Normal human oral keratinocytes were prepared from
keratinized oral epithelial tissue.
Detached oral keratyinocytes were seeded onto
collagen-treated flaks and cultured in Keratinocyte
Growth Medium (KGM)
The replication kinetics was focused on number of
normal human oral keratinocytes (NHOK)
Established primary keratinocytes from epidermis
(NHEK)
Retroviruses expressing Bmi-1
Lentivirus-based shRNA expression plasmid pLL.3-7
capable of knocking down the expression of endogenous
Bmi-1
NKOH cultures, infected with RV-Bm1 and LV-Bmi-1i,
gave more than 90% infection.
For retroviruses, selection of cell began at 48 hours after
infection with 1 μg/ml puromycin.
It’s the process of introducing nucleic acid into cells
Genetic materials, or even protein such as antibodies,
may be transfected.
The retroviruses, RV-B0 and RV-Bmi-1, were prepared
for transfection
Two days after transfection, the virus supernatant was
collected and concentrated by ultracentrifugation.
Total RNA was isolated from cultured cells and subjected
to the optional column Dna digestion to elimitate any
contaminating genome.
Real-time PCR was performed in triplicates for each
sample. A total of 45 cycles were executed.
The PCR’ s main aim is to amplify a few copies of a piece
of DNA across several orders of magnitude; the method
relies on thermal cycling.
Analytical technique which include using antibodies to
detect protein in tissue and cell by immunostaining and
enzyme-linked immunosorbent assay (ELISA).
The membrane was incubated with primary antibodies
and probed with the respective secondary antibodies.
Antibodies used: p15, p57, p53, TGF- β1, p-Smad2/3
TGFB1 is a multifunctional peptide that controls
proliferation, differentiation and other functions in many
cell types, also acts with TGFA in traformation.
To measure secreted TGF-B1 by NHOK, supernatants
with or without Bmi-1were collected, centrifuged and
stored in -80 °C.
It’s related with Luciferase activity
Luciferase is a class of oxidative enzymes used in
bioluminescence.
Luciferase con be used for the level of cellular ATP in cell
viability assays or for kinase activity assay.
NHOK were plated onto the 12- well dish with 40000
cells per well.
After 24 hours, cells treated with o without TGF-β kinase
inhibitor.
Cells collected for cell counting or changed the medium
every two days.
I. Senescing NHOK were treated with 10 ng/ml of α-TGF-
β1 for 6 days
Dark green colors indicate the presence of SA β-Gal
activity, and it was observed under the microscope.
The percentages of β-Gal-positive cells were
obtained, counting in a random manner one thousands
cells.