Transforming growth factor beta tgf β

Berberine (B1), berberrubine (B2), 9-O-isoprenylberberrubine bromide (B3), 9-O-gernylberberrubine bromide (B4), and 9-O-farnesylberberrubine bromide (B5) were synthesized and provided by Dr. Jin-Yi Wu [7 (link)]. Dimethyl sulfoxide (DMSO), propidium iodide, crystal violet, chloroquine, rapamycin, 3-methyladenine (3-MA), paclitaxel (Taxol), and transforming growth factor beta (TGF-β) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies such as phospho-CDK2 (2351-1; Epitomics; Burlingame, CA, USA), CDK4 (sc-601; Santa Cruz Biotechnology; Dallas, TX, USA), p27 (GTX100446; GeneTex; Irvine, CA, USA), caspase-3 (#9662; Cell Signaling Technology; Beverly, MA, USA), PARP (#9542; Cell Signaling Technology), GAPDH (GTX100118; GeneTex), LC3 (AP1802a; Abgent; San Diego, CA, USA), p62 (#5114; Cell Signaling Technology), N-cadherin (#4061; Cell Signaling Technology), E-cadherin (610182; BD Biosciences; Bedford, MA, USA), claudin-1 (#4933; Cell Signaling Technology), and snail (#3879; Cell Signaling Technology) as well as secondary antibodies such as rabbit antimouse antibody (GTX26728; GeneTex) and goat antirabbit antibodies (GTX213110; GeneTex) were purchased and used in this study. All chemicals and biochemicals used in this study were of analytical grade.

Prior to culture initiation, the scaffolds were
sterilized by 70% ethanol for 10 minutes followed
by washing twice with phosphate buffered
saline (PBS) (38 (link)). Five hundred thousand
USSCs at passage-three were suspended
homogenously in 500 μl of DMEM +3% FBS
placed on the top surfaces of the scaffold
cubes. Cell/scaffold constructs were placed in
the wells of a 12-well culture plate under the
laminar hood for 1 hour during which the drop
disappeared owing to its penetration into scaffold
pores. USSCs were encapsulated in BTAG
scaffold and cultured for 3 weeks in chondrogenic
medium as chondrogenic group and in
DMEM as control group. Afterward, chondrogenic
medium containing 50 ng/ml ascorbic
acid 2-phosphate (Sigma, USA), 10 μM dexamethasone
(Sigma, USA), 10 ng/ml transforming
growth factor-beta (TGF-β, Sigma, USA),
10 ng/ml basic fibroblast growth factor (bFGF;
Sigma, USA), 0.1% Insulin-Transferrin-Selenium
(ITS, Sigma, USA) and1 mg/ml linoleic
acid (Sigma, USA) was used and incubated at
37˚C in a humidified 5% CO2 atmosphere for
21 days. First medium replacement was done on day 3, and the subsequent medium changes
were performed every 2 days.

To assess the effect of HX on the efficiency of the MD protocol, C2C12 differentiation was performed using modified MD protocols. Briefly, C2C12 cells were seeded in 24-well culture plates at a rate of 1 × 10⁴ cells per well in GM and were incubated at 37 °C in 5% CO₂ and 21% oxygen concentration. Up to 60–70% confluency, MD was induced using MD medium supplemented with either of the following elements; 1 mM dexamethasone, 5 ng/mL transforming growth factor beta (TGF β) (Sigma-Aldrich, Germany), 1 mg/mL insulin, 0.57 mg/mL transferrin and 0.5 μg/mL sodium selenite (ITS). The cells were allowed to differentiate under NX and HX conditions for evaluation at day-4 and day-14 post-induction.