (B, D and F) and two-way ANOVA with Bonferroni post-hoc analysis (H and I). **P < 0.01 vs. SPF mice; # P < 0.05; ## P < 0.01 vs. GF mice by one-way ANOVA with Bonferroni's multiple comparison test (J and K).
Gut microbiota regulate lacteal integrity by inducing VEGF-C in intestinal villus macrophages (2019)
Figure 6

Figure 6. Depletion of gut microbiota reduces both VEGF-C transcription of macrophages and number of macrophages in intestinal villi.

(A) Comparison of mRNA levels of VEGF-C in sorted CD45- stromal cells and CD45+ MHCII+ F4/80+ macrophages from jejunum and ileum of vehicle-treated mice. The mean of transcription levels of VEGF-C in CD45- stromal cells was normalized to 1, and the relative levels in macrophages were presented as fold change (n = 4 mice/group).

(B) Comparisons of VEGF-C transcription level in CD45- stromal cells (left) and CD45+ MHCII+ F4/80+ macrophages (right) in jejunum and ileum of vehicle- and ABX-treated mice. The mean of transcription levels of VEGF-C in vehicle-treated mice was normalized to 1, and the relative levels in ABX-treated mice were presented as fold change (n = 5 -6 mice/group).

(C, D) Images and comparison of F4/80+ macrophages in the villi of duodenum (DD), jejunum (JJ), and ileum (IL) from vehicle- or ABX-treated mice. Each dot indicates mean value of 5 sites in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.

(E, F) Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) or CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).

Data information: Data are represented as means ± SD. *P < 0.05, **P < 0.01 vs. vehicle-treated mice by two-tailed unpaired Student's t-test.
Gut microbiota regulate lacteal integrity by inducing VEGF-C in intestinal villus macrophages (2019)
Figure 7

Figure 7. Depletion of CX3CR1+ macrophages causes lacteal regression.

(A) Diagram for ablation of CX3CR1+ macrophages using CX3CR1-DTR (DTR) mice. DT, diphtheria toxin.

(B, C) Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).

(D) Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from WT and DTR mice (n = 5 mice/group).

(E, F) Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.

(G, H) Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.

(I, J) Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and DTR mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.

Data information: Data are represented as means ± SD. *P < 0.05, **P < 0.01 vs. WT by two-tailed unpaired Student's t-test (C, D, F and H) and by two-way ANOVA with Bonferroni post-hoc analysis (J).
Gut microbiota regulate lacteal integrity by inducing VEGF-C in intestinal villus macrophages (2019)
Figure 8

Figure 8. Deletion of MyD88 in macrophages reduces VEGF-C expression in macrophages of villi, leading to lacteal regression.
(A) Diagram for deletion of MyD88 in macrophages using MyD88ΔMP mice.

(B, C) Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group).

(D) Comparisons of mRNA levels of VEGF-C in sorted MHCII+ F4/80+ macrophages from jejunum and ileum of WT and MyD88ΔMP mice. The mean of transcription levels of VEGF-C in WT mice was normalized to 1, and the relative level in MyD88ΔMP mice were presented as fold change (n = 5 mice/group).

(E, F) Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.

(G, H) Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.

(I, J) Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and MyD88ΔMP mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.

Data information: Data are represented as means ± SD. *P < 0.05, **P < 0.01 vs. WT by two-tailed unpaired Student's t-test (C, D, F and H) and two-way ANOVA with Bonferroni post-hoc analysis (J).

EXPANDED VIEW FIGURE LEGENDS
Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis (2016)
Figure 1

Figure 1. The PRL-CNNM pathway.

A, Domain organization of CNNM magnesium transporters and PRL phosphatases. B, ITC experiment shows the CBS-pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one-to-one stoichiometry. C, Sequence alignment of the extended loop in the CBS-pair domains of CNNM proteins. The loop is a unique to the four human CNNM family members compared to related CBS-pair domain proteins: MAM3, CorC efflux protein from Haemophilus influezae, and the MgtE Mg2+ transporter from Enterococcus faecalis. Loop residues unique to CNNM proteins are highlighted in yellow. D, Structure of CBS-pair domain of CNNM2 (PDB 4IY0) with the residues DPFYE indicated. E, Schematic of catalytic cycle of PRL phosphatases. Catalysis proceeds through a long-lived phosphocysteine intermediate. Turnover of the intermediate is the rate-limiting step. F, ITC data of CBS-pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding. G, Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics. Addition of the wild-type CBS-pair domain but not the D426A mutant inhibits phosphatase activity.
Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis (2016)
Figure 2

Figure 2. Cysteine phosphorylation of PRLs prevents binding to CNNM proteins.

A, Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue, cysteine 101. Recombinant PRL2 was treated with 3-O-methylfluorescein phosphate (OMFP) and analyzed by SDS-PAGE with the Phos-tag reagent. Phosphocysteine is hydrolyzed by boiling. B, Detection of phosphorylated by electrospray mass spectrometry. OMFP-treatment increased the mass of the intact protein by 79.99 amu but had no effect on the C101A mutant. C, 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2. The broad peak is TCEP in the buffer. D, Cysteine phosphorylated PRL2 does not bind to CNNM3. Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull-down by the CBS-pair domain of CNNM3 as a GST fusion. Only unphosphorylated PRL2 was observed in the pull-down lanes while phosphorylated PRL2 was in the flow-through lanes. E, ITC measurement of the affinity of PRL2 for the CNNM3 GST-CBS-pair domain. Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200-fold. F, The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS-pair domain.
Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis (2016)
Figure 3

Figure 3. The PRL-CNNM interaction inhibits magnesium efflux.

A, Mutations in the CBS-pair loop of CNNM4 prevent binding to PRL3. Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies. B & C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni’s multiple comparison test. n.s., not significant.
Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis (2016)
Figure 4

Figure 4. Crystal structures of PRL2 in complex with the CNNM3 CBS-pair domain.

A, The CBS-pair domain (yellow) forms a dimer and binds two molecules of PRL2 via an extended loop. The positions of the PRL2 C-terminal CAAX prenylation motif [42] and CNNM3 N-terminus suggest that the membrane would sit above the complex. B, Contacts between the CNNM3 CBS2 loop and the PRL2 active site. C, Stereoview of the 2FO-FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 . D, Detail of complex with reduced PRL2, showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101. E, Model of phosphocysteine preventing CNNM binding. Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding. Residues are numbered according to the PRL1 structure (PDB 1XM2).
Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis (2016)
Figure 5

Figure 5. PRLs in cells are cysteine phosphorylated in response to Mg2+.

A, PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine. Phos-tag analysis of cell lysates shows close to 50% of transiently expressed FLAG-PRL1, -2, and -3 are phosphorylated. The phosphorylated PRL bands are marked by an asterisk. B, Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine (C104) but not the adjacent cysteine (C49) in the active site. C, Co-immunoprecipitation of Myc-PRL by FLAG-CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4. D, Endogenous PRLs in HeLa cells are cysteine phosphorylated. E, Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium.
Cysteamine-bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis (2021)
Figure 1

Figure 1. CRISPR-generated CTNS-/- ciPTEC demonstrate cystinosis phenotype.

(A) Quantification of cystine levels (nmol/mg protein) in control (CTNSWT) (n = 4), CRISPR-generated cystinotic cells (CTNS-/-)(n = 6), and patient-derived cystinotic cells (CTNSPatient) (n = 4).

(B) Quantification of transcription factor EB (TFEB )-GFP nuclear translocation in CTNSWT, CTNS-/-, and CTNSPatient, respectively. Data is demonstrated as the ratio between number of the cells with nucleus-TFEB positive over the total number of TFEB-transfected cells. The ratios were then presented as a fold change compared to control cells (n = 3).

(C) TFEB mRNA expression in CTNS-/- and CTNSPatient cells compared to control cells (n = 3).

(D, E) representative confocal micrographs and quantification of LC3-II accumulation in CTNSWT and CTNS-/- cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 hrs, respectively (n = 3). Scale bars are 20 µm.

(F, G, H) Western blotting and densitometric analyses for LC3-II/LC3-I ratio and SQSTM1 protein levels in CTNSWT, CTNS-/-, and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3).

(I, J) Representative confocal micrographs and quantification of DQ-BSA and BSA in CTNSWT, CTNS-/-, and CTNSPatient cells, respectively (n = 7-14 quantified images). Scale bars are 20 µm.

Data information: Data are expressed as mean ± SEM. *P-values < 0.05 were considered to be significant. One‐way ANOVA with Dunnett's correction (A, B, C, E, G, H, I and J) or Unpaired t‐test (E, G and H). Exact P-values and statistical tests are listed in Appendix Table S1.