The effect of IL-1β treatment on IL-6 mRNA and protein levels.

<p>(A) Primary MPCs were treated with IL-1β (0.25 ng/ml) and IL-6 mRNA was determined over 24 hours. *denotes significance (p≤0.05) compared to control. # denotes significance (p≤0.05) compared to the 2 hour time point (n = 4 per time point). (B) IL-1β treatment (1 ng/ml) also increased IL-6 protein released into the media in C2C12 myoblasts. *denotes significance (p≤0.05) compared to control (n = 4–6).</p

IL-1β and IL-6 mRNA expression post-injury to the tibialis anterior (TA).

<p>TA muscle was injured using a barium chloride injection method and muscles were collected 1, 5, 10, and 28 days post-injury and analyzed for (A) IL-1β and (B) IL-6 mRNA expression levels. Data are expressed as fold-increase +/− expected high and low expression relative to the average value <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092363#pone.0092363-Livak1" target="_blank">[51]</a>. *denotes significance (p≤0.05) compared to uninjured control (n = 3–4 per time point).</p

Histological representation of the progression of injured muscle through repair and regeneration.

<p>(A) Uninjured tibialis anterior (TA) muscle. Injured TA muscle (B) 1 days, (C) 5 days, (D) 10 days, and (E) 28 days after barium chloride (BaCl₂) injection. Bar in E = 100 μm. Images taken at 10X magnification.</p

The effects of TNF-α and IL-1β on nuclear factor-kappa B (NF-κB) activity.

<p>Transfection of myoblasts with NF-κB cis-reporter construct allowed the study of the effects of (A) TNF-α (20 ng/ml) and (B) IL-1β (1 ng/ml) on NF-κB activity. 24 hours after transfection, cells were treated with either TNF-α or IL-1β for an additional 24 hours. Data are reported as the ratio of firefly to <i>Renilla</i> luminescence *denotes significance (p≤0.05) compared to control (n = 7 for TNF-α and n = 6 for IL-1β).</p

Determining the role of NF-κB activation in IL-1β and TNF-α induced proliferation.

<p>(A) NF-κB activity was determined using the NF-κB cis-reporter construct and data are reported as the ratio of firefly to <i>Renilla</i> luminescence. C2C12 myoblasts were transfected with the NF-κB cis-reporter construct, pretreated with 50 μM PDTC for 1.5 hours, and treated with either 1 ng/ml IL-1β or 20 ng/ml TNF-α for four hours. *denotes significance (p≤0.05) compared to control (n = 4). (B) Proliferation of myoblasts was measured through BrdU incorporation in C2C12 myoblasts following a 50 μM PDTC pretreatment for 1.5 hours, and a 20 hour treatment with either 1 ng/ml IL-1β or 20 ng/ml TNF-α. *denotes significance (p≤0.05) compared to control (n = 4).</p

Determining the role of TNF-α in IL-1β mediated proliferation.

<p>(A) The effect of TNF-α on myoblast proliferation is blocked by pre-incubation of TNF-α with soluble TNF receptor I (sTNFRI) (0.3 μg/ml, 2 hours at 37°C). (B) IL-1β induced proliferation of myoblasts is not blocked by pre-incubation with sTNFRI. Data are expressed relative to control ± SEM. *denotes significance (p≤0.05) compared to control. # denotes significance (p<0.05) compared to TNF-α treated.</p

TNF-α promotes proliferation of myoblasts.

<p>BrdU incorporation was measured after a 24 hour TNF-α treatment (20 ng/ml). Data are expressed relative to control ± SEM. *denotes significance (p≤0.05) compared to control (n = 7).</p

The mitogenic effects of IL-1β.

<p>(A) An IL-1β dose response was performed on C2C12 myoblasts. Concentrations of 0.5 ng/ml to 1 ng/ml significantly increased proliferation in myoblasts. (B) An intermediate dose of IL-1β (0.25 ng/ml) was used to test the mitogenic effects of IL-1β on primary muscle precursor cells. Data are expressed relative to control ± SEM. *denotes significance (p≤0.05) compared to control (n = 3–5 per dose).</p