Journal: Diabetes

Article Title: Adiponectin reduces glomerular endothelial glycocalyx disruption and restores glomerular barrier function in a mouse model of type 2 diabetes.

doi: 10.2337/db23-0455

Figure Lengend Snippet: Figure 3—Adiponectin ameliorates TNFa-induced glycocalyx shedding. GEnCs were treated with or without 10 ng/mL TNF-a for 2 h in the presence or absence of 2.5 mg/mL gAd. A) qPCR analysis of SDC4 mRNA levels, normalized to GAPDH, are shown. Data are plotted as the mean 2-(DCT) (gene of interest/housekeeping gene) of each triplicate with the mean. B) SDC4 protein expression was quantified by ELISA in the medium of treated cells. C) Sulfated GAGs were quantified in the medium of treated cells by Alcian blue colorimetric assay. MMP2 (D) and MMP9 (E) mRNA levels in CiGEnCs treated with TNF-a (10 ng/mL) and/or gAd (2.5 mg/mL). A–E) One-way ANOVA (n = 5); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by post hoc analysis (Bonferroni). MMP2 was knocked down using three shRNA dif- ferent sequences in GEnCs (v33, v47, v49) or not, using scrambled controls. F) qPCR data analysis highlighting the decreased expression of MMP2 mRNA in CiGEnCs by all three different shRNA sequences. Data are plotted as the mean comparative cycle threshold of each triplicate. GAPDH used as the housekeeping gene control, n = 4; one-way ANOVA, ****P < 0.0001 post hoc analysis (Bonferroni). Gi) Representative Western Blot demonstrating the protein knockdown of MMP2 by shRNA clone v47. Gii) Densitometry showed significant knockdown of MMP2 protein expression by clone v47. Data are normalized to the b-actin loading control. Dots represent mean ± SEM; one-way ANOVA, ***P < 0.001 by post hoc analysis (Bonferroni). H) SDC4 mRNA levels in MMP2 knockdown CiGEnCs treated with TNF-a and/or gAd. Data are plotted as the mean comparative cycle threshold of each triplicate. GAPDH was used as the housekeeping gene control, n = 5; one-way ANOVA, ***P < 0.001 post hoc analysis (Bonferroni).

Article Snippet: Cellular levels of SDC4 were quantified using a SDC4 ELISA (R&D Systems; catalog DY2918) per the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, shRNA, Control, Western Blot, Knockdown

Journal: Diabetes

Article Title: Adiponectin reduces glomerular endothelial glycocalyx disruption and restores glomerular barrier function in a mouse model of type 2 diabetes.

doi: 10.2337/db23-0455

Figure Lengend Snippet: Figure 4—Adiponectin signals to glomeruli directly and prevents the mRNA upregulation of glycocalyx-related genes in db/db glomeruli. Hu- man (A) and mouse (B) glomeruli were isolated and treated ex vivo with 2.5 mg/mL gAd. Representative Western Blots demonstrating gAd ef- fects at 30 min in human (Ai) and mouse (Bi) ex vivo glomeruli on AMPK phosphorylation (p-). Aii and Bii) Densitometry confirmed significant phosphorylation of AMPK in response to gAd after 30 min in human and mouse glomeruli. Densitometry was performed on blots from inde- pendent repeats (n = 4) showing levels of protein of interest normalized to the b-actin loading control. Dots represent mean ± SEM; one-way ANOVA; *P < 0.05, by post hoc analysis (Bonferroni). C–F) qPCR analysis graph representing the mRNA expression of TNF (C), SDC4 (D), MMP2 (E), and MMP9 (F) in ex vivo (wild type or db/db) sieved glomeruli treated with or without adiponectin. Data are plotted as the mean comparative cycle threshold of each triplicate, n = 5; one-way ANOVA; *P < 0.05, **P < 0.01, ****P < 0.0001 by post hoc analysis (Bonferroni).

Article Snippet: Cellular levels of SDC4 were quantified using a SDC4 ELISA (R&D Systems; catalog DY2918) per the manufacturer’s instructions.

Techniques: Isolation, Ex Vivo, Western Blot, Phospho-proteomics, Control, Expressing

Journal: eLife

Article Title: Emerging role of oncogenic β-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma

doi: 10.7554/elife.95191

Figure Lengend Snippet: Figure 3. Activated ß-catenin represses SDC4 and RAB27A expression in liver cancer cells. (a–d) HepG2 cells were treated with doxycycline (DOX) to express either a control shRNA (shCtrl) or a shRNA targeting mutated ß-catenin (shßcat MUT). (a) Analysis of SDC4 and RAB27A mRNA expression by qRT-PCR. Graphs show the quantification. (b) Analysis of Rab27a protein expression by western-blot. Stain-free was used as a loading control. The graph shows the quantification. (c–d) Epifluorescence images of HepG2 shCtrl and shßcat MUT cells stained with Syndecan-4 and Rab27a antibodies

Article Snippet: The following primary antibodies were used: ß-catenin (1:400, mouse, 610154, BD Biosciences), Rab27a (1:800, rabbit, 69295, Cell signaling), SDC4 (1:200, rabbit, 12236, Cell signaling).

Techniques: Expressing, Control, shRNA, Quantitative RT-PCR, Western Blot, Staining

Journal: eLife

Article Title: Emerging role of oncogenic β-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma

doi: 10.7554/elife.95191

Figure Lengend Snippet: Figure 5. Downregulation of SDC4 and RAB27A in human hepatocellular carcinoma (HCC) samples with CTNNB1 mutations. (a) Analysis of SDC4 and RAB27A mRNA expression in ß-catenin mutated (red) and non-mutated (blue) HCC (Cbioportal cohort, n=366). (b) Analysis of SDC4 and RAB27A mRNA expression in ß-catenin mutated (red) and non-mutated (blue) HCC (Boyault et al. cohort, n=56). (c) Immunohistochemistry (IHC) analysis of glutamine synthetase (GS) and Rab27a in HCC samples presenting or not ß-catenin mutations. Scale bar: 1 mm. (T: tumoral, NT: non tumoral). The upper graphs show the quantification of the percentage of cells positive for GS or Rab27A. The analysis was split into two cohorts (circle and triangle). The lower graph shows the Pearson correlation between Rab27a and GS protein expression (n=56). Results are expressed as Mean ± SEM, two-tailed Student’s t-test analysis, or Pearson correlation test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: The following primary antibodies were used: ß-catenin (1:400, mouse, 610154, BD Biosciences), Rab27a (1:800, rabbit, 69295, Cell signaling), SDC4 (1:200, rabbit, 12236, Cell signaling).

Techniques: Expressing, Immunohistochemistry, Two Tailed Test

Journal: Scientific Reports

Article Title: Glycoprotein non-metastatic melanoma protein B is a potential biomarker for arthroplasty aseptic loosening

doi: 10.1038/s41598-025-13922-3

Figure Lengend Snippet: Transcriptome analysis of eight sex- and age-balanced BM-MSC preparations by RNA-seq. ( A ) Volcano plot emphasizing differentially expressed genes with DESeq2-based p adj -value cut-off 0.05. These genes were included in the gene set enrichment analysis in C , D . Arrows highlight GPNMB and SDC4 . ( B ) Heatmap showing differentially expressed genes with DESeq2-based p adj -value cut-off 0.05. Qlucore Omics Explorer was used for the heatmap visualisation and normalization of the data (each column has mean of 0 and variance of 1) of four primary hip surgeries vs. four revisions (2x hip, 2x knee). Arrows highlight GPNMB and SDC4 . ( C ) KEGG analyses (KEGG: https://www.kegg.jp/ ; of downregulated genes (upper panel) as identified using DESeq2. Gene ontologies for Biological Processes (middle panel) and Molecular Functions (lower panel) are included. ( D ) KEGG analyses of upregulated genes (upper panel) as identified using DESeq2. Gene ontologies for Biological Processes (middle panel) and Molecular Functions (lower panel) are included.

Article Snippet: Afterwards, sandwich ELISA was performed to analyse the secretion of GPNMB (Human Osteoactivin/GPNMB DuoSet ELISA; DY2550, R&D Systems) or SDC4 (Human Syndecan-4 DuoSet ELISA; DY2918, R&D Systems) according to the manufacturer’s protocols.

Techniques: RNA Sequencing

Journal: Scientific Reports

Article Title: Glycoprotein non-metastatic melanoma protein B is a potential biomarker for arthroplasty aseptic loosening

doi: 10.1038/s41598-025-13922-3

Figure Lengend Snippet: Transcriptome analysis of all primary vs. all revision arthroplasties at both implantation sites (28 samples). ( A ) KEGG analyses (KEGG: https://www.kegg.jp/ ; of downregulated genes (upper panel). Gene ontologies for Biological Processes (middle panel) and Molecular Functions (lower panel) are included. ( B ) KEGG analyses of upregulated genes (upper panel). Gene ontologies for Biological Processes (middle panel) and Molecular Functions (lower panel) are included. ( C ) RNA-seq data for GPNMB in BM-MSCs (normalised counts). For the statistical comparisons between primary and revision the parametric unpaired Welch’s t-test was used. Experimental data are shown as individual values. ( D , E ) GPNMB relative expression levels in BM-MSCs based on RPS29 as housekeeping gene. E indicates the levels for individual samples. ( F ) GPNMB relative expression levels in MNCs from blood (PB) and synovial fluid (SF). ( G ) RNA-seq data for SDC4 in BM-MSCs (normalised counts). For the statistical comparisons between primary and revision the parametric unpaired Welch’s t-test was used. Experimental data are shown as individual values. (H , I) SDC4 relative expression levels in BM-MSCs based on RPS29 as housekeeping gene. I indicates the levels for individual samples. ( J ) SDC4 relative expression levels in MNCs from blood (PB) and synovial fluid (SF). The black bar in C, D, G, H represents the median value. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Afterwards, sandwich ELISA was performed to analyse the secretion of GPNMB (Human Osteoactivin/GPNMB DuoSet ELISA; DY2550, R&D Systems) or SDC4 (Human Syndecan-4 DuoSet ELISA; DY2918, R&D Systems) according to the manufacturer’s protocols.

Techniques: RNA Sequencing, Expressing

Journal: Nature communications

Article Title: Dynamic catch of a Thy-1-α5β1+syndecan-4 trimolecular complex.

doi: 10.1038/ncomms5886

Figure Lengend Snippet: Figure 2 | Thy-1 forms catch bonds and binds cooperatively to a K562 cell. (a) Depiction of setup and interacting molecules. An RBC with a probe bead attached to apex (left) was aligned against a target K562 cell (right). The molecules of interest: a5b1 and Syn4 were expressed on a target K562 cell or recombinant a5b1 was coated on a target bead, and Thy-1 was coated on the probe bead. (b) Force regulation of bond lifetime of Thy-1 versus receptor- bearing targets. Bond lifetime (mean±s.e.m.) of Thy-1–K562 (black circle) and Thy-1–a5b1-Fc (grey square) bonds is plotted versus force. (c) Binding specificity. Adhesion frequencies between beads (coated with and without Thy-1 or D37E mutant) and K562 cells (with and without a5b1 or Syn4 knockdown; or HPSE treatment) in the absence and presence of 20 mg ml 1 anti-b1 mAb (AIIB2). Measurements at surface densities of 898 and 2,210 mm 2 are denoted by the white and black bars, respectively. The treatment combinations for each experiment are indicated. Data are presented as mean±s.e.m. of 3–5 pairs of cells and beads repeatedly contacting 50 times with fixed 0.5-s contact duration. *Po0.05; **Po0.01; *** ¼ Po0.001, as assessed by unpaired, two-tailed Student’s t-test. (d) The average bond numbers /nS derived from adhesion frequency Pa (same data from c) by equation 1 (see Methods) for bimolecular bond (Thy-1–a5b1 or Thy-1–Syn4) and trimolecular bond (Thy-1–a5b1 þ Syn4). The reference (grey) line depicts the /na5b1S þ /nSyn4S value (black bar).

Article Snippet: For Syn4 siRNA knockdown, human Syn4 siRNA (SC-36588; Santa Cruz Biotechnology) or scrambled control siRNA (SC-37007; Santa Cruz Biotechnology) was transfected into K562 or A375 cells using the Amaxa Nucleofector system (Lonza, Switzerland) at 10 nM, and cells were used the following day for experimentation or verification of knockdown efficiency by flow cytometry.

Techniques: Recombinant, Binding Assay, Mutagenesis, Knockdown, Two Tailed Test, Derivative Assay

Journal: Nature communications

Article Title: Dynamic catch of a Thy-1-α5β1+syndecan-4 trimolecular complex.

doi: 10.1038/ncomms5886

Figure Lengend Snippet: Figure 3 | Trimolecular bonds exhibit ‘Dynamic catch’ behaviour. (a) Representative force versus time overlapping plots showing no bond (black), non- stiffening bond (blue) and stiffening bond (red) events. Data (points, grey) were acquired at 1,200 fps and were smoothed using the Savitzky–Golay method. (b,c) Representative force versus time zoom-in plots in the ramping phase showing non-stiffening (b) and stiffening (c) signatures. The force at SP (fsp), the stiffness before SP (ksys 1 ) and after SP (ksys 2 ) are indicated. Zero forces are shown by dotted lines. (d) Mean±s.e.m. (of 450 measurements) stiffness of Thy-1–K562 molecular complexes measured by BFP, from left to right: ka5b1 obtained by Syn4 siRNA treatment, kSyn4 obtained by anti-b1 mAb and b1 shRNA treatment, respectively, knon-stiff for all non-stiffening bonds and kws calculated as the /nS weighted sum of ka5b1 and kSyn4. *Po0.05; **Po0.01 as assessed by unpaired, two-tailed Student’s t-test. (e) The relative fraction of Thy-1 bonds: the left column shows trimolecular (empty) versus bimolecular (solid) bond compositions at zero force; the right column shows stiffened (grid) versus non-stiffened (shaded) bond compositions at non-zero force in the ramping phase. (f) Scatter plots of bond lifetimes versus corresponding clamp forces. Individual lifetimes (scatter points) from a were colour- coded for non-stiffening (blue) and stiffening (red) bonds, respectively. (g) Plots of lifetime (mean±s.e.m.) versus force of Thy-1–a5b1 (/ta5b1S, Syn4 siRNA treated, purple square), Thy-1–Syn4 (/tSyn4S, b1 shRNA treated, green triangle) bimolecular bonds and total Thy-1–K562 bonds (/ttotalS, untreated, black circle) for the full force regime investigated. (h) Non-stiffening synergy zoom-in. The low force regime from g (yellow) is highlighted. Weighted sum (by respective /nS in Fig. 2d) of bimolecular bonds (/ta5b1S þ /tSyn4S, blue dashed line) and calculated trimolecular bonds (/ta5b1 þ Syn4S, red dashed line) are shown. (i) Stiffening synergy zoom-in. The high force regime from g (turquoise) is highlighted. Non-stiffening bonds ((/tnon-stiffS þ /tstiffS, green dashed line, /tnon-stiffS, blue square), stiffening bonds (/tstiffS, red triangle) and their weighted sum (by respective /nS in corresponding force regime) are shown in the high force regime.

Article Snippet: For Syn4 siRNA knockdown, human Syn4 siRNA (SC-36588; Santa Cruz Biotechnology) or scrambled control siRNA (SC-37007; Santa Cruz Biotechnology) was transfected into K562 or A375 cells using the Amaxa Nucleofector system (Lonza, Switzerland) at 10 nM, and cells were used the following day for experimentation or verification of knockdown efficiency by flow cytometry.

Techniques: shRNA, Two Tailed Test

Journal: Nature communications

Article Title: Dynamic catch of a Thy-1-α5β1+syndecan-4 trimolecular complex.

doi: 10.1038/ncomms5886

Figure Lengend Snippet: Figure 4 | Biophysical mechanism of the dynamic catch. (a) Measurement of molecular stiffness by BFP; a representative extension versus force plot showing the first kink in the force-ramping phase at B0 pN. The slope of the linear fit to the compressive loading segment (slope0 of the black dashed line) equals the stiffness kcell of a cellular spring. The slope of the linear fit to the tensile loading segment (slope1 of the blue dashed line) equals the stiffness of a system ksys that consists of the cellular spring and the molecular spring in series. The system stiffness before (ksys 1 ) and after (ksys 2 ) SP are calculated from respective tensile loading segments (slope1 of the blue dashed line and slope2 of the red dashed line) the same way as the non-stiffening bonds. (b) Stiffness (mean±s.e.m. of 450 measurements) of Thy-1–K562 molecular complexes, left to right: knon-stiff for non-stiffening bonds; kstiff 1 ( ¼ ksys 1 ) before SP for stiffening bonds; kstiff 2 ( ¼ 1/(1/ksys 2 1/kcell)) after SP for stiffening bonds; ka5b1 þ kSyn4, sum of knon-stiff obtained with Syn4 and b1 knockdown K562 cells. NS, not significant, *Po0.05; **Po0.01; ***Po0.001, as assessed by unpaired, two-tailed Student’s t-test. (c) Mechanical model of the trimolecular system corresponding to a. ka5b1 and kSyn4 are the bimolecular stiffness for Thy-1–a5b1 and Thy-1–Syn4, respectively. ka5b1 þ Syn4 ¼ ka5b1 þ kSyn4 is the trimolecular stiffness for Thy-1–a5b1 þ Syn4. (d) A model for Thy-1–a5b1 þ Syn4 dual-receptor engagement and dynamic catch. A trimolecular complex exhibiting synergistic binding is formed during initial contact of Thy-1 and the a5b1 þ Syn4-bearing cell. When tensile force is applied to the complex, the interaction is strengthened (that is, catch bond formation) and stiffened, as Syn4 contributes to the molecular rigidity of the complex. The stiffness of the trimolecular complex is equivalent to the sum of the two bimolecular bonds (Thy-1–a5b1 or Thy-1–Syn4) in parallel.

Article Snippet: For Syn4 siRNA knockdown, human Syn4 siRNA (SC-36588; Santa Cruz Biotechnology) or scrambled control siRNA (SC-37007; Santa Cruz Biotechnology) was transfected into K562 or A375 cells using the Amaxa Nucleofector system (Lonza, Switzerland) at 10 nM, and cells were used the following day for experimentation or verification of knockdown efficiency by flow cytometry.

Techniques: Knockdown, Two Tailed Test, Binding Assay

Journal: Nature communications

Article Title: Dynamic catch of a Thy-1-α5β1+syndecan-4 trimolecular complex.

doi: 10.1038/ncomms5886

Figure Lengend Snippet: Figure 5 | Syn4 engagement is required for dynamic catch and contractility-dependent adhesion maturation. (a) Scatter plot of bond lifetime versus clamp force for HPSE-treated K562 cells. The measurements and colour codes are the same as the data of Fig. 3f, which are overlaid with the same but dimmed colours for comparison. (b) Fraction of non-stiffening (blue) versus stiffening (red) Thy-1–K562 bonds at the absence and presence of anti-b1 mAb, HPSE and those with Syn4 siRNA in K562 cells. The same treatment conditions were used as Fig. 2c. (c) Immunofluorescence images of Pxn, FAK-pY397, overlays (depicting Pxn in green and FAK-pY397 in red), and ratiometric quantification of FAK activation in A375 cells spread on Thy-1 with control (Cont.) or Syn4 siRNA, and with or without 25 mM blebbistatin. Expanded views correspond to the area indicated with a red rectangle. The range of ratio values (in arbitrary values) is indicated by colour scale. Scale bar, 10 mm. (d) Distributions of FAK activation (FAK-pY397:Pxn) versus adhesion size for individual adhesions of representative Cont. (upper) or Syn4 siRNA-treated (lower) cells with or without 25 mM blebbistatin. Statistically significant Pearson’s correlation values and two-tailed P-values are indicated. (e) Mean±s.e.m. for non-treated adhesions binned into groupings o1 mm2, 41 mm2, or non-binned adhesions treated with Bleb. Data sets represent n4800 adhesions from ten cells or greater for each treatment condition. One representative of two independent experiments is shown. *Po0.05; **Po0.01; ***Po0.001; as assessed by unpaired, two-tailed Student’s t-test.

Article Snippet: For Syn4 siRNA knockdown, human Syn4 siRNA (SC-36588; Santa Cruz Biotechnology) or scrambled control siRNA (SC-37007; Santa Cruz Biotechnology) was transfected into K562 or A375 cells using the Amaxa Nucleofector system (Lonza, Switzerland) at 10 nM, and cells were used the following day for experimentation or verification of knockdown efficiency by flow cytometry.

Techniques: Comparison, Activation Assay, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

doi: 10.3390/ijms24043141

Figure Lengend Snippet: SDC4 contributes to AAV9’s entry into hCMEC/D3 cells. ( A – F ) Imaging flow cytometry assessment of SDC4 expression and AAV9-mediated gene (GFP) delivery in wild-type (WT) and SDC4 KD hCMEC/D3 cells. SDC4 KD was performed using SDC4-specific shRNA plasmids. WT and SDC4 KD hCMEC/D3 cells were treated 4 × 10 4 vg/cell AAV9-GFP for 72 h at 37 °C. SDC4 and GFP expression levels were measured with imaging flow cytometry, as shown by the representative histograms and cellular images of three independent experiments. Scale bar = 20 μm. The effect of SDC4 KD on SDC4 and GFP expression expressed as percent inhibition. The bars represent the mean ± SEM of three independent experiments. Statistical significance vs. WT was assessed with analysis of variance (ANOVA). ** p < 0.01; *** p < 0.001. ( G ) SDS-PAGE showing VP1-3 immunoprecipitated with SDC4 from AAV9-treated (4 × 10 4 vg/cell AAv9 for 6 h at 37 °C) hCMEC/D3 cells’ extracts. Lane 1: 10 6 vg recombinant AAV9; lane 2–3: immunoprecipitates of AAV9-treated WT hCMEC/D3 and SDC4 KD cells; lane 4: MW marker; lane 5–6: immunoprecipitates of WT and SDC4 KD hCMEC/D3 cells untreated with AAV9 (i.e., controls). Standard protein size markers are indicated on the right.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis® FlowSight®, Luminex Corporation, Austin, TX, USA) with APC-conjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A, 5:100 dilution) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A, 5:100).

Techniques: Imaging, Flow Cytometry, Expressing, shRNA, Inhibition, SDS Page, Immunoprecipitation, Recombinant, Marker

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

doi: 10.3390/ijms24043141

Figure Lengend Snippet: AAV9 colocalizes with SDC4 during cellular internalization. hCMEC/D3 cells were incubated with AAV9 (4 × 10 4 vg/cell) for 6 h at 37 °C. The cells were then trypsinized, fixed, permeabilized and treated with AF488-labeled AAV9 (green) and APC-labeled SDC4 (red) antibodies. AAV9’s colocalization with SDC4 was analyzed with confocal microscopy. Representative images of three independent experiments are shown. Scale bar = 10 μm. The MOC ± SEM and PCC ± SEM for the overlap and colocalization of SDC4 with AAV9 are indicated below the images.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis® FlowSight®, Luminex Corporation, Austin, TX, USA) with APC-conjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A, 5:100 dilution) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A, 5:100).

Techniques: Incubation, Labeling, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

doi: 10.3390/ijms24043141

Figure Lengend Snippet: Contribution of the various parts of the SDC4 ectodomain to AAV9 uptake. GFP-tagged SDC4 mutants incubated with AAV9 vectors at 4 × 10 4 vg/cell for 6 h were fixed, permeabilized, and treated with specific primary AAV9 and AF 633-labeled secondary antibodies. The cellular uptake of AAV9 was then analyzed with imaging flow cytometry and confocal microscopy. ( A , B ) Representative fluorescent images and flow cytometry histograms showing the intracellular fluorescence of AAV9-treated SDC4 mutants. Scale bar = 20 μm. The indicated BDS values of AAV9 and SDCs represent the mean + SEM of eight independent experiments. Statistical significance was assessed with ANOVA. ( C ) Detected fluorescence intensities were normalized to AAV9-treated transfectants expressing GFP-labeled WT SDC4 as standards. The bars represent mean + SEM of eight independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001. ( D ) Confocal microscopic visualization of AAV9-treated SDC4 mutants. Scale bar = 10 μm. MOC ± SEM for the overlap of AAV9 with the mutants was calculated by analysis of 15 images with ~10 cells in each image (from three separate samples). Statistical significance vs. AAV9-treated transfectants expressing WT SDC4 (standards) was assessed with ANOVA. *** p < 0.001.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis® FlowSight®, Luminex Corporation, Austin, TX, USA) with APC-conjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A, 5:100 dilution) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A, 5:100).

Techniques: Incubation, Labeling, Imaging, Flow Cytometry, Confocal Microscopy, Fluorescence, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

doi: 10.3390/ijms24043141

Figure Lengend Snippet: The effect of undersulfation on SDC4 expression and AAV9-mediated gene transduction. Stable SDC4 transfectants (created in K562 cells) were preincubated with or without NaClO 3 for 48 h. Effect of NaClO 3 preincubation on HS and SDC4 expression was measured with imaging flow cytometry by incubating the cells with HS- and SDC4-specific antibodies. SDC4 transfectants preincubated with or without NaClO 3 were then treated with AAV9-GFP vectors at 4 × 10 4 vg/cell. After 72 h of incubation with AAV9-GFP, GFP expression was measured with imaging flow cytometry. ( A – D ) Representative flow cytometry histograms and fluorescent cellular images showing HS, GFP, and SDC4 expression of cells preincubated with or without NaClO 3 . Scale bar = 20 μm. ( E ) Detected HS, GFP, and SDC4 expression levels in SDC4 transfectants were normalized to those untreated with NaClO 3 (controls). The bars represent the mean + SEM of six independent experiments. Statistical significance was assessed with ANOVA. *** p < 0.001. ( F ) Linear regression between SDC4 expression and AAV9-mediated GFP transduction.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis® FlowSight®, Luminex Corporation, Austin, TX, USA) with APC-conjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A, 5:100 dilution) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A, 5:100).

Techniques: Expressing, Transduction, Imaging, Flow Cytometry, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

doi: 10.3390/ijms24043141

Figure Lengend Snippet: The AAV9 interactome in SDC4 transfectants.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis® FlowSight®, Luminex Corporation, Austin, TX, USA) with APC-conjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A, 5:100 dilution) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A, 5:100).

Techniques: Membrane, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

doi: 10.3390/ijms24043141

Figure Lengend Snippet: SDC4 overexpression increases AAV9-mediated GFP transduction in SH-SY5Y cells. SDC4 transfectants (created in SH-SY5Y cells) and WT SH-SY5Y cells were incubated with AAV9-GFP (4 × 10 4 vg/cell) at 37 °C for 72 h. GFP expression was then analyzed with imaging flow cytometry. ( A – C ) Representative flow cytometry histograms and fluorescent images showing the GFP and SDC4 expression levels in WT SH-SY5Y cells and SDC4 transfectants treated with AAV9-GFP. Scale bar = 20 μm. ( D , E ) Detected SDC4 and GFP expression levels of AAV9-treated SDC4 transfectants were normalized to that of WT SH-SY5Y cells as standards. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis® FlowSight®, Luminex Corporation, Austin, TX, USA) with APC-conjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A, 5:100 dilution) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A, 5:100).

Techniques: Over Expression, Transduction, Incubation, Expressing, Imaging, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

doi: 10.3390/ijms24043141

Figure Lengend Snippet: AAV9-mediated GFP transduction is more efficient in U-87 MG than in SH-SY5Y cells. WT U-87 MG and SH-SY5Y cells were incubated with AAV9-GFP (4 × 10 4 vg/cell) for 72 h. GFP expression was then analyzed with imaging flow cytometry. ( A – C ) Representative flow cytometry histograms and fluorescent images showing the SDC4 and GFP expression levels in SH-SY5Y and U-87 MG cells treated with AAV9-GFP. Scale bar = 20 μm. ( D , E ) Detected SDC4 and GFP expression levels of AAV9-treated U-87 MG cells were normalized to that of SH-SY5Y cells as standards. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis® FlowSight®, Luminex Corporation, Austin, TX, USA) with APC-conjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A, 5:100 dilution) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A, 5:100).

Techniques: Transduction, Incubation, Expressing, Imaging, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant’s Superior Transmission

doi: 10.3390/ijms23020796

Figure Lengend Snippet: Cellular uptake of the WT spike and its Delta variant into WT K562 cells and SDC1, 4 transfectants. WT K562 cells, SDC1 and SDC4 transfectants were treated with the WT spike and its Delta variant. The cellular uptake was then analyzed with imaging flow cytometry by using fluorescently labeled specific antibodies against the His-tag of the recombinant spikes. ( A , B ) Representative flow cytometry histograms and cellular images show the intracellular fluorescence of WT K562 cells, SDC1 and SDC4 transfectants treated with either of the spike proteins. Scale bar = 20 μm. ( C ) Detected fluorescence intensities normalized to WT-spike-treated K562 cells as standards. The bars represent the mean + SEM of six independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. ( D ) Detected fluorescence intensities normalized to WT-spike-treated cells as standards. The bars represent the mean + SEM of six independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The N-terminally His-tagged recombinant SARS-CoV-2 spike protein and its Delta variant were purchased from Sino Biological (Beijing, China, cat. no. 40589-V08B1-100 and 40589-V08B16-100), while the recombinant human SDC4 were obtained from R&D Systems (Minneapolis, MN, USA; cat. no. 2918-SD).

Techniques: Variant Assay, Imaging, Flow Cytometry, Labeling, Recombinant, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant’s Superior Transmission

doi: 10.3390/ijms23020796

Figure Lengend Snippet: PSV-mediated gene transduction into WT K562 cells, SDC1 and SDC4 transfectants. The cells were incubated with SARS-CoV-2 PSV-RFP (either the WT or the Delta variant). RFP expression was analyzed 72 h later with imaging flow cytometry. ( A ) Representative flow cytometry histograms showing RFP fluorescence of WT K562 cells, SDC1 and SDC4 transfectants incubated with either the WT or the Delta SARS-CoV-2 PSV. ( B ) Cellular images of SARS-CoV-2 PSV-treated WT K562 cells and SDC transfectants as detected with imaging flow cytometry. Scale bar = 20 μm. ( C ) Detected cellular RFP intensities normalized to WT PSV-treated WT K562 cells as standards. The bars represent the mean + SEM of three independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01. ( D ) Detected RFP intensities normalized to WT PSV-treated cells as standards. The bars represent the mean ± SEM of three independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Article Snippet: The N-terminally His-tagged recombinant SARS-CoV-2 spike protein and its Delta variant were purchased from Sino Biological (Beijing, China, cat. no. 40589-V08B1-100 and 40589-V08B16-100), while the recombinant human SDC4 were obtained from R&D Systems (Minneapolis, MN, USA; cat. no. 2918-SD).

Techniques: Transduction, Incubation, Variant Assay, Expressing, Imaging, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant’s Superior Transmission

doi: 10.3390/ijms23020796

Figure Lengend Snippet: Cellular uptake of the WT and Delta spike proteins into SDC4 deletion mutants. WT SDC4 transfectants and CBD, HSA and Si4 mutants (all tagged with GFP) were treated with the Delta spike. The cellular uptake was then analyzed with imaging flow cytometry by using fluorescently labeled specific antibodies against the His-tag of the recombinant spike proteins. ( A ) Representative flow cytometry histograms showing the intracellular fluorescence of WT SDC4 transfectants, CBD, HSA and Si4 mutants treated with the Delta spike protein (along with specific fluorescent antibodies). Scale bar = 20 μm. ( B ) Detected fluorescence intensities were normalized Delta spike-treated WT SDC4 transfectants as standards. The bars represent the mean ± SEM of four independent experiments (experimental data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. *** p < 0.001. ( C ) Cellular images showing the intracellular fluorescence of WT SDC4 and CBD, HSA and Si4 mutants treated with the Delta spike protein. Scale bar = 20 μm. The indicated BDS values of SARS-CoV-2 and SDCs represent the mean + SEM of four independent experiments.

Article Snippet: The N-terminally His-tagged recombinant SARS-CoV-2 spike protein and its Delta variant were purchased from Sino Biological (Beijing, China, cat. no. 40589-V08B1-100 and 40589-V08B16-100), while the recombinant human SDC4 were obtained from R&D Systems (Minneapolis, MN, USA; cat. no. 2918-SD).

Techniques: Imaging, Flow Cytometry, Labeling, Recombinant, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant’s Superior Transmission

doi: 10.3390/ijms23020796

Figure Lengend Snippet: Internalization of the WT and Delta spike into Calu-3 cells. Calu-3 cells were treated with the WT spike and its Delta variant. The cellular uptake was then analyzed with imaging flow cytometry by using fluorescently labeled specific antibodies against the His-tag of the recombinant spike proteins. ( A ) Representative flow cytometry histograms and cellular images show the intracellular fluorescence of Calu-3 cells treated with either the WT or Delta spike. ( B ) Detected intracellular fluorescence levels normalized to those treated with the WT spike as standards. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. 293T expression levels was assessed with ANOVA. * p < 0.05. ( C ) SDS-PAGE showing the WT or Delta spike immunoprecipitated with SDC4 antibodies from extracts of untreated control or spike-treated Calu-3 cells. Lanes 1–2: 0.5 ug of WT and Delta spike proteins; Lanes 3–4: immunoprecipitate of WT or Delta spike-treated Calu-3 cells; Lane 5: immunoprecipitate of untreated Calu-3. Standard protein size markers are indicated on the left. The signal of spike proteins was detected with UVITEC Alliance Q9 Advanced Imager, and the intensity of bands were analyzed with the NineAlliance © software. ( D ) Detected band intensities were normalized to WT spike-treated Calu-3 cells as standard. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05.

Article Snippet: The N-terminally His-tagged recombinant SARS-CoV-2 spike protein and its Delta variant were purchased from Sino Biological (Beijing, China, cat. no. 40589-V08B1-100 and 40589-V08B16-100), while the recombinant human SDC4 were obtained from R&D Systems (Minneapolis, MN, USA; cat. no. 2918-SD).

Techniques: Variant Assay, Imaging, Flow Cytometry, Labeling, Recombinant, Fluorescence, Expressing, SDS Page, Immunoprecipitation, Control, Software

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant’s Superior Transmission

doi: 10.3390/ijms23020796

Figure Lengend Snippet: Effect of SDC4 knockdown (KD) or GAG inhibition on Delta spike internalization into Calu-3 cells. SDC4 KD in Calu-3 cells was performed using a lentiviral vector specific to human SDC4. ( A ) SDC4 expression levels were measured with imaging flow cytometry, as shown by the representative histograms and cellular images. Detected SDC4 levels of KD cells were normalized to WT Calu-3 cells as standards. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05. ( B,C ) SDC4 KD and WT Calu-3 cells were treated with either the Delta spike protein ( B ) or the Delta PSV ( C ). For GAG inhibition, some of the WT Calu-3 cells were preincubated with heparin (200 ug/mL, 30 min before Delta treatment). Representative flow cytometry histograms and cellular images show the intracellular fluorescence of WT or SDC4 KD Calu-3 treated with Delta spike protein or PSV in the presence or absence of heparin. Scale bar = 20 μm. The effect of SDC4 KD or heparin treatment was expressed as percent inhibition, calculated with the following formula: ((X − Y)/X) × 100, where X is the fluorescence intensity obtained on controls treated with the Delta spike or Delta PSV without SDC4 KD or heparin, and Y is the fluorescence intensity obtained on cells receiving Delta spike or Delta PSV treatment, along with SDC4 KD or heparin. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. controls was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The N-terminally His-tagged recombinant SARS-CoV-2 spike protein and its Delta variant were purchased from Sino Biological (Beijing, China, cat. no. 40589-V08B1-100 and 40589-V08B16-100), while the recombinant human SDC4 were obtained from R&D Systems (Minneapolis, MN, USA; cat. no. 2918-SD).

Techniques: Knockdown, Inhibition, Plasmid Preparation, Expressing, Imaging, Flow Cytometry, Fluorescence