Journal: The FASEB Journal

Article Title: Fluid shear stress generates a unique signaling response by activating multiple TGFβ family type I receptors in osteocytes

doi: 10.1096/fj.202001998R

Figure Lengend Snippet: FSS stimulation activates multiple distinct TGFβ family type I receptors in OCY454 cells. A, qRT‐PCR analysis of TGFβ target gene Serpine1 after 60‐minute treatment with vehicle or SB‐431542 followed by 120‐minute treatment with TGFβ or FSS stimulation as indicated (n = 4 biological replicates). All values normalized to control cells. * P < .05 compared to unstimulated cells and # P < .05 compared to SB‐treated controls; † P < .05 compared to corresponding treatment group without SB‐431542, ‡ P < .05 compared to FSS‐stimulated cells. B, C, Representative western analysis of Smad phosphorylation in control cells and cells pretreated (60 minutes) with vehicle or an inhibitor of a subset of TGFβ type I receptors (SB‐431542, ALK4/5/7 inhibitor; LDN‐193189, ALK1/2/3/6 inhibitor; LDN‐214117, ALK1/2 inhibitor, as shown in B), followed by treatment (30 minutes) with TGFβ or FSS (C) (n = 3 biological replicates). D, Representative western analysis of cells pretreated (60 minutes) with ALK1Fc followed by treatment (30 minutes) with TGFβ, BMP4, or FSS (n = 2 biological replicates, non‐flow conditions were collected from cells grown in well plates)

Article Snippet: Except where noted in the figures, cells were treated as indicated with TGFβ1 (5 ng/mL), BMP4 (50 ng/mL) (both from Peprotech); 1d11 (1.25 μg/mL, Clone 1d11.16.8, BioXCell); Noggin (100 ng/mL, SRP3227, Sigma Aldrich); SB‐431542 (10 μM), LDN‐193189 (1 μM), LDN‐214117 (1 μM), SC‐79 (10 μM) (all from Selleckchem); recombinant mouse ALK1Fc (100 ng/mL, R&D Systems); and LY294002 (50 μM, Calbiochem).

Techniques: Quantitative RT-PCR, Control, Western Blot, Phospho-proteomics