Fig 2: Matrine alleviates mice myofibers atrophy by down-regulating the expression of E3 ubiquitin ligase. (A) Hematoxylin-eosin staining of myofibers in mice gastrocnemius. (B) The statistical results of myofiber CSA distribution in each group (n>180 per group). (C) Mean muscle fibers CSA. (D) Reverse transcription-quantitative polymerase chain reaction detected MAFbx and MuRF1 mRNA expression in mice Ga (upper panels) and TA (lower panels) (n=6). (E) Representative western blot images of MuRF1 and MAFbx in mice Ga. (F and G) Statistical results of the western blot analysis (n=3). Data are presented as the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. ##P<0.01, ###P<0.001, vs. NC. *P<0.05, **P<0.01, ***P<0.001 vs. CC. NC, negative control; Mat, matrine; CC, cancer cachexia; CSA, cross-sectional area; Ga, gastrocnemius; TA, tibialis anterior; MuRF1, muscle RING-finger containing protein-1; MAFbx, muscle atrophy Fbox protein.

Fig 3: LL/2 altered expression of innate immune cell markers in skeletal muscles, but did not affect protein expression of muscle protein specific E3 ubiquitin ligases. Quantifications of western blots in Figure 6. (A) tryptase, (B) Mas-related gene X2 (MrgX2), (C) CD68, (D) myeloperoxidase (MPO), (E) muscle-specific RING finger-1 (MuRF1), (F) muscle atrophy F-box (MAFbx), and (G) fast myosin skeletal heavy chain (MyHC). Mean ± SEM. * P = 0.05, ** P = 0.01, *** P = 0.001 vs. sham-injected group (Student's t-test).

Fig 4: Immunohistochemistry analysis of slow-twitch SOL muscle from WT and Kir6.1wt/VM mice. (A) Immunostaining (IMS) for NF?B p50 (E-10) on endomysia in SOL section from Kir6.1wt/VM (arrow) (40X, Bar 100 µm), (B) negative reaction in WT mice (40X, Bar 100 µm). (C,D) Nuclear IMS for MAFbx (F-9) in the myofibrils of the Kir6.1wt/VM mice (C, 40X, Bar 100 µm; D, 100X, Bar 100 µm); (E) negative IMS in the myofibrils of the WT mice (E, 40X, 100 µm). (F) IMS for BNIP-3 (ANa40) on endomysia in SOL section from Kir6.1wt/VM (40X, Bar 100 µm); (G) negative IMS in the WT mice section (40X, Bar 100 µm). (H) Negative IMS for caspase-3 (E-8) in the Kir6.1wt/VM mice section (40X, Bar 100 µm). (I) Representative sample gel of PCR analysis: in SOL muscle, FBX O 32, TRIM63 and BNIP3, genes encoding atrogin-1, MuRF1 and BNIP3, are markedly up-regulated in Kir6.1wt/VM SOL muscle. Casp3 is not detected in SOL from both phenotypes.

Fig 5: Immunohistochemistry analysis of slow-twitch SOL muscle from WT and Kir6.1wt/VM mice. (A) Immunostaining (IMS) for NF?B p50 (E-10) on endomysia in SOL section from Kir6.1wt/VM (arrow) (40X, Bar 100 µm), (B) negative reaction in WT mice (40X, Bar 100 µm). (C-D) Nuclear IMS for MAFbx (F-9) in the myofibrils of the Kir6.1wt/VM mice (C, 40X, Bar 100 µm; D, 100X, Bar 100 µm); (E) negative IMS in the myofibrils of the WT mice (E, 40X, 100 µm). (F) IMS for BNIP-3 (ANa40) on endomysia in SOL section from Kir6.1wt/VM (40X, Bar 100 µm); (G) negative IMS in the WT mice section (40X, Bar 100 µm). (H) Negative IMS for caspase-3 (E-8) in the Kir6.1wt/VM mice section (40X, Bar 100 µm). (I) Representative sample gel of PCR analysis: in SOL muscle, FBXO32, TRIM63 and BNIP3, genes encoding atrogin-1, MuRF1 and BNIP3, are markedly up-regulated in Kir6.1wt/VM SOL muscle. Casp3 is not detected in SOL from both phenotypes.