Journal: Molecular Cancer Therapeutics

Article Title: Novel Anti-LY6G6D/CD3 T-Cell–Dependent Bispecific Antibody for the Treatment of Colorectal Cancer

doi: 10.1158/1535-7163.mct-21-0599

Figure Lengend Snippet: Figure 1. LY6G6D is differentially overexpressed in colorectal cancer. A, LY6G6D differential expression in samples in TCGA RNA-seq dataset. Gene expression is displayed in normalized Reads Per Kilobase of exon model per Million mapped reads (nRPKM). B, LY6G6D expression in MSS/MSI-L/MSI-H colorectal cancer samples in TCGA dataset. MSS: n ¼ 108; MSI-L: n ¼ 36; MSI-H: n ¼ 35. C, LY6G6D expression in human normal tissue samples in the GTEx RNA-seq dataset. A–C, Boxes represent the interquartile range (IQR, 25th to 75th percentile), the horizontal line is the median. Whiskers extend to the most extreme data point that is within 1.5IQR of the 25th and 75th percentiles. Data points beyond the whiskers represent outliers. D, Representative images of LY6G6D IHC staining in normal colon and primary MSS colorectal cancer samples (n ¼ 107). E, Representative images of LY6G6D IHC staining in colorectal cancer liver metastases (n ¼ 66). Numbers of positive samples under each IHC score are derived at tumor cell expression cutoff of 50%, which requires that at least 50% of the tumor cells stained positive for LY6G6D at the denoted levels. IHC score: 0 ¼ negative; 1þ ¼ low signal; 2þ ¼ moderate signal; 3þ ¼ strong signal.

Article Snippet: Colorectal cancer cell lines (LS1034, HT55, GP2D, and Colo320DM) were sourced from the Genentech cell line repository that was originally obtained from either the ATCC or ECACC.

Techniques: Quantitative Proteomics, RNA Sequencing, Gene Expression, Expressing, Immunohistochemistry, Derivative Assay, Staining

Journal: Molecular Cancer Therapeutics

Article Title: Novel Anti-LY6G6D/CD3 T-Cell–Dependent Bispecific Antibody for the Treatment of Colorectal Cancer

doi: 10.1158/1535-7163.mct-21-0599

Figure Lengend Snippet: Figure 4. LY6G6D-TDB isactive against colorectal cancer cell lineswith a broad range of LY6G6D level. A, IHCstainingon colorectal cancer cell pellets. B, LY6G6D-binding sites on cell surface determined by flow cytometry. C, Cell killing by 1G4/CD3H, 48-hour time point. Cell killing data are shown as means SD of triplicate wells. Representative data of assays with three PBMC donors.

Article Snippet: Colorectal cancer cell lines (LS1034, HT55, GP2D, and Colo320DM) were sourced from the Genentech cell line repository that was originally obtained from either the ATCC or ECACC.

Techniques: Binding Assay, Cytometry

Journal: PLoS ONE

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells

doi: 10.1371/journal.pone.0175585

Figure Lengend Snippet: COLO 320DM cells (A) or CHO DG44 cells (B) were transfected with the plasmid pKV or pKV-AR1 or a mixture of the plasmid and the direct (Dir) or the inverted (Inv) repeats of G5AR1, G5, or a part of λ-phage. As a control, pG5 or pΔBM d2EGFP plasmid was transfected. Two days after the transfection, 5 μg/ml blasticidin was added, and the cells were cultured for a further week. The number of large (more than ca. 150 cells) or small (ca. 50 to 150 cells) colonies per 10 5 transfected cells was counted and plotted. A representative result of three independent experiments that gave similar results is shown.

Article Snippet: The human colorectal carcinoma COLO 320DM cell line (ATCC CCL 220) was cultured in RPMI 1640 medium (Nissui Pharmaceutical Co. Ltd.) supplemented with 10% FCS.

Techniques: Transfection, Plasmid Preparation, Control, Cell Culture

Journal: PLoS ONE

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells

doi: 10.1371/journal.pone.0175585

Figure Lengend Snippet: After single transfection or co-transfection of the indicated DNA into COLO 320DM cells, the stable transformants were selected by blasticidin for 1 month. The metaphase chromosome spreads from the transformants were analyzed by FISH using a DIG-probe prepared from pG5 plasmid DNA. The hybridized probes were detected by green (FITC) fluorescence, and DNA was counterstained red with propidium iodide. Representative images for each type of amplification are shown in A. The frequency of cells having each type of extrachromosomal (B) or chromosomal (C) amplification was scored by examination of more than 30 metaphase cells in triplicate, and mean +/- standard deviations are plotted.

Article Snippet: The human colorectal carcinoma COLO 320DM cell line (ATCC CCL 220) was cultured in RPMI 1640 medium (Nissui Pharmaceutical Co. Ltd.) supplemented with 10% FCS.

Techniques: Transfection, Cotransfection, Plasmid Preparation, Fluorescence, Amplification

Journal: PLoS ONE

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells

doi: 10.1371/journal.pone.0175585

Figure Lengend Snippet: A mixture of pKV-AR1 DNA and G5 inverted (A and B) or direct (C and D) repeat DNA was co-transfected into COLO 320DM cells, and the stable transformants were selected by blasticidin for a month. Metaphase chromosome spreads (A to C) or chromatin fibers (D) were prepared from these cells. The slides were simultaneously hybridized with biotin-labeled G5 probe and DIG-labeled pKV-AR1 plasmid probe, and the hybridized probes were detected by red (Alexa594) or green (FITC) fluorescence, respectively. DNA was counterstained blue with DAPI in panels A to C. Among metaphase chromosomes, strong G5 signals and weak pKV-AR1 signals were co-localized at multiple DMs (A) or HSRs (B and C). In panel D, small pKV-AR1 signals (green arrowheads) were periodically embedded in the long stretch of G5 signals (red broken lines).

Article Snippet: The human colorectal carcinoma COLO 320DM cell line (ATCC CCL 220) was cultured in RPMI 1640 medium (Nissui Pharmaceutical Co. Ltd.) supplemented with 10% FCS.

Techniques: Transfection, Labeling, Plasmid Preparation, Fluorescence

Journal: PLoS ONE

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells

doi: 10.1371/journal.pone.0175585

Figure Lengend Snippet: After transfection of the indicated DNA into COLO 320DM or CHO DG44 cells, the stable transformants were selected by blasticidin for 1 month. The genomic DNA was isolated and subjected to real-time PCR to determine the amount of SRα promoter, G5, λ-phage, and Gapdh sequence. The PCR reaction was done in triplicate, and the mean value was used to calculate the copy number of each sequence relative to Gapdh (see graph). The deviation between triplicate reactions was too small to be represented with error bars.

Article Snippet: The human colorectal carcinoma COLO 320DM cell line (ATCC CCL 220) was cultured in RPMI 1640 medium (Nissui Pharmaceutical Co. Ltd.) supplemented with 10% FCS.

Techniques: Transfection, Isolation, Real-time Polymerase Chain Reaction, Sequencing

Journal: PLoS ONE

Article Title: Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells

doi: 10.1371/journal.pone.0175585

Figure Lengend Snippet: After transfection of the indicated DNA into COLO 320DM (A) or CHO DG44 (B) cells, the stable transformants were selected by blasticidin for 34 or 29 days, respectively. The d2EGFP expression was measured using flow cytometry, and the mean fluorescence intensity in arbitrary units was plotted. (C) The indicated COLO 320DM transformants were cultured for 34, 62, and 82 days after transfection, and analyzed by flow cytometry in the absence (blue filled line) or presence (unfilled line) of 2 mM sodium butyrate during the last 3 days. The mean fluorescence intensity for butyrate (-) and (+; parenthesized) culture was noted in each chart.

Article Snippet: The human colorectal carcinoma COLO 320DM cell line (ATCC CCL 220) was cultured in RPMI 1640 medium (Nissui Pharmaceutical Co. Ltd.) supplemented with 10% FCS.

Techniques: Transfection, Expressing, Flow Cytometry, Fluorescence, Cell Culture