Journal: bioRxiv

Article Title: Comparative analysis of wavelength-specific UV stress granule formation

doi: 10.64898/2026.03.15.711948

Figure Lengend Snippet: Representative images of SG formation by G3BP1 and TIA1 signals in wt U2OS cells treated as indicated. Cells were either untreated, treated with 250μM As III for 1h, or exposed to 15mJ/cm 2 of UV. UV samples were harvested and processed for imaging 4 hours after exposure. Yellow arrows indicate SGs. Scale bar = 20μm.

Article Snippet: The following antibodies were used in this study; G3BP1 (Proteintech® 13057 (rabbit) and 66486 (mouse)), S6 Ribosomal protein (RPS6) (ABclonal A6058), TIA1 (Proteintech® 12133), RACK1 (Proteintech® 66940), TRAF2 (Proteintech® 26846 (rabbit) 67315 (mouse)), Geminin (Proteintech® 10802), DHX9 (Proteintech®17721), cytokeratin 8 (Invitrogen, 06318), and eIF3n (Santa Cruz, 137214).

Techniques: Imaging

Journal: bioRxiv

Article Title: Comparative analysis of wavelength-specific UV stress granule formation

doi: 10.64898/2026.03.15.711948

Figure Lengend Snippet: (A) Representative images of SG formation by G3BP1 and TIA1 signals in HaCaT cells treated as indicated. Cells were either untreated, treated with 250μM As III for 1h, or exposed to 15mJ/cm 2 of UV. UV samples were harvested and processed for imaging 4 hours after exposure. (B) Quantification of SG formation by G3BP1 and TIA1 signals in wt U2OS, HaCaT, and wt MEF cells treated as indicated. Cells were either untreated, treated with 250μM As III for 1h, or exposed to 15mJ/cm 2 of UV. UV samples were harvested and processed for imaging 4 hours after exposure. At least 250 cells and 3 fields of view were scored to calculate percent SG formation. n =3; error bars are ±SD; ns P > 0.05, ** P ≤ 0.01, **** P ≤ 0.0001 by one-way ANOVA and Dunnett’s post-hoc test. (C) Quantification of SG formation by G3BP1 and TIA1 signals in wt U2OS cells treated as indicated. Cells were either untreated, treated with 250μM As III for 1h, or exposed to 150mJ/cm 2 of UV. UV samples were harvested and processed for imaging 4 hours after exposure. At least 250 cells and 3 fields of view were scored to calculate percent SG formation. n =3; error bars are ±SD; ns P > 0.05, **** P ≤ 0.0001 by one-way ANOVA and Dunnett’s post-hoc test. Scale bar = 20μm.

Article Snippet: The following antibodies were used in this study; G3BP1 (Proteintech® 13057 (rabbit) and 66486 (mouse)), S6 Ribosomal protein (RPS6) (ABclonal A6058), TIA1 (Proteintech® 12133), RACK1 (Proteintech® 66940), TRAF2 (Proteintech® 26846 (rabbit) 67315 (mouse)), Geminin (Proteintech® 10802), DHX9 (Proteintech®17721), cytokeratin 8 (Invitrogen, 06318), and eIF3n (Santa Cruz, 137214).

Techniques: Imaging

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Scheme of L -arginine-immobilized NHS magnetic beads and the enrichment strategy for arginine-binding proteins. B Volcano plot of arginine- and leucine-binding proteins. C , D GO analysis of arginine-binding proteins in A2780 cells. E Venn diagram showing arginine-binding proteins that were upregulated in both 500 μM arginine-treated A2780 ovarian cancer cells and omentum metastases. F Relative abundance of DDX3X protein captured by arginine or leucine-coupled magnetic beads. G WB detection of DDX3X in A2780 cells with arginine treatment, or lysate from omentum metastasis, and elution after purification with L -leucine- or L -arginine-immobilized NHS magnetic beads. H Molecular docking between DDX3X (HMDB ID: HMDB0000517) and L -arginine (PubChem CID: 6322) performed using Discovery Studio. I Molecular dynamics (MD) simulation was performed using Gromacs. The backbone root mean square deviation (RMSD) of the DDX3X- L -arginine complex over the 100 ns MD simulation was shown. J Surface plasmon resonance analysis for the binding of L -arginine to DDX3X. Data were shown as the mean ± SD. The p value was calculated using an unpaired two-tailed Student’s t -test ( F ). ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Magnetic Beads, Binding Assay, Purification, SPR Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Representative confocal microscopy images and immunofluorescence analysis of DDX3X in A2780 cells treated with arginine at the indicated times. Scale bars, 10 μm. B WB analyses of DDX3X, histone H3, and tubulin protein levels in cytoplasmic and nuclear fractions of A2780 cells under the indicated treatments. C Representative confocal microscopy images of DDX3X immunofluorescence in A2780 cells treated as indicated. Scale bars, 10 μm. D WB analyses of DDX3X, CRM1, histone H3, and tubulin protein levels in the cytoplasmic and nuclear fractions of A2780 cells under the indicated treatments. E Representative confocal microscopy images of Flag immunofluorescence in A2780 shDDX3X cells treated as indicated. Scale bars, 10 μm. F WB analyses of Flag, histone H3, and tubulin protein levels in the cytoplasmic and nuclear fractions of A2780 shDDX3X cells under the indicated treatments. G Quantification of the nuclear-to-cytoplasmic ratio of DDX3X fluorescence intensity in A2780 cells treated with arginine as indicated. H Quantification of the nuclear-to-cytoplasmic ratio of DDX3X fluorescence intensity in A2780 shDDX3X cells treated with arginine as indicated. I Representative images and quantification of immunohistochemical staining of DDX3X in primary tumors and omentum metastases ( n = 20). Scale bars, 50 μm. J Immunohistochemical quantification of nuclear DDX3X in primary tumors and omentum metastases ( n = 20). Scale bars,50 μm. Data were shown as the mean ± SEM. The p value was calculated using one-way ANOVA ( A , G , H ), and unpaired two-tailed Student’s t -test ( I , J ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Confocal Microscopy, Immunofluorescence, Fluorescence, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A GO analysis of differentially expressed genes between DDX3X knockdown and control A2780 cells. B GO analysis of differentially expressed genes between A2780 cells with 100 and 500 μM arginine. C The relative levels of reactive oxygen species (ROS) were measured using the dihydroethidium (DHE) fluorescence method in fresh primary tumor and omental tissues ( n = 5). D Representative image and quantification of immunohistochemical staining of γH2AX and 8-OHdG in primary tumors and omentum metastases ( n = 20). Scale bars, 50 μm. E WB analyses of the protein levels of H2AX, γH2AX, and GAPDH in metastases from mice fed with the indicated diets. F Representative images and quantification of γH2AX immunofluorescence staining in A2780 cells treated with cisplatin and under the indicated treatments. Scale bars, 10 μm. G Representative images and quantification of the comet assay in A2780 cells treated with cisplatin and under the indicated treatments. Scale bars, 10 μm. H Representative images and quantification of immunofluorescence staining of γH2AX in A2780 cells treated with H 2 O 2 (1 mM final concentration) and under the indicated treatments. Scale bars, 10 μm. I Representative images and quantification of comet assay in A2780 cells treated with H 2 O 2 (1 mM final concentration) and under the indicated treatments. Scale bars, 10 μm. Data were shown as the mean ± SEM. The p value was calculated using one-way ANOVA ( F - I ), and unpaired two-tailed Student’s t -test ( C , D ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Knockdown, Control, Fluorescence, Immunohistochemical staining, Staining, Immunofluorescence, Single Cell Gel Electrophoresis, Concentration Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A The bar graph shows the fold changes in DDR gene expression following treatment with 500 μM arginine or DDX3X knockdown. B , C WB analyses for the protein level of ATM, Phospho-ATM (Ser 1981), ATR, Phospho-ATR, and GAPDH in metastasis of mice treated with the indicated diets. D , E WB showing the protein level of DDX3X, ATM, Phospho-ATM (Ser 1981), CHK2, Phospho-CHK2 (Thr68), P53, Phospho-P53 (Ser15), H2AX, γH2AX, and GAPDH in A2780 or HEY A8 cells with indicated treatments. F WB analyses for the protein level of ATM, Phospho-ATM (Ser 1981), H2AX, γH2AX, and GAPDH in A2780 cells with indicated treatments. G – I Representative images and quantification of immunofluorescence staining of γH2AX, comet assay in A2780 cells treated with cisplatin and ATM inhibitors (AZD0156[10 μM] and AZ31[5 μM]). Scale bars, 10 μm. J WB analyses for the protein level of CHK2, Phospho-CHK2 (Thr68), H2AX, γH2AX, and GAPDH in A2780 cells with the indicated treatments. K – M Representative images and quantification of immunofluorescence staining of γH2AX, comet assay in A2780 cells treated with cisplatin and CHK2 inhibitors (CCT241533 [5 μM] and PHI-101[1 μM]). Scale bars, 10 μm. Data were shown as the mean ± SEM from three independent experiments. The p value was calculated using one-way ANOVA ( H , I , L , M ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Gene Expression, Knockdown, Immunofluorescence, Staining, Single Cell Gel Electrophoresis

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Experimental scheme illustrating orthotopic ovarian cancer and intraperitoneal ovarian cancer mice with arginase (10 μg/kg, intraperitoneal injection, three times a week) or DDX3X inhibitor (RK-33, 50 mg/kg, intraperitoneal injection, three times a week) administration. B Plasma arginine levels in arginase-injected mice were quantified using a biochemical assay kit at 24 h post-injection. C , D Representative IVIS bioluminescence images and quantitative luminescence analysis of mice with indicated treatments at 30 days after i.o. injection of ID8-Luc cells ( n = 5/group). E Representative images and weight of ovaries from orthotopic ovarian tumor mice with indicated treatments at 60 days after cell injection ( n = 5/group). Scale bars, 1 cm. F Representative IVIS bioluminescence images and quantitative luminescence analysis of mice with indicated treatments at 30 days after i.p. injection of ID8-Luc cells ( n = 5/group). G Kaplan–Meier analysis of mice with i.p. injection of ID8-Luc cells treated as indicated. ( n = 10/group). H – J Representative images of peritoneal metastasis ( H ), total number of metastatic deposits ( I ), and ascites volume ( J ) in mice with indicated treatments at 35 days after i.p. injection ( n = 5/group). K – M Representative images, tumor weight, and size of subcutaneous xenograft tumors in nude mice 30 days after injection of A2780 cisplatin-resistant cells, with the indicated treatments ( n = 5/group). Data were shown as the mean ± SD. The p value was calculated using unpaired two-tailed Student’s t -test ( A ), two-way ANOVA ( D – F , I , J , L ), and one-way ANOVA ( M ). Survival curves were generated with the Kaplan–Meier method and analyzed using the log-rank test ( G ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Injection, Clinical Proteomics, Quantitative Luminescence, Two Tailed Test, Generated

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Tumor-associated antigen expression (normalized MFI, mean fluorescence intensity) in PDAC cell lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Expressing, Fluorescence

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Structure and in vitro activity of PDAC-directed T cell-engaging bispecific antibodies ( A ) IgG-[L]-scFv structure of BsAbs. CH, constant heavy chain; CL, constant light chain; scFv, single chain variable fragment; VH, variable heavy chain; VL, variabl light chain. ( B ) Flow cytometric analysis displaying fluorescent intensities of IgG-[L]-scFv BsAbs bound to human PDAC cells. IgG-[L]-scFv BsAbs evaluated were specific for human EGFR, HER2, MSLN, c-MET, and B7-H3. ( C ) Antibody-dependent T cell-mediated cytotoxicity (ADTC) as a function of increasing doses of BsAbs against PDAC cell lines. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro, Activity Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: In vitro sensitivities (EC50, pM) to target antigen-specific bispecific antibodies in PDAC cell lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: EGFR and HER2 T-BsAb heterodimerization and in vitro kinetics ( A ) Schematic of BsAb heterodimerization by controlled Fab Arm Exchange. Parental BsAbs bore 2 Fabs specific to EGFR or HER2 and 2 scFvs specific for CD3 (‘2 + 2’). Reduction of inter-H-chain disulfide bonds and subsequent heterodimerization (driven by F405L and K409R amino acid substitutions) yielded EGFRxHER2 BsAbs bearing 1 EGFR Fab, 1 HER2 Fab, and 2 CD3 scFv (‘1 + 1 + 2’). ( B ) SPR analysis of T-BsAbs binding to EGFR (left panel), HER2 (middle panel), and EGFR x HER2 (right panel) proteins. Representative normalized sensorgrams at 20nM were shown. ( C ) IHC staining of two cell-derived PDAC xenograft tumors SW1990 (top) and BxPC-3 (bottom). OCT-embedded tumor sections were stained with EGFRxEGFR, HER2xHER2, or EGFRxHER2 BsAbs. Control slides were either unstained (SW1990) or incubated with a control antibody (BxPC-3). ( D ) Antibody-dependent T cell-mediated cytotoxicity assays (ADTC) against SW1990 (left) and BxPC-3 (right). Cytotoxicity measured by Chromium-51 release. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1. EC50s (SW1990; BxPC-3): EGFRxEGFR: 165.8fM; 31.5fM, HER2xHER2: 18.5pM; 7.14pM, EGFRxHER2: 699.6fM; 128.8fM. CD33xCD33 BsAb was included as a negative control

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vitro, Binding Assay, Immunohistochemistry, Derivative Assay, Staining, Control, Incubation, Negative Control

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Avidities of EGFR and HER2 T-BsAbs

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Protein Binding

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: Heterodimeric EGFR and HER2 T-BsAbs impede PDAC growth ( A ) In vivo antitumor effect of BsAbs in the presence of human T cells against SW1990 cell line xenografts; 3 × 10 6 cells of SW1990 were subcutaneously implanted into mice. T-BsAbs were administered twice per week and 2 × 10 7 of luciferase-transduced T cells (Luc(+) T cells) were administered once per week for 2 weeks to treat the tumors. ( B ) In vivo anti-tumor effects of 10 µg EGFR and HER2 T-BsAbs. ( C ) Relative body weight of mice during treatment and ( D ) overall survival were plotted. ( E ) Bioluminescence imaging (BLI) of Luc(+) T cells. Quantitation of bioluminescence intensity in the lesions of tumors. Representative images (right) were taken on day 7

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: In Vivo, Luciferase, Imaging, Quantitation Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: T-BsAbs drive T cell infiltration, activation, and function in PDAC ( A ) Treatment of SW1990 tumor-bearing mice with EGFR and HER2 T-BsAbs; 3 × 10 6 cells of SW1990 were subcutaneously implanted into mice. Once tumor reached ∼500mm 3 , mice received a single infusion of 2 × 10 7 T cells and were treated with 10µg T-BsAb, administered twice per week. Tumors were harvested for analysis on day 10 after initiation of treatment. Mice that exhibited 50% or more reduction in tumor volume by day 10 were excluded from this and subsequent analyses. ( B ) Flow cytometric analysis of the in vivo effect of T-BsAb treatments on T cell infiltration into harvested SW1990 PDAC tumors. Infiltrating T cells were identified by detection of human CD45 + and human CD4 + or CD8 + . ( C ) Frequency of T cell activation indicated by detection of IFN-γ + TNF-α + T cells and expression of CD69 among CD4 + or CD8 + T cells. ( D ) Frequency of CD11b + intratumoral myeloid cells and prevalence of PDL1 expression

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Activation Assay, In Vivo, Expressing

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: ADTC sensitivities (EC50, pM) of SW1990 lines to EGFR and HER2 T-BsAbs

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques:

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: FACS binding (EC50, pM) of EGFR and HER2 T-BsAbs to SW1990 lines

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Binding Assay

Journal: Journal of Hematology & Oncology

Article Title: Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-024-01538-5

Figure Lengend Snippet: EGFRxHER2 T-BsAbs efficacy is lost in EGFR and HER2-single-positive PDAC ( A ) Mean fluorescent intensities determined by flow cytometry and ( B ) antibody-dependent T cell-mediated cytotoxicity (ADTC) against SW1990-EGFR-KO (left) and SW1990-HER2-KO (right) cell lines. Ratio of effector T cells to target PDAC cells (E: T ratio) was set to 10:1. ( C ) Tumor growth of subcutaneous SW1990 EGFR-KO (left) and HER2-KO (right) tumors. One infusion of 2 × 10 7 T cells was administered. Two doses of T-BsAbs (10 µg each) were given by i.v. injection

Article Snippet: Either recombinant human EGFR (R&D system: Cat# 9565-ER), HER2 (Sino Biological: Cat# 10,004-H08H), or both were immobilized on CM5 chips.

Techniques: Flow Cytometry, Injection

Journal: bioRxiv

Article Title: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices

doi: 10.1101/2022.03.27.485990

Figure Lengend Snippet: Versican co-localizes with collagen fibers in mouse extrahepatic bile duct and collagen gels. A) Schematic showing the different isoforms of versican and the location of the antibody used for IEM within the β-GAG domain. B, C) Representative IEM images showing localization of versican (black dots, indicated by teal arrows) on collagen fibers (blue arrows) in postnatal day 3 (B) and adult (C) mouse bile ducts. D) Quantification of the number of gold particles, indicating versican, per total fiber area. E, F) Representative IEM images showing the localization of versican in plugs of co-gelled collagen (blue arrows) and versican (black dots indicated by teal arrows) (E) or collagen alone (F). Data represent mean ± SD and were analyzed by unpaired t test, ****P<0.0001.

Article Snippet: Recombinant human versican isoform V3 and lumican were from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: bioRxiv

Article Title: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices

doi: 10.1101/2022.03.27.485990

Figure Lengend Snippet: Versican interacts with collagen via its G3 domain. A) Plates were coated with versican (Ver, red) or the V3 isoform (V3, blue) at 0.05, 0.1, 0.25, 0.5 and 1 μg/ml. The absorbance of collagen, added at 2.5 μg/ml and detected via a biotin-conjugated antibody, was measured colorimetrically. B) Plates were coated with 0.25 μg/ml versican (red), V3 (blue) or versican after chondroitinase ABC digestion (Ver-ChABC, orange). The binding of increasing concentrations of collagen (0.1, 0.5, 1.0, 2.5 and 5 μg/ml) was assayed. C) Plates were coated with 0.25 μg/ml recombinant G1 (teal) or G3 (pink) and the binding of increasing concentrations of collagen (0.1, 0.5, 1.0, 2.5 and 5 μg/ml) was assayed. D) Plates were coated with 0.25 μg/ml versican (red), V3 (blue), decorin (Dec, green), lumican (Lum, orange dash line) or aggrecan (Agg, purple) and the binding of increasing concentrations of collagen (0.1, 0.5, 1.0, 2.5 and 5 μg/ml) was measured. E) Plates were coated with 0.25 μg/ml V3 to which was added collagen (1 μg/ml) mixed with increasing concentrations of HA (0.1, 0.5, 1, 5 and 10 ng/ml). F) Plates were coated with 0.25 μg/ml V3 to which was added HA (10 mg/ml) mixed with increasing concentrations of collagen (0.1, 0.5, 1.0, 2.5 and 5 μg/ml). Three independent experiments were carried out for each condition; each line in an individual graph is from the same trio of experiments. Data represent mean ± SD.

Article Snippet: Recombinant human versican isoform V3 and lumican were from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Recombinant

Journal: bioRxiv

Article Title: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices

doi: 10.1101/2022.03.27.485990

Figure Lengend Snippet: Potential versican binding sites on collagen identified using the Collagen Toolkit II. A, B) Binding between Collagen Toolkit II peptides and 10 μg/ml recombinant V3 (A) or G3 (B) was tested using a solid-phase binding assay. Empty wells in the Toolkit peptide plate were coated with full-length collagen as a positive control and data were normalized to the positive control. Three independent experiments were carried out. Data represent mean ± SD. C) Binding motifs (in color, and shaded) identified by the alignment of versican-binding Toolkit peptides. Note that peptide II-5 has two instances of the potential motif.

Article Snippet: Recombinant human versican isoform V3 and lumican were from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Recombinant, Positive Control

Journal: bioRxiv

Article Title: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices

doi: 10.1101/2022.03.27.485990

Figure Lengend Snippet: Versican and its V3 isoform upregulated the deposition of collagen-rich matrices by fibroblasts and led to increased fiber alignment. A-C) Representative SHG imaging of the fibroblast-deposited matrices: control plate, with vitronectin coating (A), versican-coated plate (B), V3-coated plate (C). D) Quantification of the intensity of the SHG signals, normalized to the value for the control group. E) The distribution of collagen fiber orientation was analyzed using OrientationJ. The data were normalized to the dominant angle of each SHG image. The statistical significance of differences between conditions is shown in Table S2. F-H) Representative confocal imaging of immunostaining for fibronectin in fibroblast-deposited matrices (red - fibronectin, blue - DAPI): vitronectin coated plate as a control (F), versican coated plate (G), V3 coated plate (H). I) Quantification of the intensity of fibronectin staining. J) Quantification of cell numbers after 7 days in culture; each data point is from 9 images of each technical repeat. Four independent experiments were carried out with two technical repeats for each coating condition per experiment. Scale bar = 100 μm. Data represent mean ± SD; D, I and J were analyzed using one-way ANOVA, E was analyzed using two-way ANOVA with repeated measurements; *P<0.5, ****P<0.0001.

Article Snippet: Recombinant human versican isoform V3 and lumican were from R&D Systems (Minneapolis, MN, USA).

Techniques: Imaging, Control, Immunostaining, Staining

Journal: bioRxiv

Article Title: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices

doi: 10.1101/2022.03.27.485990

Figure Lengend Snippet: Different matrix proteoglycans have distinct effects on the mechanics of collagen networks. A) Gelation times for collagen-proteoglycan co-gels, with rheological measurements taken during gelation. Col: 2.5 mg/ml pure collagen gel; Col-Ver: 2.5 mg/ml collagen with 0.167 mg/ml versican; Col-V3: 2.5 mg/ml collagen gel with 0.167 mg/ml V3; Col-Agg: 2.5 mg/ml collagen gel with 0.167 mg/ml aggrecan; Col-Dec: 2.5 mg/ml collagen gel with 0.167 mg/ml decorin. B) The shear storage modulus (G’) for the gels in (A), measured after complete gelation. C) G’ for collagen gelled with addition of high molecular weight (1.5 MDa) HA or both HA and V3. Col-0.1HA: 2.5 mg/ml collagen gel containing 0.1 mg/ml HA; Col-0.1HA-V3: 2.5 mg/ml collagen gel containing 0.1 mg/ml HA and 0.167 mg/ml V3. D) G’ of different collagen-proteoglycan co-gels measured by shear rheometry under increasing strain stiffening after full gelation (see Table S3). E) G’ of different collagen-proteoglycan co-gels measured with the gap remaining at 1 mm and under compression to 10% (gap changed to 0.9 mm). F) G’ values at 10% compression for the different co-gels in (E) were normalized to the G’ in the non-compressed state. For measuring G’ during gelation, N=17 for Col, N=12 for Col-Ver, N=11 for Col-V3, N=15 for Col-Agg and N=11 for Col-Dec. These gels were tested with either strain sweep (N=3 for Col, N=3 for Col-Ver, N=3 for Col-V3, N=4 for Col-Agg and N=3 for Col-Dec) or compression (N=3 for Col, N=3 for Col-Ver, N=4 for Col-V3, N=4 for Col-Agg and N=3 for Col-Dec); each gel was subject to only one test. The dotted lines in (D) represent SD. Data represent mean ± SD; A, B, C, and G were analyzed using one-way ANOVA, D, E, and F were analyzed using two-way ANOVA with repeated measurements; *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Recombinant human versican isoform V3 and lumican were from R&D Systems (Minneapolis, MN, USA).

Techniques: Shear, High Molecular Weight

Journal: bioRxiv

Article Title: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices

doi: 10.1101/2022.03.27.485990

Figure Lengend Snippet: ADAMTS-5 and chondroitinase ABC treatment of rat livers alters compression stiffening behavior. A) Sulfated GAG quantification after perfusion with either enzyme. B) Representative confocal imaging of immunostaining using an antibody against DPEAAE, the epitope exposed by ADAMTS-5 cleavage of versican. DPEAAE (green), DAPI (blue). C) G’ was measured under 0%, 10%, 15%, 20% and 25% compression (with HBSS perfusion as a control). There is a significant difference at 25% compression. D) Young’s modulus ( E ) was calculated from normal force and gap changes and plotted at 5%, 12.5%, 17.5% and 22.5% compression (significance shown for 22.5% compression). E) G’ measured at increasing strain from 1% to 50%. N=3 for HBSS, N=4 for ADAMTS-5 and for ChABC perfused livers. Data did not show statistical differences. Compression and strain sweep experiments were done on the same liver samples, as was the assay for sulfated GAGs (A). Scale bar = 200 μm. Data represent mean ± SD; A was analyzed using one-way ANOVA, C, D, and E using two-way ANOVA with repeated measurements; *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Recombinant human versican isoform V3 and lumican were from R&D Systems (Minneapolis, MN, USA).

Techniques: Imaging, Immunostaining, Control

Journal: bioRxiv

Article Title: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices

doi: 10.1101/2022.03.27.485990

Figure Lengend Snippet: Model of interactions between HA, collagen, and versican. A) Collagen and HA binding sites on different versican isoforms. B) Versican may serve as a linker between collagen fibers and HA chains in collagen fibrous networks.

Article Snippet: Recombinant human versican isoform V3 and lumican were from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Electroacupuncture Pretreatment Ameliorates Perioperative Neurocognitive Disorder in Aged Mice by Inhibiting Ferroptosis Through the SIRT1 / NRF2 / GPX4 Pathway

doi: 10.1111/jcmm.71021

Figure Lengend Snippet: SIRT1/NRF2/GPX4 pathway is involved in hippocampal ferroptosis in aged mice. (A) WB images and quantification analysis of SIRT1, NRF2 and GPX4 in the hippocampus of aged mice. (B) WB images and quantification analysis of SLC7A11, TFR1, IRP2 and ferritin in the hippocampus of aged mice ( n = 3 per group). (C) qRT‐PCR expression of SIRT1, NRF2, GPX4, SLC7A11, TFR1, IRP2 and ferritin mRNA in the hippocampus of aged mice ( n = 3 per group). Values are presented as mean ± SEM. ** p < 0.01 compared with the C group; # p < 0.05 and ## p < 0.01 compared with the M group; + p < 0.05 and ++ p < 0.01 and compared with the EX group.

Article Snippet: The membrane was then incubated overnight at 4°C with primary antibodies: SIRT1 (1:850; Lot‐19G10A10; BOSTER), NRF2 (1:1500; Cat#YT3189; Immunoway), iron regulatory protein 2 (IRP2) (1:3000; Cat#YN3307; Immunoway), transferrin receptor 1 (TFR1) (1:750; LotNo‐23BP65E1; BOSTER), GPX4 (1:1500; Cat#YN3047; Immunoway), ferritin (1:3000; Cat#YT1692; Immunoway) and SLC7A11 (1:2000; Cat#YT8130; Immunoway).

Techniques: Quantitative RT-PCR, Expressing