Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

doi: 10.1074/jbc.m109.049700

Figure Lengend Snippet: FIGURE 1. Targeting TAP sequences into the GABAB1 gene on BAC citb544e14. A, sequences coding for the HA-SBP tag were introduced into the GABAB1a-specific exon 1a2 (6), 3 to the signal sequence, whereas sequences coding for the CBP-TEV-ProtA tag and a kanamycin resistance gene were inserted into exon 18 (6), 5 to the stop codon. GABAB1a-specific exons are shown in light gray, and the GABAB1b-specific region of exon 1a/b is in dark gray. B, scheme of tagged and untagged GABAB1 isoforms in the BAC transgenic mice. TMD, transmembrane domain; sushi, sushi repeat.

Article Snippet: Coimmunoprecipitation—Solubilized membrane proteins (6–18 mg) of juvenile wild-type C57BL/6 mouse brains were supplemented with 10 g of monoclonal mouse anti-GABAB1 antibody (ab55051, Abcam) or mouse IgG as a control (Sigma) and rotated overnight.

Techniques: Sequencing, Transgenic Assay

Journal: Journal of Biological Chemistry

Article Title: GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

doi: 10.1074/jbc.m109.049700

Figure Lengend Snippet: FIGURE 2. Expression of the transgenes in mouse brain. A, autoradiographic images (upper panels) and photomicrographs of the cerebellum (lower panels) depicting in situ hybridization of radioactively labeled cRNA probes detecting either the transgenic (HA-SBP, ProtA-TEV-CBP) or the wild-type (B1a, B1a/b pan) mRNAs at P12. Hc, hippocampus; Cx, cortex; Th, thalamus; Cb, cerebellum; Bo, bulbus olfactorius; Gr, granule cell layer; M, molecular layer; P, Purkinje cell layer. Scale bars, 3 mm in the upper panels; 50 m in the lower panels. B,immunoblotsdetectingthetaggedproteinsinNTAP-GABAB1a(leftpanel,anti-HA)andGABAB1a/b-CTAP(right panel, anti-GABAB1 pan sc-14006) transgenic mice. The anti-GABAB1 blot also reveals GABAB1b (bottom band in the right lane), whereas GABAB1a comigrates with GABAB1b-CBP-TEV-ProtA. wt, wild-type; tg, transgenic; NTAP- B1a, HA-SBP-GABAB1a; B1b-CTAP, GABAB1b-CBP-TEV-ProtA; B1a-CTAP, GABAB1a-CBP-TEV-ProtA.

Article Snippet: Coimmunoprecipitation—Solubilized membrane proteins (6–18 mg) of juvenile wild-type C57BL/6 mouse brains were supplemented with 10 g of monoclonal mouse anti-GABAB1 antibody (ab55051, Abcam) or mouse IgG as a control (Sigma) and rotated overnight.

Techniques: Expressing, In Situ Hybridization, Labeling, Transgenic Assay

Journal: Journal of Biological Chemistry

Article Title: GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

doi: 10.1074/jbc.m109.049700

Figure Lengend Snippet: FIGURE 4. Tandem affinitypurificationofGABAB receptorsfromtransgenic mouse brains. A, left panel, immunoblot of NTAP-GABAB1a purification steps stained with an anti-HA antibody; post STV, extract after incubation with the streptavidin (STV) matrix; STV eluate, protein eluted with biotin from the STV matrix. A, right panel, immunoblot of GABAB1a/b-CTAP purification steps stained with an anti-GABAB1 pan antibody (AB1531). post IgG, extract after incubation with the IgG matrix; pre TEV, IgG-bound protein prior to TEV protease cleavage; postTEV,eluateoftheIgGmatrixafterincubationwithTEVprotease.Anti-GABAB1 AB1531 strongly reacted with protein A revealing primarily the TAP-tagged vari- ants (compare with Fig. 2B). B, immunoblots of input and final TAP fractions pre- pared from the transgenic lines (tg) and wild-type (wt) control animals. B2, GABAB2; PSD-95, postsynaptic density protein 95.

Article Snippet: Coimmunoprecipitation—Solubilized membrane proteins (6–18 mg) of juvenile wild-type C57BL/6 mouse brains were supplemented with 10 g of monoclonal mouse anti-GABAB1 antibody (ab55051, Abcam) or mouse IgG as a control (Sigma) and rotated overnight.

Techniques: Western Blot, Purification, Staining, Incubation, Transgenic Assay, Control

Journal: Journal of Biological Chemistry

Article Title: GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

doi: 10.1074/jbc.m109.049700

Figure Lengend Snippet: FIGURE 6. GRP78 and KCTD12 are components of mouse brain GABAB1 complexes. A and B, immunoblots of input and final TAP fractions prepared from transgenic (tg) and wild-type (wt) animals. B1a, GABAB1a; B1, GABAB1. A, right panel, immunoblot with GRP78 antibody showing that extracts of NTAP- GABAB1a and wild-type mouse brains contain comparable amounts of GRP78; as a loading control the blot was incubated with an antibody to N-ethylma- leimide-sensitive factor (NSF). C, immunoblots of GRP78 and KCTD12 coim- munoprecipitated (IP) with GABAB1 from wild-type mouse brains. Input: 0.1% in A,B; 0.5% in C. Control (ctrl).

Article Snippet: Coimmunoprecipitation—Solubilized membrane proteins (6–18 mg) of juvenile wild-type C57BL/6 mouse brains were supplemented with 10 g of monoclonal mouse anti-GABAB1 antibody (ab55051, Abcam) or mouse IgG as a control (Sigma) and rotated overnight.

Techniques: Western Blot, Transgenic Assay, Control, Incubation

Journal: The Journal of Cell Biology

Article Title: Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity

doi: 10.1083/jcb.202404079

Figure Lengend Snippet: RhoA downstream signaling is impaired in galectin-9–depleted DCs. (A) Heat map of proteins involved in cytoskeletal rearrangements analyzed by RPPA. Whole-cell lysates obtained from mature WT, gal9 KD, or gal-9 KD +rGal9 DCs were denatured, arrayed on nitrocellulose-coated slides, and probed with antibodies against the specified protein targets. Data shown represent mean protein expression normalized against the loading control. (B) Metascape pathway enrichment analysis from all proteins found to be differentially regulated by RPPA between WT and gal9 KD moDCs. (C and D) WT, gal9 KD, or gal9 KD + rGal9 mature DCs were embedded in 3D collagen gels and stained for pMLC, actin (phalloidin), and nucleus (Hoechst). Box-and-whisker plots show mean (as +) pMLC (C) or actin (D) intensity ratio at the rear over the front of the cell (based on the nucleus localization) for 4 independent donors (10–15 cells analyzed per donor). Whiskers are generated using the Tukey method. Representative high-resolution Airyscan images of WT, gal9 KD, or gal9 KD + rGal9 moDCs are shown. Scale bar = 10 µm. Two-way ANOVA followed by Dunnett’s test for multiple comparisons was performed in all panels. (E) Average actin intensity for the entire width of WT (black), gal9 KD (red), and gal9 KD + rGal9 (blue) moDCs at each position forward from the rearmost point of the cell (left) and backward from the frontmost point of the cell (right) for the rear and front 25 μm. 15 cells were analyzed across three independent donors with bar representing SEM; all values were normalized to the maximum actin intensity of each cell. (F) Distance of the nucleus to the rear of the cell in WT, gal9 KD, and gal9 KD + rGal9 moDCs. Box-and-whisker plot shows mean (as +) nuclear distance for four independent donors (10–15 cells analyzed per donor). Whiskers are generated using the Tukey method. Representative high-resolution Airyscan images of WT, gal9 KD, or gal9 KD + rGal9 moDCs stained for actin (phalloidin) and nucleus (Hoechst) are shown. Scale bar = 10 µm. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. n.s., P > 0.05; ***P < 0.001; ****P < 0.0001. n.s., P > 0.05; *P < 0.05; ***P < 0.01; ****P < 0.001.

Article Snippet: Then, cells were incubated with Duolink Antibody Diluent with 2% human serum supplemented with primary antibodies against RhoA (1:300, #sc-418; Santa Cruz and 1:200, #2117S; Cell Signaling), GEF-H1 (1:600, #GTX125893; GeneTex), and CD44 (1:500, #8E2F3; Novus Biologicals) for 30 min at 37°C.

Techniques: Expressing, Control, Staining, Whisker Assay, Generated

Journal: The Journal of Cell Biology

Article Title: Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity

doi: 10.1083/jcb.202404079

Figure Lengend Snippet: Galectin-9 interaction with CD44 regulates DC migration. (A) IP of galectin-9 or isotype negative control in whole-cell lysates obtained from day 7 matured moDCs. Co-IP complexes were resolved and probed against galectin-9, Vamp-3, and CD44. Graph shows quantification of the CD44 signal. Data shown are representative of three independent experiments. (B) CD44 expression in WT, gal9 KD, and gal9 KD + rGal9 moDC lysates. Tubulin was used as a loading control. Graph shows mean CD44 expression of four independent donors (black symbols), and immunoblot is representative of four independent experiments. (C) Super-resolution dSTORM images of the basal membranes of day 7 matured WT, gal9 KD, and gal9 KD + rGal9 moDCs stained for CD44. ROI (red square) in the upper panel is depicted in the zoomed bottom panels (5,000 × 5,000 nm) to visualize CD44 nanoscale organization. (D) Mean ± SEM CD44 cluster diameter in WT, gal9 KD, and gal9 KD + rGal9 was calculated per cell (gray dots) based on pair correlation analysis for 4 independent donors. One-way ANOVA with Tukey’s multiple comparisons was performed. (E) RhoA_GFP was incubated with either whole DC lysates from three independent donors pulled together (+) or nothing (−), and immunocomplexes were resolved by western blot and probed with specific antibodies against GEF-H1 and RhoA. Western blot is representative of two independent experiments. (F) Confocal microscopy of PLA assessing the proximity of CD44 to RhoA and GEF-H1 in mature moDCs. RhoA-GEF-H1 and isotypes were used as a positive and negative control, respectively. Scale bar: 5 µm. (G) Quantification of the number of PLA foci per cell area (black symbols) from images shown in F. Data represent the mean ± SEM from four independent donors (15–20 cells were analyzed/condition). One-way ANOVA with Dunn’s multiple comparison correction was performed. (H) Quantification of the number of PLA foci (CD44-RhoA) per cell (black symbols) in WT or gal9 KD moDCs untreated or treated with 5 µg/ml of RhoA activator II. Data represent the mean ± SEM from two independent donors. 25–30 cells were analyzed per condition. One-way ANOVA was performed. (I) Quantification of the number of PLA foci (RhoA-GEF-H1) per cell (black symbols) in WT or gal9 KD moDCs. Data represent the mean ± SEM from three independent donors (20 cells were analyzed per condition). An unpaired t test was used to assess significance. n.s, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001. IP, immunoprecipitation. Source data are available for this figure: .

Article Snippet: Then, cells were incubated with Duolink Antibody Diluent with 2% human serum supplemented with primary antibodies against RhoA (1:300, #sc-418; Santa Cruz and 1:200, #2117S; Cell Signaling), GEF-H1 (1:600, #GTX125893; GeneTex), and CD44 (1:500, #8E2F3; Novus Biologicals) for 30 min at 37°C.

Techniques: Migration, Negative Control, Co-Immunoprecipitation Assay, Expressing, Control, Western Blot, Staining, Incubation, Confocal Microscopy, Comparison, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity

doi: 10.1083/jcb.202404079

Figure Lengend Snippet: Graphical abstract. Galectin-9 controls cell rear contractility. Left: Galectin-9 binds to glycosylated residues on CD44 inducing CD44 clustering. This facilitates a functional interaction between CD44 and RhoA-GDP, priming RhoA for activation by GEF-H1. RhoA activation leads to actin contractility at the cell rear and migration. In the absence of galectin-9 (right panel), CD44 clustering is deficient, which results in defective RhoA activation and impaired uropod retraction and migration. Created in BioRender. Franken, G. (2025) https://BioRender.com/9zh1jyh .

Article Snippet: Then, cells were incubated with Duolink Antibody Diluent with 2% human serum supplemented with primary antibodies against RhoA (1:300, #sc-418; Santa Cruz and 1:200, #2117S; Cell Signaling), GEF-H1 (1:600, #GTX125893; GeneTex), and CD44 (1:500, #8E2F3; Novus Biologicals) for 30 min at 37°C.

Techniques: Functional Assay, Activation Assay, Migration

Journal: Cancer immunology research

Article Title: B7-H3 Suppresses Antitumor Immunity via the CCL2-CCR2-M2 Macrophage Axis and Contributes to Ovarian Cancer Progression.

doi: 10.1158/2326-6066.CIR-21-0407

Figure Lengend Snippet: Figure 4. B7-H3 suppression downregulates CCL2 production, in part, via the STAT3 pathway in ovarian cancer cells. A, Differential gene expression based on RNA sequencing (RNA-seq) between controls (n ¼ 3) and B7-H3 KO (n ¼ 3) groups in HM-1 and ID8 cells and HM-1 intradermal tumors. CCL2 protein in the culture supernatants (B) and tumors derived from HM-1 B7-H3 KO (top), ID8 B7-H3 KO (bottom), and their respective controls (n ¼ 5; C). D, CCL2 protein in the culture supernatants of the OVCAR3 shB7-H3 (top), OVCA420 shB7-H3 (bottom), and their respective controls (n ¼ 5). Representative Western blot analysis of nuclear phosphorylated STAT3 (p-STAT3) in HM-1 and ID8 (E) and OVCAR3 and OVCA420 (F) cells. Bottom bar graphs show the signal intensity of p-STAT3 relative to the TBP control in HM-1 and ID8 cells (both n ¼ 10) and OVCAR3 and OVCA420 cells (both n ¼ 7). qPCR for expression of Ccl2 in HM-1 and ID8 cells (G) and CCL2 in OVCAR3 and OVCA420 cells (H) treated with 1.25 and 5 mmol/L of the STAT3 inhibitors C188-9 (G) and Stattic (H), respectively. DMSO was used as the control (n ¼ 3). Data are presented as the mean SEM; , P < 0.05; , P < 0.01; , P < 0.001; N.S., not significant, unpaired t test in B, C, D (OVCA420), E, and F and one-way ANOVA with Tukey multiple comparisons test in D (OVCAR3), G, and H.

Article Snippet: Mouse bone marrowmonocytes (isolated and cultured as described in the “Macrophage and monocyte selection” section) were plated in the upper compartment of 0.4 mm cell culture inserts (catalog no. 353095, Corning) at a density of at 1 105, and 500 mL of TCM of HM-1-B7-H3 KO cells, ID8-B7-H3 KO cells or their control cells (generated as described in the “ELISAs” section) supplemented with 20 mg/mL anti-mouse CCL2 (catalog no. AB-479-NA, RRID: AB_354366; R&DSystems) or goat IgG control (catalog no. AB-108-C, RRID:AB_354267; R&D Systems) was added to the lower compartment.

Techniques: Gene Expression, RNA Sequencing, Derivative Assay, Western Blot, Control, Expressing

Journal: Cancer immunology research

Article Title: B7-H3 Suppresses Antitumor Immunity via the CCL2-CCR2-M2 Macrophage Axis and Contributes to Ovarian Cancer Progression.

doi: 10.1158/2326-6066.CIR-21-0407

Figure Lengend Snippet: Figure 5. The CCL2–CCR2–M2 macrophage axis contributes to B7-H3–mediated tumor progression. A, Chemotaxis of mouse monocytes (left) and generated M2 macrophages (right) in response to HM-1 control or B7-H3 KO TCM. Monocytes or M2 macrophages were pretreated with 2 mmol/L RS504393 (CCR2 antagonist) or DMSO before plating. MEM-Alpha was used as the negative control (n ¼ 3). B, Differentiation of mouse monocytes into M2 macrophages in response to HM-1 control or B7-H3 KO TCM supplemented with 20 mg/mL anti-CCL2 or control IgG. MEM-Alpha was used as the negative control (n ¼ 3). Ab, antibody. C, Tumor growth in mice intradermally injected with HM-1 B7-H3 KO cells or control cells and treated with RS504393 (2 mg/kg body weight) or DMSO daily following tumor inoculation. D, Flow cytometric analysis of the treated tumors in C (n ¼ 6). Data are presented as the mean SEM (, P < 0.05; , P < 0.01; , P < 0.001; N.S., not significant, one-way ANOVA with Tukey multiple comparisons test in A and B, and unpaired t test in C and D).

Article Snippet: Mouse bone marrowmonocytes (isolated and cultured as described in the “Macrophage and monocyte selection” section) were plated in the upper compartment of 0.4 mm cell culture inserts (catalog no. 353095, Corning) at a density of at 1 105, and 500 mL of TCM of HM-1-B7-H3 KO cells, ID8-B7-H3 KO cells or their control cells (generated as described in the “ELISAs” section) supplemented with 20 mg/mL anti-mouse CCL2 (catalog no. AB-479-NA, RRID: AB_354366; R&DSystems) or goat IgG control (catalog no. AB-108-C, RRID:AB_354267; R&D Systems) was added to the lower compartment.

Techniques: Chemotaxis Assay, Generated, Control, Negative Control, Injection

Journal: Cancer immunology research

Article Title: B7-H3 Suppresses Antitumor Immunity via the CCL2-CCR2-M2 Macrophage Axis and Contributes to Ovarian Cancer Progression.

doi: 10.1158/2326-6066.CIR-21-0407

Figure Lengend Snippet: Figure 6. B7-H3 expression associates with an M2 macrophage–rich, IFNgþCD8 T cell–poor TME and a poor prognosis in patients with HGSOC. A, Correlation between CCL2 expression and B7-H3 protein in primary HGSOC tumors (n ¼ 28) analyzed by Spearman correlation. B, Representative images of HGSOC samples showing B7-H3 expression and CD206þ, CD8þ, and IFNgþCD8þ cells. Cells were stained with hematoxylin and eosin (H&E) or immunostained with the corresponding antibodies. Scale bar, 100 mm. Positive cells were stained brown in IHC, and red (CD8), green (IFNg), and blue (DAPI) in immunofluorescence slides. The boxed area represents the zoomed image shown in the top right corner. C, Correlation between the infiltration of CD206þ cells and B7-H3 expression in the corresponding primary tumors of HGSOC (n ¼ 62) analyzed using Jonckheere–Terpstra test. Data are presented as the medianwith 95% confidenceintervals. D, Correlation between tumor-infiltrating CD8þ and IFNgþCD8þ T cells (n ¼ 62) analyzed using Spearman correlation. Blue and pink dots represent B7-H3–low and B7-H3–high expression cases, respectively. E, Comparisons of the number of tumor-infiltrating CD8þ T cells (left) and IFNgþCD8þ T cells (right) in B7-H3–low and B7-H3–high groups by immunostaining (n ¼ 62). Data are presented as the medianwith 95% confidence intervals; , P < 0.05; N.S., not significant, Mann–Whitney U test. F, PFS (left) and OS(right) of patients with HGSOC (n ¼ 62). Patients were classified into a B7-H3–low group (n ¼ 31) and a B7-H3–high group (n ¼ 31). , P < 0.05; N.S., not significant, log-rank test.

Article Snippet: Mouse bone marrowmonocytes (isolated and cultured as described in the “Macrophage and monocyte selection” section) were plated in the upper compartment of 0.4 mm cell culture inserts (catalog no. 353095, Corning) at a density of at 1 105, and 500 mL of TCM of HM-1-B7-H3 KO cells, ID8-B7-H3 KO cells or their control cells (generated as described in the “ELISAs” section) supplemented with 20 mg/mL anti-mouse CCL2 (catalog no. AB-479-NA, RRID: AB_354366; R&DSystems) or goat IgG control (catalog no. AB-108-C, RRID:AB_354267; R&D Systems) was added to the lower compartment.

Techniques: Expressing, Staining, Immunostaining, MANN-WHITNEY