Fig. 2
From: A two-pore channel protein required for regulating mTORC1 activity on starvation
Developmental delay of tpc2− cells. Parental Ax2 and tpc2− cells were harvested from exponential growth and washed twice with HKC-LoCa buffer. a Agarose development. 3.84 × 106 cells plated on 1% agarose HKC-LoCa buffer in a 6-well plate (final cell density 4 × 105 cells/cm2), incubated at 22 °C. b Under buffer development, 3.8 × 106 cells plated on 1% agarose HKC-LoCa buffer in a 6-well plate (final cell density 4 × 105 cells/cm2), incubated at 22 °C. Developmental structures were observed at the times shown and photographed using a Coolpix P310 digital camera mounted on a light microscope (Motic AE21 Inverted Phase Microscope). Scale bars, 200 μm. Cells were developed as A (c-I) after 9 h or B (c-II) after 20 h, and representative images at low magnification are shown. Scale bars: c-I 200 μm, c-II 1 cm. Following development under buffer for 20 h (c-II) tpc2− cells form 96 ± 4 aggregates/cm2 compared to 20 ± 2 aggregates/cm2 for Ax2 cells (average ± SEM of three independent experiments). d Cells were resuspended at 1.25 × 107 cells/ml HKC-LoCa buffer and shaken for 2 h then pulsed with or without cAMP (50 nM every 6 mins) for 4 h, all at 22 °C. Cells were allowed to settle for 15 min then observed using DeltaVision Elite microscope and photographed using a EMCCD camera mounted on the microscope. Scale bar, 50 μm