Journal: Toxins

Article Title: Purification and Characterization of His-Tagged Recombinant Bacteroides fragilis Toxin-2 Variants In Vitro and In Vivo

doi: 10.3390/toxins18040189

Figure Lengend Snippet: Functional characterization of His-tagged BFT variants: ( a ) E-cadherin cleavage activity of A-hBFT from three His-rETBF clones. A-hBFT cleaves full-length E-cadherin (120 kDa) while serum-free medium (SFM) and imidazole controls do not. Clone 1 was selected for subsequent experiments. ( b ) Serial dilution analysis (1:10 1 to 1:10 8 ) comparing biological activity of His-tagged and wild-type BFT culture supernatants. ( c ) Cross-species validation in mouse (CMT93) and human (HT29/c1) epithelial cells. A-hBFT cleaves E-cadherin in both cell types while I-hBFT serves as negative control. Soluble 80 kDa E-cadherin ectodomain (sECAD) appears in culture supernatants following A-hBFT treatment. ( d ) RT-qPCR analysis of inflammatory cytokine response. A-hBFT significantly upregulates KC / cxcl1 (CMT93) and IL-8 / cxcl8 (HT29/c1) mRNA expression. Data represent mean ± SEM. ** p < 0.01, *** p < 0.001. ns = not significant.

Article Snippet: Human colonic epithelial cell line HT29/c1 (#HTB-38, ATCC, Manassas, VA, USA; passage 130) and mouse rectal epithelial cell line CMT93 (#CCL-223, ATCC, VA, USA; passage 75) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, #MD10000A, Bandio Bio Science, Pocheon, Republic of Korea) supplemented with 5% fetal bovine serum (FBS, #35-015-CV, Corning, Palo Alto, CA, USA), 20 mM HEPES (#15630-080, Gibco, Miami, FL, USA), without penicillin-streptomycin [ , ].

Techniques: Functional Assay, Activity Assay, Clone Assay, Serial Dilution, Biomarker Discovery, Negative Control, Quantitative RT-PCR, Expressing

Journal: Toxins

Article Title: Purification and Characterization of His-Tagged Recombinant Bacteroides fragilis Toxin-2 Variants In Vitro and In Vivo

doi: 10.3390/toxins18040189

Figure Lengend Snippet: BFT receptor-dependent cellular binding patterns across mouse intestinal epithelial cell lines: Confocal microscopy of mouse intestinal epithelial cells treated with SFM, I-hBFT, or A-hBFT. Green fluorescence indicates hBFT detection via anti-6× His tag. ( a ) CMT93 cells (BFT-responsive) show binding only with A-hBFT treatment; ( b ) MSIE cells (BFT-non-responsive) show no binding under any condition; ( c ) YAMC cells (BFT-non-responsive) similarly show no binding signals. Differential binding patterns suggest BFT responsiveness depends on specific receptor presence rather than direct E-cadherin interaction. Scale bar = 20 μm.

Article Snippet: Human colonic epithelial cell line HT29/c1 (#HTB-38, ATCC, Manassas, VA, USA; passage 130) and mouse rectal epithelial cell line CMT93 (#CCL-223, ATCC, VA, USA; passage 75) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, #MD10000A, Bandio Bio Science, Pocheon, Republic of Korea) supplemented with 5% fetal bovine serum (FBS, #35-015-CV, Corning, Palo Alto, CA, USA), 20 mM HEPES (#15630-080, Gibco, Miami, FL, USA), without penicillin-streptomycin [ , ].

Techniques: Binding Assay, Confocal Microscopy, Fluorescence

Journal:

Article Title: Stimulation of Chondrocyte Hypertrophy by Chemokine Stromal Cell-Derived Factor 1 in the Chondro-osseous Junction during Endochondral Bone Formation

doi: 10.1016/j.ydbio.2010.02.033

Figure Lengend Snippet: To determine whether SDF-1 can diffuse into cartilage, 17-day-chicken embryonic sternal cartilage was incubated with SDF-1 (100ng/mL) or without SDF-1 for 1h, 3h, and 24h. 10 µm frozen sections were used to detect SDF-1 by immuno-fluorescent staining with mAb against SDF-1. Fluorescence microscopy showed a progressive increase in SDF-1 staining (red color) surrounding chondrocytes during the 24 h time course (A, B,C) compared to control at 24 h (D). Scale bar = 20 µm.

Article Snippet: Before collecting samples for experiment, the cells were stimulated with SDF-1 (100ng/mL; Cat# 351-FS, R&D Systems, Inc. Minneapolis, MN) for 24h or pretreated with AMD3100 for 2 h (5ug/mL; Cat# 155148–31–5, Sigma-Aldrich, St. Louis, MO), a specific inhibitor for CXCR4, before stimulation with SDF-1.

Techniques: Incubation, Staining, Fluorescence, Microscopy, Control

Journal:

Article Title: Stimulation of Chondrocyte Hypertrophy by Chemokine Stromal Cell-Derived Factor 1 in the Chondro-osseous Junction during Endochondral Bone Formation

doi: 10.1016/j.ydbio.2010.02.033

Figure Lengend Snippet: To confirm that SDF-1 induces chondrocyte hypertrophy in growth plates, 12-day-old chicken tibia growth plates were cultured in the presence of SDF-1 (100ng/mL) or in the absence of SDF-1 for 2, 4, and 6 days. 10 µm frozen sections were used to detect Type X collagen expression by immuno-fluorescent staining with mAb against type X collagen. A progressive increase in the size of the hypertrohic growth plate based on Type X collagen staining was seen. The ratio of the length of the hypertrophic zone to that of the total growth plate was calculated at the different time points (B). (* p<0.05). Scale bar = 100 µm.

Article Snippet: Before collecting samples for experiment, the cells were stimulated with SDF-1 (100ng/mL; Cat# 351-FS, R&D Systems, Inc. Minneapolis, MN) for 24h or pretreated with AMD3100 for 2 h (5ug/mL; Cat# 155148–31–5, Sigma-Aldrich, St. Louis, MO), a specific inhibitor for CXCR4, before stimulation with SDF-1.

Techniques: Cell Culture, Expressing, Staining