2. 
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)


3. 



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.

4. 



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.

5. 


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.

6. 


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

7. 

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.

8. 


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.

9. 


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.

10. 


I. Senescing NHOK were treated with 10 ng/ml of α-TGFβ1 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.