Journal: Gene therapy

Article Title: Retrofitting of a satellite repeat DNA-based murine artificial chromosome (ACes) to contain loxP recombination sites.

doi: 10.1038/sj.gt.3301757

Figure Lengend Snippet: Figure 1 Generation of murine ACes cell line 5B11-12. (a) Shown is pFKPuro, used in generation of 5B11-12 ACes. The murine rDNA insert comprises a portion of a 45S pre-rRNA gene as indicated (see Csonka et al8). Black boxes within the 45S pre-rRNA transcript indicate position of transcribed spacers which flank the regions coding for rRNA subunits. ∗Indicates location of PCR primer used in screening for targeted events. (b) FISH analysis of puromycin resistant LMTK clone 5B11-12 showing an ACes (indicated by arrow) hybridizing strongly with a digoxigenin (DIG)-labeled mouse major satellite repeat probe (red signal). This probe also hybridizes with the pericentric heterochromatin of all other LMTK- chromosomes. A biotinylated pFKPuro probe hybridized to two regions of the ACes (green signal). (c–e) Split images of ACes depicted in (b). (c) DAPI stain. (d) DAPI stain and DIG-labeled mouse major satellite probe (red signal). (e) DAPI stain and biotinylated pFKPuro probe (green signal). Note that the mouse major satellite probe hybridizes to the regions staining most intensely with DAPI and the two regions of lower intensity DAPI staining co-localize to regions of pFKPuro hybridization. (f) FISH analysis of flow sorted 5B11-12 ACes (population R1 in (i)) using DIG- labeled mouse major satellite probe (red signal) and biotinylated pFKPuro probe (green signal). (g) FISH analysis of flow sorted 5B11-12 ACes using DIG- labeled mouse major satellite probe (red signal) and biotinylated mouse minor satellite probe (green signal). (h) Flow karyogram of meta- phase chromosomes from wildtype LMTK cells. Larger chromosomes bind proportionately more chromomycin A3 and Hoechst 33258 dye mol- ecules. Note that most chromosomes fall on a diagonal line indicative of roughly equimolar binding of chromomycin A3 and Hoechst 33258 dyes. (i) Flow karyogram of metaphase chromosomes from 5B11-12 cells. Due to their relative AT richness, the ACes (circled region R1) binds more Hoechst 33258 dye than chromomycin A3 and is easily resolved from other LMTK chromosomes. Methods: pFKPuro is derived from pFK161,8 a cosmid containing a 8.2 kb mouse rDNA insert, by replacement of the neomycin resistance gene with the puromycin resistance gene of pPUR (Clontech, Palo Alto, CA, USA) using standard cloning methods.16 Cul- tures of LMTK cells (ATCC CCL-1.3) were maintained as previously described.4 1.5 × 106 LMTK cells were transfected with 20 g of SpeI- linearised pFKPuro by calcium phosphate co-precipitation.17 Drug selec- tion was carried out in medium supplemented with 10 g/ml Puromycin (Sigma, St Louis, MO, USA). After 10 days of selection, puromycin resist- ant colonies were picked and individually expanded for subsequent analy- sis and freeze down. Puromycin resistant LMTK clones were analyzed by FISH as described. pFKPuro was labeled with biotin-dUTP using a nick-translation kit (Roche Diagnostics, Laval, QC, Canada). DIG-labeled mouse major satellite probe and biotinylated mouse minor satellite probe were generated by PCR using sequence-specific primers and DIG-dUTP or biotin-dUTP, as appropriate (5’ mmajor: 5’-ATACTCTTCAG- GACCTGGAATATGGCGAG-3’, 3’ mmajor: 5’-ATACTCTTCGTCC TTCAGTGTGCATTTCTCATTTTTC-3’; 5’ mminor: 5’- GGAAAAT- GATAAAAACCTAC-3’, 3’ mminor: 5’- ATGTTTCTAATT GTAACTCAT-3’). All primers were synthesized by Invitrogen (Burlington, ON, Canada). Preparation and flow cytometry analysis of metaphase chromosomes was carried out as previously described.2

Article Snippet: Methods: pFKPuro is derived from pFK161,8 a cosmid containing a 8.2 kb mouse rDNA insert, by replacement of the neomycin resistance gene with the puromycin resistance gene of pPUR (Clontech, Palo Alto, CA, USA) using standard cloning methods.16 Cultures of LMTK cells (ATCC CCL-1.3) were maintained as previously described.4 1.5 × 106 LMTK cells were transfected with 20 g of SpeIlinearised pFKPuro by calcium phosphate co-precipitation.17 Drug selection was carried out in medium supplemented with 10 g/ml Puromycin (Sigma, St Louis, MO, USA).

Techniques: Labeling, Staining, Hybridization, Binding Assay, Derivative Assay, Cloning, Transfection, Selection, Clone Assay, Nick Translation, Generated, Sequencing, Synthesized, Cytometry

Journal: bioRxiv

Article Title: Rat hepatitis E virus (HEV) cross-species infection and transmission in pigs

doi: 10.1101/2023.07.06.547957

Figure Lengend Snippet: (A) Workflow of HEV capping and transfection of target cells. LMTK, A549, Huh7, ST cell lines were transfected with in vitro transcribed capped HEV RNA (rat HEV). (B) Flow cytometry quantification of LMTK, A549, Huh7, ST cells transfected with capped RNA transcripts of rat HEV; Huh7 transfected with cell culture adaptive Kernow-C1 genotype 3 P6 strain and non-cell culture adaptive genotype 1 Sar55 and Kernow-C1 genotype 3 P1 strain. Sar55, P6 and P1 belong to Paslahepevirus is used as the control to determine the replication ability of rat HEV. The assay was performed in the cells harvested on day 5 post transfection. Samples were fixed in methanol and probed with rabbit anti-ORF2 followed by goat anti-rabbit alexa fluor 594 antibodies. Each bar (mean ± SD) represents separate transfections stained in parallel and displays the mean of two independent biological experiments with three replicates per sample. (C) Immunofluorescence detection of HEV ORF2 antigen in methanol fixed LMTK, A549, Huh7, ST cells after 5 days post transfection. Cells are stained with goat anti-rabbit IgG H&L combined with anti-rabbit Alexa fluor 594 (red), and 4’ s, 6-diamidino-2-phenylindole (DAPI) (blue). (D) RT-qPCR data from the supernatants collected from day 5 of replication assay. (E) RT-qPCR data from the cell lysates collected from day 5 of replication assay.

Article Snippet: LMTK (isolated from mouse subcutaneous tissue, ATCC:CCL-1.3), A549 (isolated from human lung carcinoma, ATCC:CRM-CCL-185), Huh7 (isolated from human liver carcinoma), ST (isolated from swine testicular, ATCC:CRL-1746) and Huh7 (human hepatoma cells) S10-3 subclone ( , ) were used for the studies.

Techniques: Transfection, In Vitro, Flow Cytometry, Cell Culture, Control, Staining, Immunofluorescence, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α

doi: 10.3389/fonc.2021.692282

Figure Lengend Snippet: The cytoplastic expression of LMTK3 with significant difference between EEC tumor and para-tumor groups of tissue microarray. (A) Low and high staining of LMTK3 in endometrioid adenocarcinoma tissues; scale bar: 50 µM. (B) Scatter plot of cytoplastic LMTK3 expression with significant difference between EEC tumor and para-tumor groups, * P < 0.05.

Article Snippet: The following antibodies were used: ERα rabbit monoclonal (#8644, Cell Signaling) and LMTK3 mouse monoclonal (#M06A, Abnova and #110516, Abcam).

Techniques: Expressing, Microarray, Staining

Journal: Frontiers in Oncology

Article Title: Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α

doi: 10.3389/fonc.2021.692282

Figure Lengend Snippet: Effects of LMTK3 knockdown on cell viability in Ishikawa cells by CCK-8 (n=3, ** P < 0.01, *** P < 0.001).

Article Snippet: The following antibodies were used: ERα rabbit monoclonal (#8644, Cell Signaling) and LMTK3 mouse monoclonal (#M06A, Abnova and #110516, Abcam).

Techniques: Knockdown, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α

doi: 10.3389/fonc.2021.692282

Figure Lengend Snippet: Effects of LMTK3 knockdown on cell cycle arrest in Ishikawa cells by flow cytometry. (A) Representative diagrams of LMTK3 knockdown on cell cycle arrest in Ishikawa cells. (B) Statistical histogram of LMTK3 knockdown on cell cycle arrest in Ishikawa (n=3, * P < 0.05, *** P < 0.001). ns represents no statistical significance).

Article Snippet: The following antibodies were used: ERα rabbit monoclonal (#8644, Cell Signaling) and LMTK3 mouse monoclonal (#M06A, Abnova and #110516, Abcam).

Techniques: Knockdown, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α

doi: 10.3389/fonc.2021.692282

Figure Lengend Snippet: Effects of LMTK3 knockdown on apoptosis in Ishikawa cells by flow cytometry. (A) Representative diagrams of LMTK3 knockdown on apoptosis in Ishikawa cells. (B) Statistical histogram of LMTK3 knockdown on apoptosis in Ishikawa (n=3, ** P < 0.01).

Article Snippet: The following antibodies were used: ERα rabbit monoclonal (#8644, Cell Signaling) and LMTK3 mouse monoclonal (#M06A, Abnova and #110516, Abcam).

Techniques: Knockdown, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α

doi: 10.3389/fonc.2021.692282

Figure Lengend Snippet: A positive correlation between the mRNA expression of LMTK3 and ESR1 .

Article Snippet: The following antibodies were used: ERα rabbit monoclonal (#8644, Cell Signaling) and LMTK3 mouse monoclonal (#M06A, Abnova and #110516, Abcam).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α

doi: 10.3389/fonc.2021.692282

Figure Lengend Snippet: Knockdown of LMTK3 reduces the mRNA and protein levels of ERα. (A, B) Effects of LMTK3 knockdown on the mRNA level of ERα (n=3, ** P < 0.01, *** P < 0.001), ns represents no statistical significance). (C) Representative image of LMTK3 knockdown on the protein level of ERα. (D, E) Effects of LMTK3 knockdown on the protein level of ERα (n=3, *** P < 0.001). siControl represents nontargeting siRNA. Mock represents nontreatment. ns represents no statistical significance).

Article Snippet: The following antibodies were used: ERα rabbit monoclonal (#8644, Cell Signaling) and LMTK3 mouse monoclonal (#M06A, Abnova and #110516, Abcam).

Techniques: Knockdown

Journal:

Article Title: Toward the development of multi-epitope p53 cancer vaccines: an in vitro assessment of CD8 + T-cell responses to HLA class I-restricted wild-type sequence p53 peptides

doi: 10.1016/j.clim.2007.05.015

Figure Lengend Snippet: Characteristics of the HLA-A2 and/or A24-positive patients with SCCHN tested in this study

Article Snippet: All subjects were HLA-A2 + and/or HLA-A24 + , as determined by flow cytometry using HLA-A2-specific antibodies (MA2.1 and BB7.2) and an HLA-A24-specific antibody (anti-HLA-A23, A24 mAb, IgG2b) purchased from American Type Culture Collection (ATCC, Rockville, MD) and One Lambda, Inc. (Canoga Park, CA), respectively. table ft1 table-wrap mode="anchored" t5 caption a7 patient age/sex HLA typing A2/A24 tumor site stage histology disease status at time of evaluation a SCCHN-1 53M +/− Oral cavity III SCC (mod) Local Rec.

Techniques: Immunopeptidomics

Journal:

Article Title: Toward the development of multi-epitope p53 cancer vaccines: an in vitro assessment of CD8 + T-cell responses to HLA class I-restricted wild-type sequence p53 peptides

doi: 10.1016/j.clim.2007.05.015

Figure Lengend Snippet: HLA-A2 and -A24 restricted wt-p53 derived peptides used in this study

Article Snippet: All subjects were HLA-A2 + and/or HLA-A24 + , as determined by flow cytometry using HLA-A2-specific antibodies (MA2.1 and BB7.2) and an HLA-A24-specific antibody (anti-HLA-A23, A24 mAb, IgG2b) purchased from American Type Culture Collection (ATCC, Rockville, MD) and One Lambda, Inc. (Canoga Park, CA), respectively. table ft1 table-wrap mode="anchored" t5 caption a7 patient age/sex HLA typing A2/A24 tumor site stage histology disease status at time of evaluation a SCCHN-1 53M +/− Oral cavity III SCC (mod) Local Rec.

Techniques: Derivative Assay, Sequencing

Journal:

Article Title: Toward the development of multi-epitope p53 cancer vaccines: an in vitro assessment of CD8 + T-cell responses to HLA class I-restricted wild-type sequence p53 peptides

doi: 10.1016/j.clim.2007.05.015

Figure Lengend Snippet: Healthy donor CD8+ T-cell responses to wt p53-derived peptides

Article Snippet: All subjects were HLA-A2 + and/or HLA-A24 + , as determined by flow cytometry using HLA-A2-specific antibodies (MA2.1 and BB7.2) and an HLA-A24-specific antibody (anti-HLA-A23, A24 mAb, IgG2b) purchased from American Type Culture Collection (ATCC, Rockville, MD) and One Lambda, Inc. (Canoga Park, CA), respectively. table ft1 table-wrap mode="anchored" t5 caption a7 patient age/sex HLA typing A2/A24 tumor site stage histology disease status at time of evaluation a SCCHN-1 53M +/− Oral cavity III SCC (mod) Local Rec.

Techniques:

Journal:

Article Title: Toward the development of multi-epitope p53 cancer vaccines: an in vitro assessment of CD8 + T-cell responses to HLA class I-restricted wild-type sequence p53 peptides

doi: 10.1016/j.clim.2007.05.015

Figure Lengend Snippet: CD8+ T-cell responses to wt-p53 derived peptides in HLA-A2-positive patients with SCCHN

Article Snippet: All subjects were HLA-A2 + and/or HLA-A24 + , as determined by flow cytometry using HLA-A2-specific antibodies (MA2.1 and BB7.2) and an HLA-A24-specific antibody (anti-HLA-A23, A24 mAb, IgG2b) purchased from American Type Culture Collection (ATCC, Rockville, MD) and One Lambda, Inc. (Canoga Park, CA), respectively. table ft1 table-wrap mode="anchored" t5 caption a7 patient age/sex HLA typing A2/A24 tumor site stage histology disease status at time of evaluation a SCCHN-1 53M +/− Oral cavity III SCC (mod) Local Rec.

Techniques: Derivative Assay

Journal:

Article Title: Toward the development of multi-epitope p53 cancer vaccines: an in vitro assessment of CD8 + T-cell responses to HLA class I-restricted wild-type sequence p53 peptides

doi: 10.1016/j.clim.2007.05.015

Figure Lengend Snippet: Positive (+) and negative (-) responsiveness to HLA class I-restricted wt p53 peptides of CD8+ T cells present in PBMC obtained from HLA-A2+ and/or HLA-A24+ subjects with SCCHN following 1x IVS are plotted against (A) Th1/Th2 ratio, (B) Tc1/Tc2 ratio and (c) % Treg of subject's PBMC. As indicated in panel (A) positive wt p53 peptide-specific CD8+ T cell responses were only significant relative to Tc1/Tc2 ratios of subject's PBMC.

Article Snippet: All subjects were HLA-A2 + and/or HLA-A24 + , as determined by flow cytometry using HLA-A2-specific antibodies (MA2.1 and BB7.2) and an HLA-A24-specific antibody (anti-HLA-A23, A24 mAb, IgG2b) purchased from American Type Culture Collection (ATCC, Rockville, MD) and One Lambda, Inc. (Canoga Park, CA), respectively. table ft1 table-wrap mode="anchored" t5 caption a7 patient age/sex HLA typing A2/A24 tumor site stage histology disease status at time of evaluation a SCCHN-1 53M +/− Oral cavity III SCC (mod) Local Rec.

Techniques: