Fig. 4. Knockdown of PLD2 does not affect C2C12 differentiation. (A) C2C12 cells were infected with lentiviruses expressing two different Pld2 shRNAs or a scrambled hairpin sequence (Scram) as negative control, selected by puromycin for 3 days, and then subjected to analysis by quantitative RT-PCR. (B) Cells treated as above were subjected to in vivo PLD assay with or without stimulation by 100 nM PMA for 30 minutes. For A and B, the average results of three independent experiments are shown, with error bars representing the s.d. Student's t-test was performed to compare cells infected with Pld2 shRNA viruses and scrambled virus. ^*P0.01. (C) The shRNA-expressing cells were grown to confluence, induced to differentiate for 3 days, and immunostained for MHC (green) and DAPI (red). Similar outcomes were observed for three independent experiments, and a representative set of data is shown.