TGF-β induces Smads1, 5 and 8 in cultured C2C12 muscle cells.

<p>(A) C2C12 muscle cells were treated with TGF-β ligands for the time frames shown and then assessed for Smad1, 5, and 8 mRNA expression by qRT-PCR. Data points were expressed as a fold-increase over vehicle treated cells and represent the mean ± SE of 6–8 independent samples. ** P < 0.005; *** < 0.0005; **** < 0.0001. (B) C2C12 cells were treated with TGF-β ligands for the time frames shown and assessed for p- and t-Smad 1, 5, 8 by Western blot. The experiment was repeated one time with similar results.</p

TGF-β mRNA levels are increased at early stages of ALS in the G93A SOD1 mouse.

<p>Total RNA was isolated from G93A SOD1 mice and littermate controls (WT) at 40, 60 and 105 d (preclinical stages as previously defined [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138425#pone.0138425.ref004" target="_blank">4</a>]), and early and late clinical stages (125 and 150 d). Samples were analyzed by qRT-PCR for TGF-β1, 2, 3 and BMP4 mRNA expression. Data points represent the mean ± SE of 6–8 mice. * p < 0.05; ** < 0.005; **** < 0.0001. RQ, relative quantity.</p

TGF-β protein is elevated in G93A mouse muscle.

<p>(A) Muscle lysates from G93A SOD1 mice (M) and non-transgenic littermates (W) were assessed for TGF-β1 by ELISA and Western blot. ELISA values were determined by comparison to a standard curve. A representative Western blot of the lysates (under reducing conditions) shows expression of mature TGF-β1. ELISA data represent the mean ± SE of 3 mice. The Western blot was repeated once with the similar results. (B) Confocal photomicrographs of G93A or WT muscle sections using a TGF-β1 antibody. WGA, wheat germ agglutinin. Size marker = 50 microns. (C) A representative Western blot of TGF-β2 and β3 in mouse muscle showing increased levels of the unprocessed peptide in mutant versus control samples. Quantitative densitometry of TGF-β ligands was performed on three Western blots from three independent mouse samples. Data are shown as fold-increase over WT controls. *p < 0.05 for each age.</p

TGF-β mRNA is increased in muscle samples from ALS patients.

<p>(A) Total RNA from muscle biopsy samples was analyzed by qRT-PCR for TGF-β1, 2, and 3 mRNA expression in patients with ALS (n = 27), myopathy (n = 11), neuropathy (n = 9) or no neuromuscular disease (n = 13). RQ, relative quantity. *, p < 0.05; **, < 0.005; *** < 0.0005; **** < 0.0001. (B) Correlation of TGF-β isoform mRNA levels (expressed as the Ct value from qRT-PCR). (C) Correlation between muscle grade of biopsied ALS muscle samples (as measured by the Medical Research Council scale) and TGF-β mRNA.</p

TGF-β1 is increased in human ALS muscle.

<p><b>(</b>A) Acid activated protein lysates from human muscle biopsy samples were assessed for TGF-β1 by ELISA and compared to a standard curve. Data points are the mean ± SE. Samples include ALS (n = 12); neuropathy (n = 7); myopathy (n = 8); normal (n = 4). *, p < 0.05. (B) Confocal photomicrographs of ALS and control muscle samples labeled with an anti-TGF-β1 antibody. Bx, biopsy specimen; Aut, autopsy specimen; Ctl, normal biopsy. Size marker = 50 microns.</p

TGF-β and Smad mRNA levels correlate in ALS muscle samples.

<p>Smad1, 5 and 8 mRNA levels were determined by qRT-PCR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138425#pone.0138425.ref004" target="_blank">4</a>] and compared with TGF-β mRNA levels from the same ALS muscle biopsy sample (Ct values are shown).</p

TGF-β induces Smads1, 5 and 8 in cultured C2C12 muscle cells.

<p>(A) C2C12 muscle cells were treated with TGF-β ligands for the time frames shown and then assessed for Smad1, 5, and 8 mRNA expression by qRT-PCR. Data points were expressed as a fold-increase over vehicle treated cells and represent the mean ± SE of 6–8 independent samples. ** P < 0.005; *** < 0.0005; **** < 0.0001. (B) C2C12 cells were treated with TGF-β ligands for the time frames shown and assessed for p- and t-Smad 1, 5, 8 by Western blot. The experiment was repeated one time with similar results.</p

Smad2 and 3 are increased in ALS muscle.

<p>(A) Total RNA from human muscle biopsy samples was analyzed by qRT-PCR for Smad2 and 3 mRNA expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138425#pone.0138425.g001" target="_blank">Fig 1</a>. (B) Smad2 and3 mRNA levels in the G93A mouse were determined by qRT-PCR. Data points represent the mean ± SE of 3–4 mice. (C) Western blot of C2C12 cells treated with TGF-β ligands for the times shown. Antibodies are shown to the left. RQ, relative quantity. *, P < 0.05; ** < 0.005; ****< 0.0001.</p