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Published: September 11, 2022 (2 weeks ago)
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Download Latest Hindi HD 1080p Films and Latest Hindi Movies HD Download Full Version Hindi Movies Bollywood Hollywood movies,2% FCS in DMEM) and treated with 5 ng/ml TGF-β1 for 24 h. The cells were then incubated in DMEM containing 10% FCS overnight. Cells were transiently transfected with siRNA using Lipofectamine 3000. The siRNA sequences were as follows: Smad2, 5′-GAGGAAUGAAGUUAAACUG-3′; Smad3, 5′-CAGGCGGCUCUUAGAAUAA-3′; Smad4, 5′-GCCAAGUGUUCAAUAAUAU-3′; TGFBR2, 5′-CGGGUUCGUAGUUUUUCAU-3′; and TGFBR1, 5′-CCGUUCUAUAUCUCCAAGU-3′.

CCK-8 assay
———–

Cells were seeded in 96-well plates at a density of 3 × 10^3^/well and incubated for 24 h. The cells were then treated with 5 ng/ml TGF-β1 for 48 h and were incubated for 2 h with a CCK-8 solution (Dojindo, Kumamoto, Japan). The optical density (OD) of each well at 450 nm was measured with an ELISA plate reader (Spectramax M2, Molecular Devices, USA).

Apoptosis assay
—————

Cell apoptosis was evaluated using a commercial kit (BD Pharmingen, CA, USA) based on a protocol provided by the manufacturer. Stained cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, CA, USA).

Colony formation assay
———————-

Approximately 100 cells were plated per well in triplicate into six-well tissue culture plates and cultured in DMEM containing 10% FCS, which was replaced every 4 days. After 10 days of culture, the plates were stained with crystal violet and colonies were counted.

RNA isolation and qRT-PCR
————————-

Total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized from the RNA using a RevertAid First Strand cDNA Synthesis Kit

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Hindi Movies 720p Meri Pyaari Bindu
Hindi Movies 720p Meri Pyaari Bindu
Hindi Movies 720p Meri Pyaari Bindu
Hindi Movies 720p Meri Pyaari Bindu
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