Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: Sorting of miR-200c from cells to exosomes via Rab1A in breast cancer. ( A ) Dig-conjugated miR-200c and let-7a complex pulled down from whole cell extracts of 4T1 lung metastasis using anti-digoxin beads and analyzed by electrophoresis followed by Coomassie blue staining. ( B ) MALDI-TOF-MS analysis of tryptic peptides from the band indicated by red arrows in ( A ). ( C ) Western blot analysis of Rab1A proteins from before (top panel) and after anti-Dig Alpha Donor beads pull-down (bottom panel) in 4T1 lung metastasis lysate. ( D ) ChIP analysis of the interaction between Rab1A and miR-200c using anti-Rab1A antibody pull-down exosomal RNAs and access the miR-200c with miRNA qPCR. ( E ) SPR analysis of the interaction between Rab1A recombinant protein and biotinylated (Bio-) miR-200c or miRNA scramble control covalently immobilized onto the streptavidin-coated sensor chip. ( F ) Rab1A knockout (KO) by transfection of Rab1A CRISPR/Cas9 plasmid with and without miR-200c inhibitor in 4T1 cells. qPCR analysis of miR-200c in exosome and donor cell 3 days after transfection. ( G ) Representative Western blot analysis. ( H ) Quantification of cell number after 48 h culture in 24-well plate. **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: To further confirm the direct interaction, DIG-conjugated miR-200c or let-7a (Takara Biomed) was incubated with whole cell extracts of 4T1 lung metastasis, then the miRNA complex was pulled down using anti-Dig Alpha Donor beads (PerkinElmer) in binding buffer (0.1% NP-40, 5 mM EDTA, 0.5 M NaCl, 0.01% SDS).

Techniques: Electrophoresis, Staining, Western Blot, Recombinant, Knock-Out, Transfection, CRISPR, Plasmid Preparation

Journal: Chemical research in toxicology

Article Title: Quantification of acylfulvene- and illudin S-DNA adducts in cells with variable bioactivation capacities.

doi: 10.1021/tx300430r

Figure Lengend Snippet: Figure 1. CNL-MS3 data-dependent analysis of products of reactions of ctDNA with illudin S in the presence of PTGR1 and NADPH. Data are normalized to the base peak in each spectra, and the relative intensity is given as a normalized level (NL). (A) Full-scan MS (NL 1.55E6). (B) Full- scan MS of m/z 384 (NL 9.88E5). (C) Triggered MS2 of m/z 384.44 (NL 1.25E6). (D) Mass spectra of the peak in panel C with major fragment m/z 336 and minor fragments m/z 249 and 201 (inset).

Article Snippet: Stable transfection of 2 × 10 5 SW-480 cells was performed in a six-well plate with 1 μg of Myc-DDK tagged PTGR1 cDNA (OriGene Technologies, Rockville, MD) with Lipofectamine reagents (Invitrogen, Inc.) and selected with Geneticin (Invitrogen, Inc.).

Techniques:

Journal: Chemical research in toxicology

Article Title: Quantification of acylfulvene- and illudin S-DNA adducts in cells with variable bioactivation capacities.

doi: 10.1021/tx300430r

Figure Lengend Snippet: Figure 3. Cell viability of drug-treated SW-480 and PTGR1-480 cells (48 h of treatment time). (A) AF-treated cells (n = 4). (B) Illudin S- treated cells (n = 3).

Article Snippet: Stable transfection of 2 × 10 5 SW-480 cells was performed in a six-well plate with 1 μg of Myc-DDK tagged PTGR1 cDNA (OriGene Technologies, Rockville, MD) with Lipofectamine reagents (Invitrogen, Inc.) and selected with Geneticin (Invitrogen, Inc.).

Techniques:

Journal: Chemical research in toxicology

Article Title: Quantification of acylfulvene- and illudin S-DNA adducts in cells with variable bioactivation capacities.

doi: 10.1021/tx300430r

Figure Lengend Snippet: Figure 2. Western blot analysis of PTGR1 levels in SW-480 and PTGR1-480 cells. Lanes 1, 6, and 10, marker; lanes 2−4, SW-480 cells; lane 5, SW-837 cells (positive control); and lanes 7−9, PTGR1-480 cells. All lanes are loaded with 70 μg of protein, except lane 4 (62 μg).

Article Snippet: Stable transfection of 2 × 10 5 SW-480 cells was performed in a six-well plate with 1 μg of Myc-DDK tagged PTGR1 cDNA (OriGene Technologies, Rockville, MD) with Lipofectamine reagents (Invitrogen, Inc.) and selected with Geneticin (Invitrogen, Inc.).

Techniques: Western Blot, Marker, Positive Control

Journal: Chemical research in toxicology

Article Title: Quantification of acylfulvene- and illudin S-DNA adducts in cells with variable bioactivation capacities.

doi: 10.1021/tx300430r

Figure Lengend Snippet: Figure 5. Time-dependent formation of AF- or illudin S-derived Ade adducts in drug-treated SW-480 and PTGR1-480 cells. (A) 3-AF−Ade, cells treated with 500 nM AF (n = 3). (B) Illudin S−Ade, cells treated with 20 nM illudin S (n = 3). Asterisks denote statistically significant differences between levels in SW-480 vs PTGR1-480 cells (p < 0.05).

Article Snippet: Stable transfection of 2 × 10 5 SW-480 cells was performed in a six-well plate with 1 μg of Myc-DDK tagged PTGR1 cDNA (OriGene Technologies, Rockville, MD) with Lipofectamine reagents (Invitrogen, Inc.) and selected with Geneticin (Invitrogen, Inc.).

Techniques: Derivative Assay

Journal: Chemical research in toxicology

Article Title: Quantification of acylfulvene- and illudin S-DNA adducts in cells with variable bioactivation capacities.

doi: 10.1021/tx300430r

Figure Lengend Snippet: Figure 4. Concentration-dependent formation of Ade adducts in drug- treated SW-480 and PTGR1-480 cells (24 h of treatment time). (A) 3- AF−Ade DNA adducts (n = 4). (B) Illudin S−Ade DNA adducts (n = 3). Asterisks denote statistically significant differences between levels in SW-480 vs PTGR1-480 cells (p < 0.05).

Article Snippet: Stable transfection of 2 × 10 5 SW-480 cells was performed in a six-well plate with 1 μg of Myc-DDK tagged PTGR1 cDNA (OriGene Technologies, Rockville, MD) with Lipofectamine reagents (Invitrogen, Inc.) and selected with Geneticin (Invitrogen, Inc.).

Techniques: Concentration Assay

Journal: Chemical research in toxicology

Article Title: Quantification of acylfulvene- and illudin S-DNA adducts in cells with variable bioactivation capacities.

doi: 10.1021/tx300430r

Figure Lengend Snippet: Figure 6. Ade adduct formation by AF and illudin S in SW-480 and PTGR1-480 cells as a function of fold-IC50.

Article Snippet: Stable transfection of 2 × 10 5 SW-480 cells was performed in a six-well plate with 1 μg of Myc-DDK tagged PTGR1 cDNA (OriGene Technologies, Rockville, MD) with Lipofectamine reagents (Invitrogen, Inc.) and selected with Geneticin (Invitrogen, Inc.).

Techniques:

Journal: Chemical research in toxicology

Article Title: Quantification of acylfulvene- and illudin S-DNA adducts in cells with variable bioactivation capacities.

doi: 10.1021/tx300430r

Figure Lengend Snippet: Figure 7. Model illustrating the steps required to form a DNA adduct from a bioactivated drug. This model is used to explain how different levels of PTGR1 do not influence illudin S−DNA adduct levels.

Article Snippet: Stable transfection of 2 × 10 5 SW-480 cells was performed in a six-well plate with 1 μg of Myc-DDK tagged PTGR1 cDNA (OriGene Technologies, Rockville, MD) with Lipofectamine reagents (Invitrogen, Inc.) and selected with Geneticin (Invitrogen, Inc.).

Techniques: