Figure 1.
Schematic diagram for EST data analysis.
Figure 2.
KOG analysis of ESTs in MSC, MFC and ALC.
The functions of genes were categorized and each function is represented by the symbols given below: .
[J] Translation, ribosomal structure and biogenesis, [A] RNA processing and modification, [L] replication, recombination and repair, [B] chromatin structure and dynamics, [Y] nuclear structure, [V] defense mechanisms, [T] signal transduction mechanisms, [M] cell wall/membrane/envelope biogenesis, [N] cell motility, [Z] cytoskeleton, [W] extracellular structures, [U] intracellular trafficking, secretion, and vesicular transport, [O] posttranslational modification, protein turnover, chaperones, [C] energy production and conversion, [G] carbohydrate transport and metabolism, [E] amino acid transport and metabolism, [F] nucleotide transport and metabolism, [H] coenzyme transport and metabolism, [I] lipid transport and metabolism, [P] inorganic ion transport and metabolism, [R] general function prediction only, [S] function unknown. The X-axis represents percentage and Y axis represents functional category.
Figure 3.
Biochemical pathway analysis of highly expressed gene list.
Representative KEGG pathway for A) MSC, B) MFC and C) ALC.
Figure 4.
mRNA expression of genes identified with higher EST numbers.
C2C12 cells treated with differentiation and transdifferentiation media at 80% confluence were harvested at different time points. Real time PCR was carried out with cDNA synthesized from1µg of total RNA. mRNA expression analysis of most of the genes showed more than 2 fold induction during MFCs (A) and ALCs (B) formation, respectively. Western blot analysis of DDAH2 shows a gradual increase in protein expression with time (C). Cellular localization of DDAH2 by immunocytochemistry during myogenesis. The first column shows cell pictures at Day 0 and Day 3. The second column shows expression of DDAH2 and the third column shows a merged image of DAPI-stained nuclei and DDAH2 IF (D).Western blot analysis of HBA2 expression during transdifferentiation confirms its protein level expression (E). Cytoplasmic localization of HBA2 by immunocytochemistry during transdifferentiation. The first column shows cell pictures at Day 0 and Day 2, the second column shows expression of HBA2 IF and the third column shows a merged image of DAPI-stained nuclei and HBA2 IF (F). DDAH2 and HBA2 immunohistochemistry of bovine skeletal muscle (G and H). Day 0 represents control (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.
Figure 5.
DDAH2 knockdown in C2C12 cells during myogenesis.
mRNA expression of DDAH2 and MYOG after DDAH2kd during differentiation in C2C12 at Day 2 (A). Representative cell picture showing morphological changes in DDAH2kd cells (B). Mock represents control (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.
Figure 6.
HBA2 knockdown in C2C12 cells during transdifferentiation.
(A) mRNA expression of HBA2 after HBA2kd during transdifferentiation in C2C12 at Day 1, and (B) HBA2kd effect on mRNA expression of different adipogenic marker genes.(C) Cell picture following intracellular lipid staining by O-R-O on Day 5 during transdifferentiation in HBA2kd and Mock cells. Quantification of O-R-O at 510 nm. Control represents Mock (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.