Journal: Scientific Reports
Article Title: Impaired MC3T3-E1 osteoblast differentiation triggered by oncogenic HRAS is rescued by the farnesyltransferase inhibitor Tipifarnib
doi: 10.1038/s41598-025-91592-x
Figure Lengend Snippet: Farnesyl transferase (FTase) inhibitor Tipifarnib rescued early osteoblast differentiation by blocking HRAS membrane localization and downstream activation of MAPK. ( a ) Schematic representation of signaling molecules that were targeted with inhibitors during osteoblast differentiation. ( b ) Differentiation of MC3T3-E1 cells in response to DOX and inhibitors treatment: FTase inhibitor (Tipifarnib; 0.5 µM), RUNX2 inhibitor (CADD522; 50 µM), SHP2 inhibitor (TNO155; 1 µM), MEK1/2 inhibitor (U0126; 10 µM) or EGFR inhibitor (erlotinib; 5 µM). Alp activity was measured 7 days post osteogenic induction using Alp assay kit and was normalized to cell viability measured by Presto Blue assay kit ( n = 3 independent experiments). ( c ) Microscopy analysis of HRAS localization in cells treated with Tipifarnib (0.5 µM) for 24 h. EYFP-HRAS fusion protein was visualized by immunofluorescence microscopy at 488 nm using Leica DMI6000B at 20x magnification. Nuclei were stained using DAPI. ( d , e ) Immunoblotting analyses of MC3T3-E1 subclones in the presence or absence of DOX and Tipifarnib (0.5 µM) after 24 h. ( d ) Activation of endogenous Hras (Hras ENDO ) and recombinant HRAS (HRAS REC ) was monitored by pull down (PD) assay using as bait the RAS-binding domain (RBD) of RAF1 kinase coupled to glutathione-S-tranferase (GST), GST-RAF1-RBD. PD samples were analyzed by immunoblotting for HRAS activation using HRAS antibody, while immunoblots using same antibody in total cell lysates (TCLs) were used as controls to monitor HRAS REC and Hras ENDO protein abundance. Original blots are presented in Supplementary Fig. 5. ( e ) Craf-Mek1/2-Erk1/2 activation was monitored with specific antibodies for activating phosphorylations of Craf, Mek1/2 and Erk1/2. Craf, Mek1/2 and Erk1/2 immunoblots were used as control for their phospho-forms, whereas vinculin (Vcl) and GFP served as loading and recombinant protein expression controls, respectively. Original blots are presented in Supplementary Fig. 5.
Article Snippet: The osteogenic induction media was further supplemented with DOX (100 μg/ml) for induction of recombinant protein expression and the indicated inhibitors: RUNX2 inhibitor (CADD522; Selleckchem, #S0790) 50 μM, FTase inhibitor (Tipifarnib; Selleckchem, #S1453) 0.5 μM, SHP2 inhibitor (TNO155; Selleckchem, #S8987) 1 μM, MEK1/2 inhibitor (U0126; Selleckchem, #S1102) 10 μM, PI3K inhibitor (LY294002; Selleckchem, #S1105) 5 μM, EGFR inhibitor (Erlotinib; Selleckchem, #S7786) 5 μM.
Techniques: Blocking Assay, Membrane, Activation Assay, Activity Assay, ALP Assay, Microscopy, Immunofluorescence, Staining, Western Blot, Recombinant, Binding Assay, Quantitative Proteomics, Control, Expressing
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