Journal: Cells

Article Title: Fixed-Bed Bioreactor Culture Enhances Yield and Reparative Properties of hTERT Mesenchymal Stem Cell Extracellular Vesicles

doi: 10.3390/cells15070654

Figure Lengend Snippet: EV function and signaling activity. ( a ) Primary keratinocytes were exposed to IR and treated with EVs from either flask or bioreactor cultures. Cell viability was assessed at 24, 48, and 72 h using the CellTiter-Glo assay. ( b ) Non-irradiated keratinocytes were treated with EVs from flask or bioreactor cultures, and viability was assessed after 72 h using the same assay. * = p < 0.05; *** = p < 0.001; **** = p < 0.0001. ( c ) Western blot of cell cycle proteins associated with hTERT MSC EVs produced from both the bioreactor and flask-based cultures. ( d ) In vitro kinase assay of EV-associated CDK2. EVs were immunoprecipitated with CDK2 antibodies or IgG control and kinase activity was assessed using Histone H1 as substrate.

Article Snippet: Normal primary epidermal keratinocytes (ATCC ® PCS-200-011TM) were maintained in Dermal Cell Basal Medium (ATCC ® PCS-200-030TM) supplemented with Keratinocyte Growth Kit (ATCC ® PCS-200-040TM).

Techniques: Activity Assay, Glo Assay, Irradiation, Western Blot, Produced, In Vitro, Kinase Assay, Immunoprecipitation, Control

Journal: Biology Direct

Article Title: BRD4 sustains p63 transcriptional program in keratinocytes

doi: 10.1186/s13062-024-00547-1

Figure Lengend Snippet: BRD4 and p63 regulates keratinocytes proliferation and differentiation. a, b, e, f Cell cycle analysis performed in HEKn comparing SCR condition and sip63 and siBRD4 conditions comparing untreated cells (DMSO) and JQ1 treated cells (10 µM). Cells were stained with Propidium Iodide (50 µg/ml) for 1 h and then analysed by flow cytometry. In a, e representative plot of cell cycle analysis performed in HEKn is shown, while in b, f a quantification of cell cycle analysis. Graphs present means ± SD of three independent experiments. c, g EdU-incorporation assay of HEKn cells transfected with siSCR, sip63 and different BRD4 (siBRD4#1, BRD4#2) siRNAs or treated with DMSO and JQ1 (10 µM). Data are shown as mean ± SD of N = 3 experiments. (Unpaired Student’s t test). d, h Growth curve of HEKn cells transfected with siSCR, sip63 and siBRD4#1, or treated with DMSO and JQ1 (10 µM). The cell confluency has been determined using the Incucyte real-time video imaging system. Each data points indicate mean ± SEM. i – n Western blot analysis was carried out with specific antibodies against K10, and β-actin was used as loading control. ImageJ program was used to quantitate the protein levels. The blot is representative of three independent experiments. DD, days of differentiation

Article Snippet: Primary Normal Human Epidermal Keratinocytes, neonatal (HEKn) (Gibco, catalog no. C-001-5C) and human TERT-immortalized keratinocytes (Ker-CT) (ATCC, CRL4048, lot. no. 0213) were cultured in EpiLife medium with the addition of Human Keratinocyte Growth Supplements (HKGS, Life Technologies).

Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Transfection, Imaging, Western Blot, Control

Journal: Biology Direct

Article Title: BRD4 sustains p63 transcriptional program in keratinocytes

doi: 10.1186/s13062-024-00547-1

Figure Lengend Snippet: Keratinocytes lacking for p63 and BRD4 have similar expression profiles. Gene expression analysis performed using nanoString technology on HEKn cells after p63 and BRD4 silencing and JQ1 treatment. a Venn diagram represents common dysregulated genes in the different conditions. b – d Pathway gene ontology of common dysregulated genes. e – g Expression levels of the shared dysregulated genes from the top five enriched categories of the GO-pathway analysis

Article Snippet: Primary Normal Human Epidermal Keratinocytes, neonatal (HEKn) (Gibco, catalog no. C-001-5C) and human TERT-immortalized keratinocytes (Ker-CT) (ATCC, CRL4048, lot. no. 0213) were cultured in EpiLife medium with the addition of Human Keratinocyte Growth Supplements (HKGS, Life Technologies).

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

doi: 10.1038/s41598-018-30562-y

Figure Lengend Snippet: Keap1 interacts with MCM3 in mammalian cells. ( a ) Western blots with antibodies against indicated proteins either with nuclear (‘N’) or cytoplasmic (‘C’) extracts of the FLAG-MCM3 expressing CHO-EBNALT85 cells (‘input’), or in MCM3 complexes immunoprecipitated with anti-FLAG affinity beads (‘flag IP’). Histone H3 and GAPDH were used as fractionation controls. See Supplementary Fig. for full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels (top panels) and Western blots with antibodies against indicated proteins (bottom panels) showing distribution of FLAG-MCM3 immunoprecipitated nuclear and cytoplasmic protein complexes in the Superdex 200 size exclusion chromatography. ‘flag’ depicts the lanes with input material. Co-elution of molecular weight markers is indicated at the bottom. See Supplementary Fig. for full-length gels and blots. ( c ) Proximity ligation analysis (PLA) of the Keap1 - MCM3 interaction in human primary epithelial keratinocytes (HPEK). The images of red PLA channel alone are shown in the left column, and combined with blue DAPI staining of nuclei in the right column. ‘Keap1 + MCM3’ indicates the images with interaction specific signals, other images correspond to the control experiments with single antibodies. Shown are the maximum intensity projection images of the Z stacks from confocal microscopy; white scale bar = 10 µM. ( d ) Scatter dot plot of the quantified data of nuclear and cytoplasmic Keap1 + MCM3 PLA signals (M3 + K1) compared to negative control with MCM3 antibody alone (M3). Each data point represents an average number of nuclear or cytoplasmic PLA dots per cell from one micrograph. Bars represent the mean and standard deviation of combined data from two independent PLA experiments, one slide analysed in first and two in second experiment and three different micrographs quantified from each slide. The significance values (***p < 0.0005) are derived from unpaired two-tailed t test. ( e ) Immunofluorescence images of the overall distribution of MCM3 and Keap1 proteins in HPEK cells. The images from green protein channel alone are in the left column, and combined with the blue nuclear DAPI staining in the right column. White scale bar = 10 µM.

Article Snippet: Human primary epidermal keratinocyte progenitor cells (single juvenile donor, passage 2) were purchased from CELLnTEC Advanced Cell Systems and grown for no more than 8 passages in CnT-Prime media provided by the supplier.

Techniques: Western Blot, Expressing, Immunoprecipitation, Fractionation, Staining, SDS Page, Size-exclusion Chromatography, Co-Elution Assay, Molecular Weight, Ligation, Control, Confocal Microscopy, Negative Control, Standard Deviation, Derivative Assay, Two Tailed Test, Immunofluorescence

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