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First published online 15 January 2008
doi: 10.1242/jcs.022566

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PLD regulates myoblast differentiation through the mTOR-IGF2 pathway

Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601 S. Goodwin Avenue B107, Urbana, IL 61801, USA

View larger version (24K): [in this window] [in a new window]   Fig. 1. PLD is upregulated during C2C12 differentiation. Confluent C2C12 cells were induced to differentiate by serum withdrawal. Cells were lysed every 24 hours, and the lysates were subjected to (A) quantitative RT-PCR and (B) western analysis. (C) In vivo transphosphatidylation assays were performed with differentiating C2C12 cells. The average results of three independent experiments are shown, with error bars representing s.d. Student's t-test was performed to compare the data from day 1 to 3 with that of day 0. *P<0.05; **P<0.01.

View larger version (40K): [in this window] [in a new window]   Fig. 2. 1-butanol inhibits C2C12 differentiation. C2C12 myoblasts were induced to differentiate for 3 days in the presence or absence of 0.5% 1-butanol, 0.5% 2-butanol, or 50 nM rapamycin. (A) The cells were immunostained for MHC (green) and DAPI (red). (B) Fusion index of cells shown in A. (C) Cells were lysed and subjected to western analysis for MHC, myogenin and tubulin. Three independent experiments were performed with similar outcomes; the result of a representative experiment is shown.

View larger version (45K): [in this window] [in a new window]   Fig. 3. Knockdown of PLD1 leads to decreased myogenic differentiation. C2C12 cells were infected with lentiviruses expressing two different Pld1 shRNAs or the empty vector, selected by puromycin for 3 days, and then subjected to analysis by (A) quantitative RT-PCR to measure levels of Pld1 and Pld2 mRNA, and (B) western blotting for PLD1. (C) The shRNA-expressing cells were grown to confluence and induced to differentiate for 3 days, followed by immunostaining for MHC. Three independent experiments were performed in each case with similar outcomes, and representative data are shown, except for A, where the average data of all experiments are shown with the error bars representing the s.d. Student's t-test was performed to compare cells infected with Pld1 shRNA viruses and empty vector virus. *P0.01.

View larger version (45K): [in this window] [in a new window]   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.

View larger version (46K): [in this window] [in a new window]   Fig. 5. PLD1 does not function through the actin cytoskeleton in C2C12 differentiation. C2C12 cells were infected with lentiviruses expressing two different Pld1 shRNAs or a scrambled hairpin sequence as a negative control, selected with puromycin for 3 days and induced to differentiate. Separately, the non-infected cells were subjected to 0.5% 1- or 2-butanol treatment during differentiation. At 0, 12 hours and 24 hours differentiation, the cells were fixed and labeled with Rhodamine-phalloidin. The samples to be compared were stained simultaneously, with image capture performed with an identical exposure time.

View larger version (27K): [in this window] [in a new window]   Fig. 6. PLD1 regulates the mTOR-IGF2 pathway during C2C12 differentiation. (A) C2C12 cells were infected with lentiviruses expressing two different Pld1 shRNAs, the empty vector or a scrambled hairpin sequence (Scram), selected with puromycin, and then induced to differentiate for 3 days. The cell lysates were analyzed by western blotting. (B) Total RNA was extracted from cells expressing Pld1 shRNAs or treated with 0.5% 1-butanol or 50 nM rapamycin, and Igf2 mRNA levels were measured by quantitative RT-PCR. (C) IGF2 protein levels in the conditioned media from cells in A were analyzed by ELISA. (D) Cells stably expressing H19-luc-ME were infected with lentiviruses as above, induced to differentiate for 3 days, and then subjected to luciferase assays. For B-D, the average results of three independent experiments are shown, with error bars representing the s.d. Student's t-test was performed to compare PLD1 shRNA with the negative control (vector or Scram). *P<0.05; **P<0.01.

View larger version (39K): [in this window] [in a new window]   Fig. 7. PLD1 regulates myogenesis through IGF2 production. C2C12 cells were infected with lentiviruses expressing various shRNAs, selected with puromycin, and then induced to differentiate for 3 days with or without exogenous IGF2 (300 ng/ml), followed by (A) western analysis of the cell lysates, or (B) immunostaining of MHC (green) and DAPI (red). (C) The average results of fusion index calculated from three independent experiments in B. Error bars represent s.d.

View larger version (8K): [in this window] [in a new window]   Fig. 8. A proposed model: PLD regulates skeletal myogenesis by controlling the amino-acid-sensing mTOR-IGF2 pathway.