Fig 1.
Conditioned medium from human cancers stimulate C/EBPβ expression in myoblasts.
(A) C2C12 myoblasts were incubated with conditioned medium from indicated human cancers or unconditioned medium (UM) mixed 1:1 with fresh myoblast medium for 48 hours. C/EBPβ expression was assessed by western blot. β-actin is a loading control. (B) Cebpb mRNA expression in myoblasts treated with PC-3 medium or unconditioned medium for 48 hours. *p<0.05, n = 5. (C) Il1b expression in SKOV3 and PC-3 cancer cells. *p<0.05, n = 5.
Fig 2.
Pre-treatment with conditioned media from a prostate tumor inhibits skeletal muscle differentiation and upregulates C/EBPβ expression.
(A) Schematic of the treatment procedure for the tissue culture model of cachexia. Conditioned media from PC-3 cells was mixed 1:1 with fresh media, and was added onto proliferating C2C12 myoblasts for 48 hours after which cells were induced to differentiate in fresh DMEM containing 2% horse serum for 5 days. (B) Immunocytochemistry staining for myosin heavy chain expression in C2C12 cultures treated with conditioned media from PC-3 or DU145 prostate cancers or unconditioned media (UM) as in (A). DAPI stains nuclei blue. (C) C2C12 cultures were induced to differentiate as in (A) and the fusion index (#myonuclei/myotube) and differentiation index (#myonuclei/# total nuclei) was calculated, and shown relative to UM. Actual values are shown in the bars. *p<0.05, n = 7. (D) Western blot analysis of C/EBPβ, Pax7, and MyoD expression in proliferating C2C12 cells on day 2 after incubation with conditioned medium. Actin is a loading control. (E) Western blot analysis of myogenic marker expression in differentiated C2C12 cells on day 7. Actin is a loading control. (F) qRT-PCR analysis of Myod1, Myog and neonatal myosin heavy chain (Myh1, Myh2, Myh8, Myh13) expression, shown relative to cells treated with unconditioned medium (UM) on day 7. *p<0.05, n = 4.
Fig 3.
Inhibition of myogenesis correlates with induction of C/EBPβ expression in myoblasts.
(A) Immunocytochemistry staining of myosin heavy chain expression in C2C12 myoblasts pre-treated with conditioned media from SKOV3 or PC-3 cells mixed 1:1 with fresh media, for 48hrs, and induced to differentiate for an additional 5 days. DAPI stains nuclei blue. (B) C2C12 cultures were induced to differentiate as in (A) and the differentiation and fusion indices were calculated as relative to SKOV3-treated cells. Actual values are shown in the respective bars. *p<0.05, n = 5. (C) C/EBPβ, and myogenic marker protein expression in C2C12 myoblasts treated as in (A). Cyclophilin B (CyPB) is shown as a loading control. (D) Immunocytochemistry staining of myosin heavy chain (MYH) expression in primary myoblasts pre-treated with conditioned media from SKOV3 or PC-3 cells mixed 1:1 with fresh media, for 48hrs, and induced to differentiate for an additional 48 hours in DMEM containing 10% horse serum. DAPI stains nuclei blue. (E) Primary myoblast cultures were induced to differentiate as in (D) and the differentiation and fusion indices were calculated as relative to SKOV3-treated cells. Actual values are shown in the respective bars. **p<0.01, n = 4. (F) Percentage of Pax7+ cells relative to total nuclei remaining in C2C12 cells cultured in conditioned medium and differentiated as in (D) as determined by immunocytochemistry. *p<0.05, n = 4. (G) C/EBPβ, and myogenic marker protein expression in primary myoblasts treated as in (D). Cyclophilin B (CyPB) is shown as a loading control.
Fig 4.
LLC tumor graft increases C/EBPβ expression in myoblasts and prevents myogenesis.
(A) 5x105 Lewis Lung Carcinoma (LLC) cells or PBS (Sham) was injected into the flank of C57BL/6 mice and allowed to engraft for 3 weeks to induce cachexia. (B) Immunocytochemistry for myosin heavy chain expression in primary myoblasts isolated from sham-injected and LLC-injected mice and differentiated for 2 days. (C) Differentiation (DI) and fusion (FI) indices from cultures isolated and differentiated as in (B). *p<0.05, n = 5. (D) Western analysis of C/EBPβ and myogenic marker expression in cells isolated from healthy or cachectic mice as in (A) and after culture expansion for 5 days, differentiated for 2 days. Cyclophilin B (CyPB) is a loading control.
Fig 5.
Defective muscle regeneration in cachectic animals after acute injury.
(A) Schematic representation of the injury model. Cachexia was induced by engrafting LLC cancer cells subcutaneously and allowing them to grow for 3 weeks. Once cachexia was established, animals were injured by injecting cardiotoxin (CTX) into the TA muscle. Sham injury was achieved using PBS. Injury was allowed to repair for one week before analysis. (B) H&E-stained TA cross-sections from sham and LLC-injected mice as in (B) 7 days after CTX injury. The contralateral TA was injected with PBS alone. Scale bar = 20μm. (C) Average fiber XSA of PBS and CTX-injured sham and LLC mice as in (B). *p<0.05, **p<0.01, ***p<0.001, n = 4. (D) TA mass in PBS and CTX-injured sham and LLC mice as in (B). *p<0.05, **p<0.01, ***p<0.001, n = 5. (E) Percentage of Pax7+ cells (relative to total DAPI+ nuclei) in PBS and CTX-injured TA of sham and LLC animals. **p<0.01, ***p<0.001, n = 5. (F) Percentage of C/EBPβ+/Pax7+ cells (relative to uninjured sham animals) in PBS and CTX-injured TA of sham and LLC animals. *p<0.05, ***p<0.001, n = 3.
Fig 6.
C/EBPβ null primary myoblasts are insensitive to conditioned medium from the PC-3 tumor.
(A) Indirect immunofluorescence of myosin heavy chain (MyHC) expression in primary myoblasts isolated from Cebpbfl/flPax7+/+ (WT) and Cebpbfl/flPax7CreER/+ (cKO) mice and cultured with SKOV3 or PC-3 conditioned media for 2 days, and induced to differentiate for an additional 2 days. (B) Differentiation index (#myonuclei/#nuclei) of primary myoblasts cultures in (A). *p<0.05, ***p<0.001, n = 6. (C) Fusion index (#myonuclei/myotube) for cells differentiated as in (A). *p<0.05, **p<0.01, n = 6. (D) Percentage of Pax7+ cells relative to total nuclei in cultures from (A) following differentiation, designated as reserve cells. *p<0.05, **p<0.01, n = 6. (E) Western blot analysis of C/EBPβ and myogenic marker expression in primary myoblasts treated as in (A). Cyclophilin B (CyPB) is a loading control.