FIG. 2.

Effects of depletion of NET25, emerin, and MAN1 on cell cycle in C2C12 cultures. (A) Immunofluorescent micrographs from a BrdU incorporation assay of C2C12 cultures (red, BrdU; blue, DNA) transfected with a nontargeting control siRNA or with siRNAs targeting NET25 (siNET25), emerin (siEmd), or MAN1 (siMAN1). Images shown correspond to the 4-h DM time point in panel B. (B) Quantification of BrdU-positive nuclei in control and NET25-, emerin-, or MAN1-depleted C2C12 cultures over a time course of 8 h after shift from PM to DM. (C) Western blot analysis (WB) confirming knockdown of the target proteins by siRNA. Protein extracts were prepared at the time of shifting cultures to DM. Left lane, protein levels after transfection with nontargeting control siRNA (non-targ.); right lane, protein levels after transfection with specific siRNA (spec.) targeting NET25, emerin, or MAN1. Blots were probed with antibodies recognizing the siRNA targets as indicated on the left. A nonspecific band on the MAN1 Western blot is marked by an asterisk. A GAPDH loading control representative of even loading in all panels is shown at the bottom. (D) Cell cycle profiles of control and siMAN1-transfected C2C12 cells in PM, 36 h after transfection. Representative examples are shown. Labels mark the cell populations in the G₁ (left peak), S (saddle), and G₂ (right peak) phases of the cell cycle based on their DNA content. (E) Summary of cell cycle distributions in control and siRNA-transfected C2C12 cells. Percentages shown are averages of the results for at least four experiments ± the standard deviations. Asterisks mark values with statistically highly significant differences in comparison to the control (P ≤ 0.035).