Journal: bioRxiv
Article Title: 5-Iodotubercidin inhibits Epithelial to Mesenchymal Transition by inhibiting IKK/NFκB-dependent gene expression
doi: 10.64898/2026.02.13.705677
Figure Lengend Snippet: A . A549 cells were treated with TGFβ in combination with the inhibitors 5-ITu and SB42 for 24h and lysates were probed with pS 536 -p65, total p65 and IκBα to show the effect of 5-ITu on the NFκB pathway. B . PANC1 and A549 cells were treated as indicated for 24 h and images were acquired using a phase-contrast 10x objective. Scale bars indicate 50µm. C-D . A549 cells were stimulated with TGFβ in combination with indicated inhibitors or solvent control (DMSO) and EMT marker expression was monitored by qPCR (mean±SD plotted) ( C ) and immunoblotting ( D ). E . HEK293T cells transfected with luciferase reporter plasmid and Renilla luciferase were treated as indicated for 6h. Luminescence was measured in microplate reader and calculated normalized relative luciferase activity. F . In vitro kinase assay performed by incubating the kinase IKKβ and the substrate IκBα along with 3 different concentrations of 5-ITu and BMS345541 as indicated for 30 mins. SDS gel was run for the reaction mixes and probed with pS32/S36-IκBα versus total IκBα and with GST. (* denotes p-value ≤ 0.05, ** denotes p-value ≤ 0.01, *** denotes p-value ≤ 0.001).
Article Snippet: The source and working concentrations used for the stimulants and inhibitors were: recombinant human TGFβ (#100-21-10, Peprotech, 5 ng/mL) and recombinant human TNFα (#PHC3016, Gibco, 101ng/ml), 5-Iodotubercidin (#HY-15424, 0.1-10 μM), IKKα/β inhibitor BMS345541 Hydrochloride (#HY-10518, 5 μM) (IKKβ IC 50 = 0.3 μM, IKKα IC 50 = 4 μM) , RIPK1 inhibitor Nec-1 (#HY-15760, 50 μM), GSK481 (HY-100131, 5 μM), ABT-702 dihydrochloride (#HY-103161, 5-10 μM) were obtained from MedChemExpress.
Techniques: Solvent, Control, Marker, Expressing, Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay, In Vitro, Kinase Assay, SDS-Gel
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