Finite element model of an adherent cell to identify vibration induced nuclear displacements.

<p>The elastic FE model comprised the cell membrane, cytoplasm and nucleus (top). Vibration induced fluid shear and accelerations were evaluated in separate dynamic simulations. Fluid shear was simulated by applying dynamically oscillating forces to the cell membrane (bottom left). Oscillatory accelerations were applied directly to the cell contact surface (bottom right).</p

Vibration induced GJIC is controlled by Akt signaling.

<p>One hour after vibration treatment, (A) Cx43 mRNA levels remained unchanged. (B) Cx43 protein levels were also unaffected by vibrations (0.15 g–100 Hz) without and with Akt inhibition but (C) vibrations increased Akt phosphorylation (ser 473) 2.4-fold. (D) Inhibition of Akt activation also inhibited the vibration induced increase in calcein transference. ***: p<0.001 against control.</p

Nuclear displacements induced by either vibration induced accelerations or vibration induced fluid shear.

<p>Nuclear displacements induced by either vibration induced accelerations or vibration induced fluid shear.</p

Vibration induced calcein transference is gap junction specific.

<p>When gap junction function was blocked with 75 µM of 18α-GA, GJIC+ cell number was significantly reduced compared to non-blocked groups (Normal) and none of the four vibration frequency/acceleration combinations increased the number of GJIC+ cells. ***: p<0.001 against control.</p

C/EBPβ is involved in ER stress in MSC.

<p>A, C3H10T1/2 cells were transfected with 1 µg of C/EBPβ versus empty vector. 48 hours after transfection, RNA analysis was performed via real time PCR (n = 5). B, C3H10T1/2 cells transiently transfected with C/EBPβ versus empty vector at indicated doses and 48 hours after transfection, protein analysis was performed. A representative blot is shown, n = 3. C, Densitometry for (B). D, Immunoblot of C3H10T1/2 cells transfected with siRNA to C/EBPβ or control siRNA shown at 72 hours after transfection. Tunicamycin at 1 µg/ml was added for the final 6 hours. E, ChIP-seq analysis of C/EBPβ, β-catenin, TCF4 binding at the mouse <i>CHOP</i> and <i>BiP</i> gene loci. MC3T3 cells were treated for 3 hours with vehicle or 50 mM LiCl prior to ChIP-seq assay. The genomic interval on the indicated mouse chromosome and the location of the <i>CHOP</i> or <i>BiP</i> transcription unit including the direction of transcription (arrow) is shown at the top. Tracks indicate tag densities (normalized to 10⁷ reads) for vehicle or LiCl treated β-catenin or TCF4 binding. Note the scale for peak height is different for each track to highlight peak activities.</p

C/EBPβ overexpression does not rescue adipogenesis from mechanical inhibition.

<p>A, C3H10T1/2 cells cultured in adipogenic media and analyzed on indicated days by real time PCR for C/EBPβ, PPARγ, and adiponectin (APN) n = 3, **p<0.01 relative to day zero B, C3H10T1/2 cells cultured in adipogenic media and blotted for C/EBPβ, markers of adipogenesis. C, Immunoblot of C3H10T1/2 cells transfected with siRNA to C/EBPβ. 24 hours after transfection, adipogenic medium was added for 72 hours. D, C3H10T1/2 cells were transfected with 1 µg of overexpression vector for C/EBPβ or control empty vector. 24 later adipogenic media was added and cells were analyzed at 48 hours by immunoblot for C/EBPβ, adiponectin (APN) or aP2. E, C3H10T1/2 cells overexpressing C/EBPβ as in A were subjected to strain for 3 days and analyzed by real-time PCR for C/EBPβ, PPARγ2 and adiponectin mRNA expression. F, experiment as described in (E) analyzed by immunoblot. G, C3H10T1/2 cells were transiently transected with a C/EBPβ overexpression vector. 24 hours later adipogenic media +/−SB41528 was added and maintained for 3 days prior to protein analysis for markers of adipogenesis and C/EBPβ.</p

C/EBPβ is a mechanical target in MSC.

<p>A, mRNA of C3H10T1/2 cells in adipogenic media subjected to strain, analyzed via real time PCR for C/EBPβ, adiponectin (APN), and PPARγ2, n = 3, *p<0.05. B, Immunoblot of day 3 C3H10T1/2 cells in adipogenic media subjected to strain. C, Densitometry from (B) n = 4, * p<0.05, ** p<0.01. D, mRNA of mdMSC in adipogenic media treated with strain, analyzed via real time PCR for C/EBPβ, adiponectin (APN), and PPARγ2, n = 3,*p<0.05. E, Representative immunoblot of day 2 mdMSC cells in adipogenic media subjected to strain, n = 3. F, C3H10T1/2 cells transiently transfected with siRNA to β-catenin. 24 hr later, induction of adipogenesis was initiated with adipogenic media. Strain was applied and immunoblot performed for β-catenin, active β-catenin, as well as C/EBPβ, and adiponectin (APN).</p

Mechanical strain ameliorates tunicamycin-induced ER stress in MSC.

<p>A, mdMSC were cultured in the presence of tunicamycin (TM) at indicated doses for 6 hours and analyzed via immunoblot for C/EBPβ, BiP and CHOP. B, as in (A) mdMSC cultured in the presence of tunicamycin 1 µg/ml for 6 hours and analyzed via conventional PCR for XBP1. C, C3H10T1/2 cells were seeded on the same day and treated with mechanical strain for the indicated number of days. Tunicamycin (TM) was added for the final 6 hours prior to protein analysis (representative blot, n = 3).</p

Mechanical strain increases ER capacity in MSC.

<p>A<b>,</b> MSC seeded on the same day were treated with mechanical strain for indicated time prior to mRNA (n = 3 * P<0.05, ** P<0.01) (A) or protein analysis (B) performed with C3H10T1/2 cells and mdMSC, as indicated. Control cultures were seeded on the same day as strained cultures and housed in similar conditions for 6 days prior to analysis.</p