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: Molecular Therapy Oncolytics

Article Title: Whole cell vaccination using immunogenic cell death by an oncolytic adenovirus is effective against a colorectal cancer model

doi: 10.1038/mto.2016.31

Figure Lengend Snippet: Schematic structure of adenoviral vectors and cytotoxic efficiencies of oncolytic adenoviruses in colorectal cancer (CRC) cells. ( a ) Schematic structure of adenoviral vectors. Schematic structures of Ad881 and Ad884 are shown. ITR, adenovirus inverted terminal repeat sequence; ψ, packaging signal; pA, polyadenylation signal; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein; CMV, cytomegalovirus promoter; Mdk, midkine; HSV-TK, herpes simplex virus-thymidine kinase. ( b )/( c ) Cyotopathic assays using Ad884 in CRC cells and normal fibroblasts. CRC cell lines (DLD-1, CMT93, and CT26) and fibroblasts were infected with Ad884 ( b ) or Ad881 ( c ) at various multiplicity of infections (MOIs) (0, 0.1, 1, 10, 100, or 1,000). On day 8, cytotoxicity was assessed by the extent of crystal violet staining. ( d ) Virus progeny production in CRC cells. DLD-1, CMT93, and CT26 cells, as well as fibroblasts were infected with Ad881 or Ad884 at an MOI of 100. At 48 hours after infection, cells and media were harvested to determine the viral titer in transducing units by EGFP expression using flow cytometry. Virus production levels were normalized to the baseline value in fibroblasts. Data are representative of three independent experiments all yielding similar results. Ad881: black bar; Ad884: hatched bar. ( e ) Time-dependent cytotoxicity. DLD-1 (triangle) and CT26 (circle) cells (1 × 10 4 /well) were cultured as multiple replicates in 96-well plates and infected with Ad881 (closed) or Ad884 (open) at an MOI of 100. On the indicated days, the number of surviving cells was analyzed by a colorimetric method using Alamar blue. Data shown are the mean ± SD of triplicates.

Article Snippet: Mouse CRC cell lines Colon-26 (CT26) and CMT93 were purchased from RIKEN BRC Cell Bank (Ibaraki, Japan) and DS Pharma Biomedical (Osaka, Japan), respectively.

Techniques: Sequencing, Virus, Infection, Staining, Expressing, Flow Cytometry, Cell Culture

Journal: Molecular Therapy Oncolytics

Article Title: Whole cell vaccination using immunogenic cell death by an oncolytic adenovirus is effective against a colorectal cancer model

doi: 10.1038/mto.2016.31

Figure Lengend Snippet: Infection efficacy of the oncolytic adenovirus Ad881 in CT26 cells. Representative bright field (top) and enhanced green fluorescence protein (EGFP) fluorescence (bottom) images of CT26 cells that were left uninfected (left) or infected with Ad881 at an multiplicity of infection (MOI) of 10 (middle) or 1,000 (right).

Article Snippet: Mouse CRC cell lines Colon-26 (CT26) and CMT93 were purchased from RIKEN BRC Cell Bank (Ibaraki, Japan) and DS Pharma Biomedical (Osaka, Japan), respectively.

Techniques: Infection, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Whole cell vaccination using immunogenic cell death by an oncolytic adenovirus is effective against a colorectal cancer model

doi: 10.1038/mto.2016.31

Figure Lengend Snippet: Assessment of vaccine conditions. ( a ) The tumor formation rate in mice following subcutaneous injection with 1 × 10 5 Ad881-infected CT26 cells that had been infected for 10 hours at multiplicity of infections (MOIs) of 0, 10, 100, or 1,000. ( b ) The tumor formation rate in mice following subcutaneous injection of 1 × 10 4 or 1 × 10 5 Ad881-infected CT26 cells that had been infected for 24 hours at MOIs of 0 or 1,000.

Article Snippet: Mouse CRC cell lines Colon-26 (CT26) and CMT93 were purchased from RIKEN BRC Cell Bank (Ibaraki, Japan) and DS Pharma Biomedical (Osaka, Japan), respectively.

Techniques: Injection, Infection

Journal: Molecular Therapy Oncolytics

Article Title: Whole cell vaccination using immunogenic cell death by an oncolytic adenovirus is effective against a colorectal cancer model

doi: 10.1038/mto.2016.31

Figure Lengend Snippet: Assessment of vaccine efficiency. ( a ) Schema of the vaccination protocol used in this study. ( b ) The tumor rejection rate in mice that had been previously vaccinated with 1 × 10 4 of Ad881-infected (MOI: 0 or 1,000) CT26 cells or no previous treatment when these mice were injected with 1 × 10 4 uninfected CT26 as a challenge. ( c ) The tumor rejection rate following challenge with 1 × 10 5 uninfected CT26 cells in mice that were vaccinated with 1 × 10 4 Ad881-infected CT26 cells once or twice (repeatedly). MOI, multiplicity of infection.

Article Snippet: Mouse CRC cell lines Colon-26 (CT26) and CMT93 were purchased from RIKEN BRC Cell Bank (Ibaraki, Japan) and DS Pharma Biomedical (Osaka, Japan), respectively.

Techniques: Infection, Injection