Fig 2: Ratio of microtubule-associated protein light chain 3 (LC3)-II to total LC3 (A), and relative abundance of atrogin-1 (B) and muscle RING-finger protein 1 (MuRF1; C) in the longissimus dorsi (LD) muscle of preterm pigs provided intermittent bolus (INT) or continuous (CONT) feeding for 21 days. Atrogin-1 and MuRF1 abundances were normalized to GAPDH abundance. Representative immunoblots are shown for each treatment group. LD muscle from pigs fed by intermittent bolus feeding was sampled in the postabsorptive state (INT-PA, before feeding) and in the postprandial state (INT-PP, 60 min after feeding). Data were analyzed by one-factor ANOVA followed by the Tukey’s procedure. Values are least squares means ± SE; individual data are shown; CONT, n = 14 (5 male and 9 female); INT-PA, n = 13 (2 male and 11 female); INT-PP, n = 16 (5 male and 11 female). Means without a common superscript letter differ, P < 0.05. AU, arbitrary units.

Fig 3: Examination of NF-?B p65 and MURF1 protein expression in Model and BYHWT treatment group. (A) Western blot assay for NF-?B p65 and MURF1 expression. (B) Semi-quantification of protein expression. *P<0.05 vs. normal control group. #P<0.05 vs. model group. BYHWT, Buyang Huanwu Tang; NF-?B, nuclear factor-?B; MURF1, muscle RING-finger protein-1.

Fig 4: MuRF1 protein expression in 6-days denervated atrophic hind-limb muscles and in 6-days denervated hypertrophic hemidiaphragm muscle. MuRF1 expression in 6-days denervated (Den) atrophic anterior tibial (A), atrophic pooled gastrocnemius and soleus muscles (B), and in hypertrophic hemidiaphragm muscle (C) compared to innervated (Inn) and sham operated controls. Representative images of Western blots are shown together with densitometric quantifications. One innervated sample from the respective muscle type was loaded onto all gels as a reference. All samples were measured relative to this reference. The data were normalized to give a mean value of 100.0 in innervated muscles. Mean values ± standard error of the mean. *p < 0.05, **p < 0.01, n = 8 (A and C) or 7 (B) muscles per group.

Fig 5: FPS-ZM1 treatment preserved hind-limb grip strength and decreased the expression of skeletal muscle atrophy markers in hSOD1G93A mice. A, Analysis of hind-limb grip strength combining both sexes together (vehicle group n = 20 [12 females and 8 males], and FPS-ZM1 group n = 29 [19 females and 10 males]). In order to combine both sexes together, data are presented as the ratio of hind-limb grip strength to body weight (mean ± SEM). B, Analysis of hind-limb grip strength in male mice (n = 8 in vehicle, n = 10 in FPS-ZM1 group). C, Analysis of hind-limb grip strength in female mice (n = 12 in vehicle, n = 19 in FPS-ZM1 group). In (B and C), data are presented as mean ± SEM. *Significantly different from vehicle-treated mice (P = .05). For (A-C), statistical significance was determined by multiple t tests assuming populations with the same standard deviation. D and E, FPS-ZM1 treatment significantly decreased the expression of skeletal muscle atrophy markers (P = .05). Total RNA was extracted from the gastrocnemius muscle of 17-week-old vehicle- or FPS-ZM1-treated mice and Fbxo32 (D) and Trim63 (E) mRNA levels were determined by real-time PCR and corrected by Rplp0 mRNA levels (n = 4 mice per group). Data are expressed as percentage of vehicle-treated mice (mean ± SD). F, Western blot analysis of FBX32 and TRIM63 protein levels in the gastrocnemius muscle of 17-week-old vehicle- or FPS-ZM1-treated mice. G and H, Quantification of FBX32 (G) and TRIM63 (H) protein expression after correction by ACTIN levels (n = 4 mice per group). Data are expressed as percentage of vehicle-treated mice (mean ± SD)