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

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

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