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.