Figure 1
Growth and cell cycle regulation during endothelial differentiation. a) Rate of cell proliferation. RoSH2 cells were labeled with CFDA, a cell-permeable fluorescent dye, and then cultured for 24 hours. They were then plated on gelatin-coated plates under non-differentiating condition or on matrigel-coated plate under differentiating condition. At 0, 24, 48 and 72 after plating, cells were harvested, median cellular fluorescence was measured by flow cytometry and the number of cell divisions was calculated as a function of the loss in fluorescence for every twenty-four hour; b) the cell numbers were counted and normalized against cell count at time 0. Cell number at time 0 was designated as one.
Figure 2
Effects of inhibiting PI3 K/PI3K/Akt or Raf/MEK/Erk on growth of RoSH2 cells. RoSH2 cells were pre-labeled with CFDA, cultured on gelatin-coated plates for 24 hours. One quarter of the plates were not treated with any inhibitor (N), one quarter with 10 μM Ras inhibitor, FTase inhibitor III (FTI), one quarter with 50 μM MEK inhibitor PD98059 (PD), and the last quarter with LY 294002 (LY). Each of the media with the appropriate inhibitors was replaced every twenty-four hours. At 0, 24, 48 and 72 hours after plating, cells were harvested and were assayed for cellular fluorescence by flow cytometry. The number of cell cycles for every 24 hours was calculated as described in Materials and Methods. b) Effects of FTI and PD98059 on cell numbers. Twenty-four hours after treatment with FTI, PD98059 or a combination of both drugs, the cell numbers were counted and normalized against that of untreated cells. Cell number at time 0 was designated as one.
Figure 3
Growth and cell cycle regulation during endothelial differentiation and rapamycin treatment. a) Rate of cell division. RoSH2 cells were labeled with CFDA, a cell-permeant fluorescent dye, cultured for 24 hours and re-plated on gelatin-coated plates to be maintained as undifferentiated cells (Undif) or on matrigel to induce differentiation (Dif). Cells were harvested at 0, 24, 48 and 72 hours. At 48 hours after replating, half of the remaining plates of cells under undifferentiating condition or differentiating condition were treated with 50 ηM rapamycin (R undif and Rdif, respectively). Median cellular fluorescence of the harvested cells was measured by flow cytometry and the number of cell divisions was calculated as a function of the loss in fluorescence; b) Cell cycle progression during endothelial differentiation. RoSH2 cells were plated on either gelatin-coated plate (self-renewing) or matrigel (differentiating) and labeled with BrdU for 16 hours. After removing BrdU, half of the gelatin-coated plates were treated with 50 ηM rapamycin. At 0, 6 and 12 hours, cells were harvested, stained with anti-BrdU and PI. DNA content of BrdU-labeled cells as measured by PI was analyzed by flow cytometry.
Figure 4
Phosphorylation of mTOR targets and expression of endothelial markers during differentiation and rapamycin treatment. At 0, 6, 24 and 48 hours after RoSH2 cells were induced to differentiate by plating on matrigel-coated plates or treated with 50 ηM rapamycin, the cells were harvested for western blot analysis and probed for a, d) endothelial markers, Tie-2 and Flk-1; b, c) Eif4ebp1 and phosphorylated Rps6kb1. Tsc2 protein was used as an internal control for loading between lanes and between blots.
Figure 5
PI3K and Raf/MEK/Erk signaling in RoSH2 cells. a) Effects of PI3 K/Akt inhibitors. Undifferentiated RoSH2 cells were plated and cultured for 24 hours, then serum-starved for 20 hours before culturing in media with 50 μM LY294002 dissolved in DMSO, 100 nM wortmannin or 0.1% (v/v) DMSO for 15 min and then challenged with 100 nM insulin for 20 minutes. Cell lysates were prepared and assayed by western blot analysis; b) Raf/MEK/Erk signaling during differentiation. At 0, 6, 24 and 48 hours after plating RoSH2 cells on matrigel, cell lysates were prepared and analyzed by western blot assay for phosphorylated ERK1/2.. c) Effects of inhibiting Raf/MEK/Erk signalingRoSH2 cells were treated with 10 μM or 50 μM Ras inhibitor FTase inhibitor III, (FTI) and MEK inhibitor PD98059 for three hours and cell lysates were analyzed by western blot assays.
Figure 6
Translational control during endothelial differentiation and rapamycin treatment. a) Preparation of total RNA and ribosome-enriched RNA. RoSH2 cells, RoSH2 induced to differentiate by plating on matrigel for 48 hours and RoSH2 cells treated with 50 ηM rapamycin for 48 hours were harvested. Total and ribosome-enriched RNAs were quantitated by absorbance at 260 nm. 2 μg of RNA from each of the two cellular fractions were separated on a 1.0% agarose gel, stained with ethidium bromide and visualized under UV illumination. Top panel Representative RNA samples from total and ribosome-enriched RNAs prepared from undifferentiated (Undiff), differentiated (Diff) and rapamycin-treated (Rapa) RoSH2 cells are shown. Bottom panel For each sample, 1 μl of serially diluted RNA sample mixed with 1 μl of 0.5 ηg/μl ethidium bromide was visualized under UV illumination to verify RNA loading in each lane; RNA was isolated from total cellular extract (total) and ribosome-enriched subcellular fraction (ribo) prepared from RoSH2 cells before and 48 hours after induction of endothelial differentiation by plating matrigel-coated plates; b) Distribution of rpL5 mRNA in total and ribosome-enriched RNAs before and after differentiation. RT-PCR using oligo-dT-primed cDNA, and Rpl5 and Fkbp12 specific primers was performed on 10 fold serial dilution i.e. 1×, 10× and 100× of RNAs.; c) RT-PCR analysis of transcript abundance in RoSH2 cells before and after differentiation. RoSH2 cells were induced to undergo endothelial differentiation by plating on matrigel. Total and ribosome-enriched RNA were purified at 0 and 48 hours and analyzed by RT-PCR for transcript abundance of G1 cyclins and endothelial receptors; d) Western blot analysis. RoSH2 cells were induced to undergo endothelial differentiation by plating on matrigel and at 0, 6, 24 and 48 hours, cell lysates were prepared and assayed by western blot analysis. Tsc2 protein was used as an internal control for loading between lanes and between blots; e) Effect of rapamycin on Rpl5, cyclinD2 and Tek transcript abundance in total and ribosome-enriched RNA. Total and ribosome-enriched RNA were isolated from RoSH2 cells treated for 0 and 48 hours with 50 ηM rapamycin treatment and analyzed by RT-PCR; f) Western blot analysis. Cell lysates from rapamycin-treated RoSH2 cells at 0, 6, 24 and 48 hours were assayed by western blot analysis for cyclinD2 and Tek. Tsc2 protein was used as an internal control for loading between lanes and between blots.