Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Involvement of HMGB1-targeted receptors in the CIPN caused by bortezomib. Bortezomib at 0.4 mg/kg or vehicle was administered i.p. on days 0, 2, 5, 7, 9, and 12. ( A , B ) Preventive ( A ) or therapeutic ( B ) effect of FPS-ZM1, TAK-242, and AMD3100, antagonists of RAGE, TLR4, and CXCR4, respectively. The mice received repeated i.p. administration of FPS-ZM1 at 1 mg/kg, TAK-242 at 3 mg/kg, AMD3100 at 8 mg/kg, or vehicle, 30 min before each dose of bortezomib ( A ), or single i.p. administration of each of them on day 14 ( B ). ( C , D ) Protein levels of RAGE, TLR4, and CXCR4 in the dorsal root ganglion ( C ) and sciatic nerves ( D ) on day 14 after the onset of bortezomib treatment. ( E , F ) Lack of preventive ( E ) and therapeutic ( F ) effects of TH1020, a TLR5 antagonist. The mice received repeated ( E ) or single ( F ) i.p. administration of TH1020 at 1 mg/kg or vehicle, in mice treated with bortezomib according to the above-mentioned schedules. Data show the mean with S.E.M for 5–6 ( A , B , F ), 5–7 ( C ), 6–7 ( D ), or 6 ( E ) mice. V, vehicle; BTZ, bortezomib; DRG, dorsal root ganglion; * p < 0.05, ** p < 0.01 vs. V ( C ) or V in V-treated mice ( A , B , E , F ). † p < 0.05, †† p < 0.01 vs. V in BTZ-treated mice.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques:

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Scheme for bortezomib-induced caspase-dependent HMGB1 release from macrophages and CIPN development, in contrast to caspase-independent mechanisms for paclitaxel. ( A ) Inhibition of proteasome by bortezomib causes caspase-dependent apoptosis of macrophages followed by the release of HMGB1, which in turn causes neuronal sensitization via activation of RAGE and acceleration of CXCL12/CXCR4 signals, leading to CIPN. ( B ) Paclitaxel causes HMGB1 release from macrophages through activation of the ROS/p38MAPK/NF-κB pathway , independently of caspase (see D), and the extracellular HMGB1 develops CIPN in a manner dependent on RAGE and CXCR4 , as shown in the CIPN caused by bortezomib.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques: Inhibition, Activation Assay

Journal: Nature Communications

Article Title: CTRP6 is an endogenous complement regulator that can effectively treat induced arthritis

doi: 10.1038/ncomms9483

Figure Lengend Snippet: ( a – c ) Complement activation was determined by C3b deposition. ( a ) The CP, LP and AP in 10% WT and C1qtnf6 −/− mice serum were activated by the OVA/anti-OVA immune complex, mannans and LPS, respectively (WT, KO: n =8 each). * P <0.05, ** P <0.01 and *** P <0.001. Student's t -test. ( b ) Complement activation in 10% WT and C1qtnf6 Tg mouse serum (WT, Tg: n =8 each). ( c ) The AP in 10% C1qtnf6 −/− serum was activated by LPS in the presence of rhCTRP6 ( n =8). ( d ) AP C3 convertase activity. RhCTRP6 was incubated with a mixture of human C3, factor B and factor D. The reaction mixtures were subjected to SDS–PAGE and were visualized by CBB staining (upper). AP C3 convertase activity (lower) was calculated by the intensity of the Bb protein band. Images have been cropped for presentation. Full-size images are presented in . ( e ) After incubation of human C3(H 2 O) with human factor B and rhCTRP6, C3 was immunoprecipitated with anti-C3 antibody, and immunoprecipitates were subjected to SDS–PAGE. After staining with CBB (upper), the factor B band intensity (lower) was quantified. Images have been cropped for presentation. Full-size images are presented in . ( f ) RhCTRP6 (+) or dilution buffer (−) was added to C1qtnf6 −/− serum, and rhCTRP6-associated proteins were immunoprecipitated with anti-CTRP6 antibody. Immunoprecipitates were subjected to SDS–PAGE, and C3 and CTRP6 were detected with anti-C3 and anti-CTRP6 antibody, respectively. Images have been cropped for presentation. Full-size images are presented in . ( g ) C3–CTRP6 interaction was detected by surface plasmon resonance analysis. Human C3 (0, 15.625, 31.25, 62.5, 250 and 500 nM) was flowed over a rhCTRP6-immobilized sensor chip. The response is expressed in resonance response units (RUs). All data were reproduced in another independent experiment. Average and s.e.m. are shown.

Article Snippet: Membranes were blocked with 5% skim milk/Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h, followed by incubation overnight at 4 °C with rat mAb against C3 (Abcam, ab11862, 1:1,000 dilution) or rabbit polyclonal antibody against CTRP6 (Anaspec, 54562, 1:1,000 dilution).

Techniques: Activation Assay, Activity Assay, Incubation, SDS Page, Staining, Immunoprecipitation, SPR Assay

Journal: Nature Communications

Article Title: CTRP6 is an endogenous complement regulator that can effectively treat induced arthritis

doi: 10.1038/ncomms9483

Figure Lengend Snippet: ( a ) The CTRP6 levels in the serum of healthy controls (HC) and RA patients (RA) were measured by ELISA (HC: n =22, RA n =30). * P <0.05. Student's t -test. ( b ) Synovium from a RA patient was stained with anti-CTRP6 antibody or isotype IgG using diaminobenzidine tetrahydrochloride staining. Scale bar, 50 μm. ( c – f ) Cryostat sections of synovia from RA patients were stained with anti-CTRP6 antibody and 4′,6-diamidino-2-phenylindole (nucleus), and either anti-CD3 antibody ( c ), or anti-CD20 antibody ( d ), or anti-CD68 antibody ( e ), or anti-Hsp47 antibody ( f ). Scale bar, 20 μm. ( g ) Expression of C1qtnf6 in primary fibroblast-like synoviocytes stimulated with IL-1α ( n =3) is shown. * P <0.05. Student's t -test. Average and s.e.m. are shown.

Article Snippet: Membranes were blocked with 5% skim milk/Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h, followed by incubation overnight at 4 °C with rat mAb against C3 (Abcam, ab11862, 1:1,000 dilution) or rabbit polyclonal antibody against CTRP6 (Anaspec, 54562, 1:1,000 dilution).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Expressing

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: Mice bearing Cer2-OVA MMTV-PyMT lung metastases were treated with vehicle (0.5% HPMC) or 75 mg/kg VS-4718 p.o. for 2 weeks starting 4 weeks post-implantation. ( A & B ) Representative images and quantification of collagen VIIIα1 and laminin 5α in metastatic versus non-metastatic lung regions at 6 weeks (H-score; each dot represents one lesion; n = 5 mice/group). Kruskal-Wallis test and Dunn’s multiple comparison. ( C & D ) Collagen VIIIα1 and laminin 511 suppress CD8⁺ T cell activation in vitro . CD8⁺ T cells were plated on 1 μg/mL ECM components, stimulated with CD3/CD28 + IL-2 for 48 h, and assessed by flow cytometry for CD25, granzyme B, IFNγ, and TNFα expression (n = 3-5). ( E ) ECM-mediated inhibition of CD8⁺ T cell migration. Transwell inserts coated with laminin 511, or collagen VIIIα were used to assess migration toward serum-rich media for 5 h (n = 3-4). ( F ) ECM-dependent CD8⁺ T cell adhesion. Adhesion to laminin 511, or collagen VIIIα-coated plates was quantified after 90 min and normalized to uncoated plates (n = 3-4). (G & H) Kaplan-Meier curves showing progression-free survival (PFS) of patients with invasive breast carcinoma stratified by (G) COL8A1 and (H) LAMA5 expression levels. Patients were divided into high and low expression groups based on the median RNA-seq expression. Point-wise z-test using Greenwood’s standard error. Data source = Breast invasive carcinoma TCGA. ( I-K ) Spearman’s Correlation of Metastatic burden signature correlated to FAK-dependent ECM score (I ), COL8A1 ( J ) and LAMA5 ( K ) expression in triple negative breast cancer patients of the SCAN-B dataset . Spearman’s correlation of cytotoxic CD8 + T cells with COL8A1 ( L ) and LAMA5 ( M ) expression in triple negative breast cancer patients of the SCAN-B dataset. ( L & M ). Log 2 mRNA abundance of COL8A1 and LAMA5 in triple negative breast cancer patients with complete response (pCR) or residual disease (RD) in biopsies taken before treatment with either Palclitaxel and Pembrolizumab ( O ) or Palclitaxel, ABT888 and Carboplatin ( N ) in ISPY-2 trial.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Comparison, Activation Assay, In Vitro, Flow Cytometry, Expressing, Inhibition, Migration, RNA Sequencing

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: Mice carrying Cer2-OVA MMTV-PyMT metastatic lesions or without Cer2-OVA MMTV-PyMT injection were treated for two weeks with either vehicle control (0.5% HPMC) or VS-4718 (75 mg/kg). Quantification (H-score) of concentration of (A) Collagen VIIIα1 and (B) Laminin 5α in metastatic lesions and tumor adjacent areas at 5 weeks post implantation of Cer2-OVA MMTV-PyMT cells. Each dot represents a metastatic lesion. n = 5 mice/group using whole lung slide scans. One-way ANOVA for normal distributed with Tukey’s multiple comparison test. Mean ± SEM; ** = p < 0.01.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Injection, Control, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: (A) Representative images of collagen VIIIα1 in metastatic lesions. Scalebar = 50 μm. (B) Quantification (H-score) of collagen VIIIα1 in metastatic lesions and tumor adjacent areas. (C) Representative images of laminin 5 α in metastatic lesions and tumor adjacent areas. Scalebar = 50 μm. (D) Quantification (H-score) of laminin 5 α in metastatic lesions and tumor adjacent areas. Dots represent metastatic lesions. n = 5 mice/group using whole lung slide scans. Kruskal-Wallis test and Dunn’s multiple comparison for not normal distributed. Mean ± SEM; **** = p < 0.0001.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Comparison

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: (A) Mean migration velocity and (B) directional change rate/ average turning frequency of activated CD8⁺ T cells migrating on laminin 511 or collagen VIIIα1 (1 µg/cm²). T cells were CellTracker™ Deep Red-labeled and imaged for 6 h at 5-min intervals; tracks with >11 spots were analyzed using TrackMate . Laminin-511 reduced velocity and increased turning frequency, whereas collagen VIIIα1 had no significant effect. n = 3 independent experiments with 3-5 field of views per group. Unpaired t-tests. Mean ± SEM; **** = p < 0.0001.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Migration, Labeling

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: FAK inhibition reduces laminin-α5 and collagen VIIIα1 within metastatic lesions, weakening basement-membrane-derived physical and inhibitory barriers, thereby improving CD8⁺ T-cell infiltration, migration, tumor-cell engagement, and cytotoxic activity to promote metastatic regression.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Inhibition, Membrane, Derivative Assay, Migration, Activity Assay