Journal: bioRxiv

Article Title: The level of endothelial glycocalyx maturity modulates interactions with charged nanomaterials

doi: 10.1101/2024.09.10.611831

Figure Lengend Snippet: (A) Representative confocal microscope image of endothelial cells exposed to AuNP+ (3 μg/mL; white) for 2 h and counterstained for the (A) plasma membrane (red; CellMask™), (B) syndecan-1 (green; antibody clone DL-101), and CD44 (green; antibody clone Hermes-1) and (C) HS (green; antibody clone 10E4), HA (green; HA binding protein) and glycans (WGA; red) and nuclei (blue; Hoechst-33342). Scale bar is 40 μm. Manders’ colocalization coefficient (MCC) analysis for the extent of AuNP+ (white pixels) colocalized with (A) cell membrane, (B) glycocalyx proteins or (C) glycans. Data are mean ± SD (n=9).

Article Snippet: The cells were then washed three times in PBS and blocked with goat serum (1% v/v) in PBST (PBS with Tween-20 (0.05% w/v); blocking solution) at room temperature (RT) for 1 h. Cells were then incubated with anti-heparan sulfate (HS) (2 μg/mL, clone 10E4, Amsbio), anti-syndecan-1 (2 μg/mL, clone DL-101, Santa Cruz Biotechnology), anti-CD44 (2 μg/mL, clone Hermes-1, Developmental Studies Hybridoma Bank), or anti-VE-cadherin (1 μg/mL, Abcam) antibodies or biotinylated hyaluronan (HA) binding protein (5 μg/ml, Sigma-Aldrich) diluted in the blocking solution for 16 h at 4 °C then washed twice with PBST.

Techniques: Microscopy, Membrane, Binding Assay

Journal: Stem Cell Research & Therapy

Article Title: Glioma-associated mesenchymal stem cells-mediated PD-L1 expression is attenuated by Ad5-Ki67/IL-15 in GBM treatment

doi: 10.1186/s13287-022-02968-z

Figure Lengend Snippet: Identification of MSCs derived from human glioma tissues. A and B Adherent growth patterns and fibroblastic morphology of GA-MSCs cultured in MSC media (× 100, scale bars = 1000 µm). C Immunofluorescence showed that GA-MSCs expressed MSC specific markers CD44 and CD105 (× 400, scale bars = 500 µm)

Article Snippet: The cells were incubated with primary antibodies against CD44 and CD105 (1:100, Proteintech, China), then overnight at 4 °C.

Techniques: Derivative Assay, Cell Culture, Immunofluorescence

Journal: International journal of molecular sciences

Article Title: Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

doi: 10.3390/ijms25168907

Figure Lengend Snippet: Figure 1. Effects of inhibiting CD44-ICD on LPS-induced hepatic inflammation and histological change in mice. Twenty mice were divided into four groups. N group mice were given only saline; D group mice were given DAPT alone; L group mice were given LPS alone; LD group mice were given DAPT 30 min before LPS. After 6 h, (A) AST levels, (B) ALT levels in serum, and (C) liver histopathological changes (10×, scale bar—100 µm; magnified 20×, scale bar—50 µm) by H&E staining were determined. Data are expressed as mean ± SD (n = 5). * p < 0.05 compared with N and D groups. # p < 0.05 compared with the L group.

Article Snippet: After blocking, the blots were incubated with iNOS (1:1000; BD Bioscience, Franklin Lakes, NJ, USA, 610432), p-IKK α/β (1:1000; Genetex, Washington, DC, USA, GTX9039), p-IκB (1:1000; Genetex, GTX00967), nuclear NF-κB (1:500; Genetex, GTX102090), IL-1β (1:1000, Santacruz, Dallas, TX, USA, sc-127420), CD44 (1:500; proteintech, 15675-1-AP), nuclear CD44-ICD (1:500; Cosmobio, Tokyo, Japan, #KAL-K0604), GAPDH (1:5000; abcam, ab9485), β tubulin (1:1000; abcam, ab6046), and Lamin B1 (1:1000; abcam, ab16048) in 5% nonfat skim milk.

Techniques: Saline, Staining

Journal: International journal of molecular sciences

Article Title: Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

doi: 10.3390/ijms25168907

Figure Lengend Snippet: Figure 2. Effects of inhibiting CD44-ICD on pro-inflammatory mediators in LPS-induced hepatic inflammation in mice and cells. Twenty mice were divided into 4 groups. N group mice were given only saline; D group mice were given DAPT alone; L group mice were given LPS alone; LD group mice were given DAPT 30 min before LPS. (A) hepatic IL-1β, (B) NO, and (C) iNOS expressions were determined six hours after LPS. The Chang cells were divided into five groups. N group, cells were treated only DMSO; L group, cells received LPS (100 ng/mL); LD5 group, cells received 5 mM DAPT 1 h before LPS; LD10 group, received 10 mM DAPT 1 h before LPS; LD 20 group, cells were received 20 mM DAPT 1 h before LPS. (D) IL-1β levels in Chang cells were determined 6 h after LPS was given. Data are expressed as mean ± SD (n = 5). * p < 0.05 compared with N and D groups. # p < 0.05 compared with the L group.

Article Snippet: After blocking, the blots were incubated with iNOS (1:1000; BD Bioscience, Franklin Lakes, NJ, USA, 610432), p-IKK α/β (1:1000; Genetex, Washington, DC, USA, GTX9039), p-IκB (1:1000; Genetex, GTX00967), nuclear NF-κB (1:500; Genetex, GTX102090), IL-1β (1:1000, Santacruz, Dallas, TX, USA, sc-127420), CD44 (1:500; proteintech, 15675-1-AP), nuclear CD44-ICD (1:500; Cosmobio, Tokyo, Japan, #KAL-K0604), GAPDH (1:5000; abcam, ab9485), β tubulin (1:1000; abcam, ab6046), and Lamin B1 (1:1000; abcam, ab16048) in 5% nonfat skim milk.

Techniques: Saline

Journal: International journal of molecular sciences

Article Title: Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

doi: 10.3390/ijms25168907

Figure Lengend Snippet: Figure 3. Effects of inhibiting CD44-ICD on NF-κB signaling-related protein expressions in LPS- induced hepatic inflammation in mice. Twenty mice were divided into 4 groups. N group mice were given only saline; D group mice were given DAPT alone; L group mice were given LPS alone; LD group mice were given DAPT 30 min before LPS. (A) Western blotting analysis of p- IKK, (B) immunohistochemical staining analysis of p-IKK, (C) Western blotting analysis of p-IκB, (D) immunohistochemical staining analysis of p-IκB, and (E) Western blotting analysis of nuclear NF-κB expression in liver tissue was determined 6 h after LPS. The immunohistochemical positive expression were observed under a microscope (10× with a scale bar of 100 µm). Data are expressed as mean ± SD (n = 5). * p < 0.05 compared with N and D groups. # p < 0.05 compared with the L group.

Article Snippet: After blocking, the blots were incubated with iNOS (1:1000; BD Bioscience, Franklin Lakes, NJ, USA, 610432), p-IKK α/β (1:1000; Genetex, Washington, DC, USA, GTX9039), p-IκB (1:1000; Genetex, GTX00967), nuclear NF-κB (1:500; Genetex, GTX102090), IL-1β (1:1000, Santacruz, Dallas, TX, USA, sc-127420), CD44 (1:500; proteintech, 15675-1-AP), nuclear CD44-ICD (1:500; Cosmobio, Tokyo, Japan, #KAL-K0604), GAPDH (1:5000; abcam, ab9485), β tubulin (1:1000; abcam, ab6046), and Lamin B1 (1:1000; abcam, ab16048) in 5% nonfat skim milk.

Techniques: Saline, Western Blot, Immunohistochemical staining, Staining, Expressing, Microscopy

Journal: International journal of molecular sciences

Article Title: Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

doi: 10.3390/ijms25168907

Figure Lengend Snippet: Figure 4. Effects of inhibiting CD44-ICD on CD44 expression in LPS-induced hepatic inflammation in mice and cells. Twenty mice were divided into 4 groups. N group mice were given only saline; D group mice were given DAPT alone; L group mice were given LPS alone; LD group mice were given DAPT 30 min before LPS. (A) Western blotting analysis of hepatic CD44 expression and (B) immunofluorescence staining analysis of hepatic CD44 expression was determined 6 h after LPS. Positive immunofluorescence reaction for CD44 (red color) and DAPI nucleic acid staining (blue color) was observed (20×) under a microscope. The Chang cells were divided into five groups. N group, cells were treated with only DMSO; L group, cells received LPS (100 ng/mL); LD5 group, cells received 5 mM DAPT 1 h before LPS; LD10 group received 10 mM DAPT 1 h before LPS; LD 20 group, cells received 20 mM DAPT 1 h before LPS. (C) CD44 and (D) nuclear CD44-ICD expression in Chang cells were determined 6 h after LPS was given. Data are expressed as mean ± SD (n = 5). * p < 0.05 compared with N and D groups. # p < 0.05 compared with the L group.

Article Snippet: After blocking, the blots were incubated with iNOS (1:1000; BD Bioscience, Franklin Lakes, NJ, USA, 610432), p-IKK α/β (1:1000; Genetex, Washington, DC, USA, GTX9039), p-IκB (1:1000; Genetex, GTX00967), nuclear NF-κB (1:500; Genetex, GTX102090), IL-1β (1:1000, Santacruz, Dallas, TX, USA, sc-127420), CD44 (1:500; proteintech, 15675-1-AP), nuclear CD44-ICD (1:500; Cosmobio, Tokyo, Japan, #KAL-K0604), GAPDH (1:5000; abcam, ab9485), β tubulin (1:1000; abcam, ab6046), and Lamin B1 (1:1000; abcam, ab16048) in 5% nonfat skim milk.

Techniques: Expressing, Saline, Western Blot, Immunofluorescence, Staining, Microscopy

Journal: bioRxiv

Article Title: Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation

doi: 10.1101/2025.08.12.669988

Figure Lengend Snippet: N- deacetyl HS and N- sulfo HS are in part separately clustered on cell membranes in the neural plate. (A) Strategy for double staining of N -deacetyl and N -sulfo HS chains in the neural plate. Stage 14 embryos were stained with anti- N- deacetyl HS (JM-403, mouse IgM) and a Cy3-conjugated monovalent Fab fragment of anti-mouse IgM to avoid cross-reaction. Then, embryos were stained with anti- N- sulfo HS (HepSS-1, mouse IgM) conjugated with Alexa Fluor 488. (B) Localization of N -deacetyl and N -sulfo HS in the neural plate (stage 14). The cyan dotted arrow indicates a cell boundary for the line plot of N -deacetyl and N -sulfo HS intensities in (C). (C) Comparison of localization of N -deacetyl and N -sulfo HS chains. The magenta dotted line indicates the normalized intensity of N- deacetyl HS staining and the green line indicates the normalized intensity of N- sulfo HS staining along the cell boundary shown in (B). Magenta and green arrowheads indicate positions where N -deacetyl and N -sulfo HS are highly accumulated, respectively. Fluorescent intensities were normalized with Min-Max normalization in the plot. (D) Correlation coefficients between localization of N -deacetyl HS and N -sulfo HS in the neural plate. Numbers of embryos (N) and numbers of cell boundaries (n) are as indicated. Scale bar, 10μm.

Article Snippet: Primary antibodies and their dilutions were as follows: anti-Wnt11 (rabbit polyclonal IgG, in-house preparation, 1/2000, or mouse monoclonal IgG2b, in-house preparation, 1/10 ), anti-Vangl2 (HPA027043, Sigma, rabbit polyclonal IgG, 1/200), anti-Fzd7 (ab64636, Abcam, rabbit polyclonal IgG, 1/200), anti-Phospho-Vangl2-T78/S79/S82 (AP1206, ABclonal, rabbit polyclonal IgG, 1/1000), anti-HA (11867423001, Roche, 3F10, rat monoclonal IgG1, Roche, 1/2000), anti-C-Cadherin (6B6, Developmental Studies Hybridoma Bank, mouse monoclonal IgG1, 1/50), anti-ZO1 (33-9100, Invitrogen, ZO1-1A12, mouse monoclonal IgG1, 1/200) anti- N -acetyl HS (in-house preparation, NAH46, mouse monoclonal IgM, 1/50 ), anti- N -sulfo HS (in-house preparation, HepSS-1, mouse monoclonal IgM, 1/400 ), and anti- N -deacetyl HS (370730-1, Amsbio, JM-403, mouse monoclonal IgM, 1/200).

Techniques: Double Staining, Staining, Comparison

Journal: bioRxiv

Article Title: Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation

doi: 10.1101/2025.08.12.669988

Figure Lengend Snippet: Polarized localization of HS chains in the neural plate, depends on PCP formation. (A and B) Spatio-temporal distribution of HS chains in the neural plate at Stages 12 (A) and 14 (B), corresponding to a 2-3-h interval. A relatively anterior region is presented (see also for the observed region). Polarization of HS chains (red rose diagram) was compared with WGA (blue rose diagram). Statistical analysis was performed with the Kuiper 2 sample test. Numbers of embryos (N) and numbers of cells (n) are as indicated. (C) Comparison of HS-chain polarization between Stages 12 and 14. Statistical analysis was performed with the Kuiper 2 sample test. (D) Effect of vangl2 knockdown on HS chains in the neural plate (Stage 14). MOs and vangl2 mRNA with tracer (FITC-dextran; 8.3 ng/embryo) were co-injected into the right dorsal blastomere at the 4-cell stage. Boundaries of tracer-negative and -positive area are indicated with dashed lines. (E) Quantification of membrane localization of HS chains with vangl2 knockdown (D). For statistical analysis, the Wilcoxon rank-sum test was performed. Numbers of embryos (N) and numbers of sub regions (n) are as indicated. (F) Increase of N -deacetyl and N -sulfo HS chains on Vangl2 overexpressing cells (Stage 14). mRNA of vangl2 and membrane tracer (mRuby2-KRas) were co-injected into the right dorsal blastomere of 4-cell embryos. (G) Quantification of membrane localization of HS chains with Vangl2 overexpression (F). For statistical analysis, the Shapiro-Wilk test was initially performed to assess data normality, and t -test was performed after the test of homogeneity of variance by F -test. Numbers of embryos (N) and numbers of cell boundaries (n) are as indicated. Amounts of mRNAs/MOs: vangl2 : 10 pg/embryo (D and E), 100 pg/embryo (F and G); mRuby2-kras : 100 pg/embryo (F and G); vangl2 MOs and std MO: 14 ng/embryo (D and E). Scale bars, 20 μm (A and B), 50 μm (D and F).

Article Snippet: Primary antibodies and their dilutions were as follows: anti-Wnt11 (rabbit polyclonal IgG, in-house preparation, 1/2000, or mouse monoclonal IgG2b, in-house preparation, 1/10 ), anti-Vangl2 (HPA027043, Sigma, rabbit polyclonal IgG, 1/200), anti-Fzd7 (ab64636, Abcam, rabbit polyclonal IgG, 1/200), anti-Phospho-Vangl2-T78/S79/S82 (AP1206, ABclonal, rabbit polyclonal IgG, 1/1000), anti-HA (11867423001, Roche, 3F10, rat monoclonal IgG1, Roche, 1/2000), anti-C-Cadherin (6B6, Developmental Studies Hybridoma Bank, mouse monoclonal IgG1, 1/50), anti-ZO1 (33-9100, Invitrogen, ZO1-1A12, mouse monoclonal IgG1, 1/200) anti- N -acetyl HS (in-house preparation, NAH46, mouse monoclonal IgM, 1/50 ), anti- N -sulfo HS (in-house preparation, HepSS-1, mouse monoclonal IgM, 1/400 ), and anti- N -deacetyl HS (370730-1, Amsbio, JM-403, mouse monoclonal IgM, 1/200).

Techniques: Comparison, Knockdown, Injection, Membrane, Over Expression

Journal: bioRxiv

Article Title: Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation

doi: 10.1101/2025.08.12.669988

Figure Lengend Snippet: Deacetyl HS and N- sulfo HS can be polarized in the ectopically established PCP directed by exogenous Wnt11. (A) Schematic image of injection. mRNAs of GFP-pk3 mixed with ha-vangl2 and wnt11 or tracer (mRuby2-KRas) were injected into adjacent animal-ventral blastomeres of 32-cell stage embryos. (B and C) Localization of GFP-Pk3, Wnt11, and N -deacetyl HS (B) or N -sulfo HS (C) with (lower panels) or without exogenous Wnt11 (upper panels) (Stage 14). Rabbit anti-Wnt11 antibody was generated and evaluated to visualize Wnt11 . Co-localization of GFP-Pk3, Wnt11, and HS chains are indicated with arrowheads. (D and E) Quantification of correlation coefficients between HS chains and Wnt11 (D) or GFP-PK3 (E) in the ectopically established PCP or Wnt11 or core PCP components-only overexpression (see also Figures S6A and S6B). All data were performed the Shapiro-Wilk test to assess data normality. For pairwise comparison, the Wilcoxon rank-sum test was performed for N -sulfo HS in (D), and for N -acetyl and N -deacetyl HS in (E). For N -acetyl and N -deacetyl HS in (D) and N -sulfo HS in (E), a t -test was performed after the test of homogeneity of variance with an F -test. For multiple comparison in (D) and in (E, core PCP components-only overexpression), the Kruskal-Wallis test for pre-analysis and the Steel-Dwass-Critchlow-Fligner test were performed. For multiple comparison in (E, ectopically established PCP), Tukey’s HSD test was performed. For (D), numbers of embryos (N) and numbers of cell boundaries (n) are as indicated. For (E), numbers of embryos (N) and numbers of cells (n) are as indicated. Amounts of mRNAs: GFP-pk3 , 100 pg/embryo (B - E); ha-vangl2 , 50 pg/embryo (B and C); vangl2 , 50 pg/embryo (D and E); wnt11 , 250 pg/embryo (B and C); wnt11-4ha , 250 pg/embryo (D and E); mRuby2-kras , 100 pg/embryo (B - E); mEGFP-kras , 100 pg/embryo (D and E). Scale bars, 50 μm (B and C).

Article Snippet: Primary antibodies and their dilutions were as follows: anti-Wnt11 (rabbit polyclonal IgG, in-house preparation, 1/2000, or mouse monoclonal IgG2b, in-house preparation, 1/10 ), anti-Vangl2 (HPA027043, Sigma, rabbit polyclonal IgG, 1/200), anti-Fzd7 (ab64636, Abcam, rabbit polyclonal IgG, 1/200), anti-Phospho-Vangl2-T78/S79/S82 (AP1206, ABclonal, rabbit polyclonal IgG, 1/1000), anti-HA (11867423001, Roche, 3F10, rat monoclonal IgG1, Roche, 1/2000), anti-C-Cadherin (6B6, Developmental Studies Hybridoma Bank, mouse monoclonal IgG1, 1/50), anti-ZO1 (33-9100, Invitrogen, ZO1-1A12, mouse monoclonal IgG1, 1/200) anti- N -acetyl HS (in-house preparation, NAH46, mouse monoclonal IgM, 1/50 ), anti- N -sulfo HS (in-house preparation, HepSS-1, mouse monoclonal IgM, 1/400 ), and anti- N -deacetyl HS (370730-1, Amsbio, JM-403, mouse monoclonal IgM, 1/200).

Techniques: Injection, Generated, Over Expression, Comparison

Journal: Circulation Research

Article Title: Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture

doi: 10.1161/CIRCRESAHA.120.317295

Figure Lengend Snippet: Protein interaction network analysis reveals numerous interactions of proteins that are differentially abundant in aortas of SR-uPA +/0 mice. A protein-protein relational network was built based on experimentally validated direct interactions. The network is comprised of 87 proteins, each portrayed as a circular node (all nodes are identified in Data Set V in the Data Supplement ). Key highly connected nodes (hubs) are labeled together with 2 members of the matrix metalloproteinase family of extracellular proteases and several extracellular matrix components. ACTB indicates beta actin; AGRN, agrin; BCAM, basal cell adhesion molecule; ELN, elastin; FBLN5, fibulin 5; FN1, fibronectin 1; HSPG2, heparan sulfate proteoglycan 2; LAMA5, laminin subunit alpha 5; LAMB2, laminin subunit beta 2; LAMC1, laminin subunit gamma 1; LTBP4, latent transforming growth factor binding protein 4; MMP2, matrix metalloproteinase 2; MMP3, matrix metalloproteinase 3; MYH9, myosin heavy chain 9; NID1, nidogen1; NID2, nidogen 2; and PLAU, urokinase-type plasminogen activator.

Article Snippet: HSPG2 was visualized with 0.375 μg/mL of HSPG2 antibody (PB9277; Boster Biological Technology) in 5% milk/PBST (phosphate-buffered saline/Tween) overnight.

Techniques: Labeling, Binding Assay