Fig 1.
TGF-β mRNA is increased in muscle samples from ALS patients.
(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.
Fig 2.
TGF-β and Smad mRNA levels correlate in ALS muscle samples.
Smad1, 5 and 8 mRNA levels were determined by qRT-PCR [4] and compared with TGF-β mRNA levels from the same ALS muscle biopsy sample (Ct values are shown).
Fig 3.
TGF-β mRNA levels are increased at early stages of ALS in the G93A SOD1 mouse.
Total RNA was isolated from G93A SOD1 mice and littermate controls (WT) at 40, 60 and 105 d (preclinical stages as previously defined [4]), 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.
Fig 4.
TGF-β protein is elevated in G93A mouse muscle.
(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.
Fig 5.
TGF-β1 is increased in human ALS muscle.
(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.
Fig 6.
TGF-β induces Smads1, 5 and 8 in cultured C2C12 muscle cells.
(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.
Fig 7.
Smad2 and 3 are increased in ALS muscle.
(A) Total RNA from human muscle biopsy samples was analyzed by qRT-PCR for Smad2 and 3 mRNA expression as described in Fig 1. (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.