Fig 2: Differentiation of C2C12 Cells after KD or Overexpression of Fkbp4 and Fkbp5(A) qRT-PCR results comparing the expression levels of indicated muscle genes during differentiation of KD cells. The value obtained with the control on day 0 was defined as 1.0.(B) MTS assay for the proliferation of undifferentiated cells after overexpression of Fkbp4 and Fkbp5. Empty vector (Emp vec) was transduced as the control.(C) MHC staining of C2C12 cells overexpressing Fkbp4 and Fkbp5 during differentiation. Scale bar, 100 µm.(D) The differentiation index of the overexpressing cells on day 1 and day 2 obtained from the observation of 1,000 nuclei.(E) Relative expression levels of muscle genes determined by qRT-PCR on day 1. The value obtained with the control cells was defined as 1.0.*p < 0.05 and **p < 0.01 with Student’s t test in comparison to the control cells. Data are presented as mean + or ± SD of technical triplicates.

Fig 3: Interacting Protein Partners of Fkbp4 and Fkbp5(A) Western blotting of immunoprecipitated proteins. Left images show immunoprecipitation (IP) of 3×FLAG-Fkbp5 with anti-FLAG antibody and co-IP of Cdk4. Right images show IP of Cdk4 and co-IP of 3×FLAG-Fkbp5.(B) IP of Hsp90a/ß and co-IP of Cdk4, Fkbp4, and Fkbp5 from C2C12 cell extracts of differentiation days 0 and 5.(C) IP of Hsp90a/ß and co-IP of Cdk4 from extracts of undifferentiated KD cells.(D) IP of cyclin D1 and co-IP of Cdk4 from extracts of undifferentiated KD cells.(E) Detection of phosphorylated Cdk4 and Rb during differentiation of the KD cells. H2B was used as a loading control.

Fig 4: Requirement of the PPIase Activity of Fkbp5 for the Inhibition of Proliferation in Myoblasts(A) Western blotting of overexpressed Fkbp5 mutants and Fkbp4 in Fkbp5-/- myoblasts.(B) EdU uptake in WT and Fkbp5-/- myoblasts transduced with the Fkbp genes before (day 0) and during differentiation (day 1).(C) EdU uptake in WT and Fkbp5-/- myoblasts transduced with the Fkbp5 genes on differentiation day 1.(D) Immunostaining of MHC in WT and Fkbp5-/- myoblasts transduced with the Fkbp5 genes on differentiation day 1.(E) The differentiation index in WT and Fkbp5-/- myoblasts transduced with the Fkbp genes before (day 0) and during differentiation (day 1).**p < 0.01 with Student’s t test in comparison to the empty vector. Data are presented as mean + SD of technical triplicates.

Fig 5: Proliferation and Differentiation of Fkbp4 and Fkbp5 KD C2C12 Cells(A) EdU uptake in undifferentiated KD cells. Two shRNA clones were used for each KD. Control shRNA encodes a scrambled non-targeting sequence.(B) MTS assay for the proliferation of undifferentiated cells after KD. Cell number and absorbance value at 492 nm were proportional in this range.(C) Flow cytometry analyses of the cell cycle phases in undifferentiated C2C12 cells with Fkbp5 KD.(D) EdU uptake in KD cells during differentiation.(E) Immunostaining of KD cells with antibodies against MHC. DNA was counterstained with Hoechst 33342. Cells were induced to differentiate with 5% horse serum. Scale bar, 100 µm.(F and G) The differentiation index (F) and the 1-nucleus index (G) on day 3 and day 5 based on the observation of 1,000 nuclei.*p < 0.05 and **p < 0.01 with Student’s t test in comparison to the control cells. Data are presented as mean + or ± SD of technical triplicates.