Journal: Toxicology and applied pharmacology

Article Title: Monoubiquitinated γ-H2AX: abundant product and specific biomarker for non-apoptotic DNA double-strand breaks

doi: 10.1016/j.taap.2018.07.007

Figure Lengend Snippet: Panels A-C used antibodies #1. (A) Impact of gel polyacrylamide percentage and transfer conditions to PVDF membranes. Treatments of H460 cells: 6 h with 3 mM hydroxyurea (HU), 6 or 16 h with 5 μM aphidicolin (Aphi) or 1 h with 5 μM bleomycin (Bleo). All γ-H2AX images had 5 s film exposures. (B) Ethanol effect in semi-dry transfer. IMR90 cells were treated with 40 μM etoposide (Eto) for 1 h. (C) Comparison of wet (pH 8.2 and pH 8.8) and semi-dry transfers using 12% ethanol-containing buffers (10 s film exposures for all γ-H2AX images). IMR90 cells were treated with 40 μM etoposide for 1 h. (D) Effectiveness of different antibodies using semi-dry transfer with 12% ethanol. Normal human keratinocytes were treated for 1 h with 30 μM etoposide or 0.5 μM bleomycin. Antibodies #1–3: 1:1000 dilutions and 5 s exposures, antibody #4: 1:1000 dilution and 60 s exposure. (E) Comparison of antibodies #1 and #5 (both at 1:1000 dilutions) in H460 cells treated with bleomycin for 1 h. (F) γ-H2AX blot (Ab#1) in control and H2AX-depleted IMR90 cells treated with 100 μM etoposide for 1h.

Article Snippet: IMR90, WI38, normal human neonatal keratinocytes (PCS-200–010), H460, A549, U2OS, mouse embryonic fibroblasts (MEFs, SCRC-1040), telomerase-immortalized human bronchial epithelial HBEC3 (CRL-4051), Daudi, HCT116, Ramos, Raji and CA46 were obtained from ATCC.

Techniques: Comparison, Control

Journal: Frontiers in Veterinary Science

Article Title: Canine Papillomavirus 2 E6 Does Not Interfere With UVB-Induced Upregulation of p53 and p53-Regulated Genes

doi: 10.3389/fvets.2021.570982

Figure Lengend Snippet: Upregulation of p53 and p53-regulated genes p21 and Bax in canine keratinocytes in response to exposure to ultraviolet B (UVB) light. Normal canine keratinocytes were exposed to UVB (10 mJ/cm 2 ) and cell lysates collected at different times (hours) post-irradiation. Proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies directed against p53, p21, and Bax. β-actin was used a loading control. One representative experiment of three is shown.

Article Snippet: Canine primary epidermal keratinocytes (CPEKs) (ZenBio, Research Triangle Park, NC) , which were isolated from normal tissue and have not been actively transformed, were infected at a multiplicity of 10 PFU/cell with retrovirus containing vector only or wild type E6.

Techniques: Irradiation, SDS Page, Western Blot, Control

Journal: Frontiers in Veterinary Science

Article Title: Canine Papillomavirus 2 E6 Does Not Interfere With UVB-Induced Upregulation of p53 and p53-Regulated Genes

doi: 10.3389/fvets.2021.570982

Figure Lengend Snippet: Upregulation of long non-coding (lnc.) RNA-p21 in canine keratinocytes in response to exposure to ultraviolet B (UVB) light. Normal canine keratinocytes were exposed to UVB (10 mJ/cm 2 ) and cell lysates collected at different times points post-irradiation for RNA isolation. Real time PCR was performed to determine expression of lncRNA-p21. Resulting Cq values were normalized to the Cq value of the reference gene and calibrated to transcript levels in non-UVB exposed keratinocytes. Experiments were performed in duplicate and repeated in three independent experiments. Results of one representative experiment is shown and expressed as mean ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Canine primary epidermal keratinocytes (CPEKs) (ZenBio, Research Triangle Park, NC) , which were isolated from normal tissue and have not been actively transformed, were infected at a multiplicity of 10 PFU/cell with retrovirus containing vector only or wild type E6.

Techniques: Irradiation, Isolation, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Veterinary Science

Article Title: Canine Papillomavirus 2 E6 Does Not Interfere With UVB-Induced Upregulation of p53 and p53-Regulated Genes

doi: 10.3389/fvets.2021.570982

Figure Lengend Snippet: Upregulation of p53 and p53-regulated genes in UVB-exposed canine keratinocytes expressing CPV2 E6. Canine keratinocytes expressing either vector only or CPV2 E6 were exposed to UVB (10 mJ/cm 2 ) and cell lysates collected at 12 and 24 h post-irradiation. (A) Proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies directed against p53, p21, Bak, and Bax. β-actin was used a loading control. One representative experiment of three is shown. (B) Real time PCR was performed to determine expression of lncRNA-p21. Resulting Cq values were normalized to the Cq value of the reference gene and calibrated to transcript levels in vector only non-UVB exposed cells. Experiments were performed in duplicate and repeated in three independent experiments. One representative experiment of three is shown. Results are expressed as mean ± SD. * p < 0.05.

Article Snippet: Canine primary epidermal keratinocytes (CPEKs) (ZenBio, Research Triangle Park, NC) , which were isolated from normal tissue and have not been actively transformed, were infected at a multiplicity of 10 PFU/cell with retrovirus containing vector only or wild type E6.

Techniques: Expressing, Plasmid Preparation, Irradiation, SDS Page, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Frontiers in Veterinary Science

Article Title: Canine Papillomavirus 2 E6 Does Not Interfere With UVB-Induced Upregulation of p53 and p53-Regulated Genes

doi: 10.3389/fvets.2021.570982

Figure Lengend Snippet: CCK8 proliferation assay in UVB-exposed canine keratinocytes expressing CPV2 E6. Canine keratinocytes expressing either vector only or CPV2 E6 were seeded in 6 well tissue culture plates, cultured overnight, and then exposed to UVB (10 mJ/cm 2 ). CCK8 assay was performed at time 0, and then 24 h post-irradiation. Experiment was performed in triplicate and repeated three independent times. Results are expressed as mean ± SEM. * p < 0.05.

Article Snippet: Canine primary epidermal keratinocytes (CPEKs) (ZenBio, Research Triangle Park, NC) , which were isolated from normal tissue and have not been actively transformed, were infected at a multiplicity of 10 PFU/cell with retrovirus containing vector only or wild type E6.

Techniques: Proliferation Assay, Expressing, Plasmid Preparation, Cell Culture, CCK-8 Assay, Irradiation

Journal: PLoS ONE

Article Title: Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

doi: 10.1371/journal.pone.0122056

Figure Lengend Snippet: The 3K3D feeders plated either alone (3T3) or co-cultured with human epidermal keratinocytes (3T3 + Kc) showed degeneration of feeders (arrowhead) and a large well circumscribed keratinocyte (K) colony surrounded by inactivated feeders. The 4K3D feeders plated alone exhibited newly formed compact proliferative feeder cells with high nucleus to cytoplasm ratio (arrows) over a background of enlarged (f) and degenerating cells (white arrow head). The co-culture with human epidermal keratinocytes showed the proliferative foci (arrows). The 3T3 alone and the co-cultures were 2 and 1 weeks old, respectively. Scale bar: 100 μm.

Article Snippet: Primary Human epidermal keratinocytes (Cat No.PH10205A, www.genlantis.com ) were co-cultured with feeder cells by the basic Rheinwald-Green (1975) technique.

Techniques: Cell Culture, Co-Culture Assay

Journal: PLoS ONE

Article Title: Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

doi: 10.1371/journal.pone.0122056

Figure Lengend Snippet: Keratinocyte cultures plated alone without the feeders showing cytological features of attached keratinocytes (K) and the contaminating non-proliferative (f) and proliferative feeders (F). The 3K3D feeder cells that came along with the keratinocytes as contaminants (Left panel) presented no dividing cells and were broad with large nuclei in phase contrast and showed coarse chromatin aggregates (arrows) that stained bright in Hoechst (inset of Hoechst image). The keratinocytes comprised of a mix of small polygonal or broad terminally differentiated cells which presented the dull small nuclei. The 4K3D contaminating feeder cells (Right panel) consisted of well circumscribed keratinocyte colonies (K) enveloped by numerous newly formed narrow-bodied feeder cells (F) with several cell divisions (inset of Hoechst image) in addition to a few broad non-proliferating feeder cells (f) showing vesicular nuclei. Scale bar: 100 μm; Left inset: 200 μm; Right inset: 450 μm.

Article Snippet: Primary Human epidermal keratinocytes (Cat No.PH10205A, www.genlantis.com ) were co-cultured with feeder cells by the basic Rheinwald-Green (1975) technique.

Techniques: Staining

Journal: PLoS ONE

Article Title: Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

doi: 10.1371/journal.pone.0122056

Figure Lengend Snippet: The 2 nd passage culture of the clone exposed to Mitomycin C and replated alone (A) exhibited the proliferative focus consisting of the newly formed compact cells (small arrows) surrounded by the broad cells (arrowhead) after 14 days of incubation. The co-culture of the growth-arrested clone cells with the human epidermal keratinocytes (B) presented the well-spread out degenerating cells (arrowhead) and the newly formed cells (small arrows) at the periphery of a large colony of keratinocytes (K) after 7 days. Scale bar: 100 μm.

Article Snippet: Primary Human epidermal keratinocytes (Cat No.PH10205A, www.genlantis.com ) were co-cultured with feeder cells by the basic Rheinwald-Green (1975) technique.

Techniques: Incubation, Co-Culture Assay