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. 2019 Oct 17;9:14910. doi: 10.1038/s41598-019-51410-7

Figure 6.

Figure 6

SM regulated autophagic flux in an PLD2 and FOXO1-dependent manner. (A) C2C12 myoblasts were incubated either under terrestrial gravity (1 g) or under SM for 36 h, lysed, and analyzed by western blotting. (B) Cells were treated as (A), with or without 100 nM AS1842856, lysed and subjected to quantitative RT-PCR (n = 6). (C) Either PLD2+/+ MEFs or PLD2−/− MEFs were incubated either under terrestrial gravity (1 g) or under SM for 36 h, lysed, and analyzed by quantitative RT-PCR (n = 4). (D) The cells were transduced with lentiviruses expressing shRNAs against PLD2 (shPLD2-1, shPLD2-2) or a scrambled sequence as control, selected with puromycin for 3 days, lysed, and analyzed by quantitative RT-PCR (n = 4). (E) Cells were treated as (A), lysed, and analyzed by western blotting. (F) Either PLD2+/+ MEFs or PLD2−/− MEFs were incubated with or without serum for 18 h, lysed, and subjected to western blotting. (G) C2C12 myoblasts were incubated either under 1 g or under SM for 24 h, after which they were induced to differentiate under 1 g for 48 h. The cells were lysed and subjected to western blotting. Abbreviations: simulated microgravity (SM); simulated microgravity in the presence of 100 nM AS1842856 (SM + AS); differentiation (Diff); simulated microgravity for 24 h before the induction of differentiation (pre-SM). All are blots representative of 3 to 5 independent experiments. Data are expressed as mean ± SD, with Mann-Whitney U test performed as indicated. **P < 0.01, *P < 0.05 versus 1 g control; ##P < 0.01, #P < 0.05 versus simulated microgravity.