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. 2020 Jul 28;39(16):e103009. doi: 10.15252/embj.2019103009

Figure 7. EVs induced by glutamine depletion promote amphiregulin‐ and ERK‐dependent growth in HCT116 recipient cells.

Figure 7

  1. Western blot analysis of ERK phosphorylation (p‐ERK) in recipient serum‐deprived HCT116 cells pre‐treated with EVs isolated by SEC from glutamine‐replete or glutamine‐depleted HCT116 cells or vehicle (PBS). Bar chart shows ratio of p‐ERK to ERK levels from triplicate independent experiments.
  2. Bar chart shows HCT116 recipient cell growth over 120 h, after pre‐treatment with EV preparations isolated as in (A) or with PBS, and then incubation for the first 24 h of culture in the presence or absence of the ERK inhibitor SCH772984 (1.00 μM). Growth curves are shown in Appendix Fig S8C.
  3. Western blot analysis showing levels of the EGFR ligand, amphiregulin (AREG) in EVs isolated by SEC of medium from HCT116 cells cultured in glutamine‐replete (2.00 mM) or glutamine‐depleted (0.15 mM) conditions for 24 h. Gel loading was normalised to cell lysate protein levels. AREG's molecular weight (approximately 26–28 kDa) suggests it is in its membrane‐associated form (see Appendix Fig S8D). In the bar chart, the levels of putative exosome proteins were normalised to cell lysate protein levels. Significantly decreased levels are in blue and increased levels are in red. (C′) Growth curves in low (1%) serum are for HCT116 recipient cells pre‐treated with the EV preparations [isolated as in (C)], which had themselves been pre‐treated with and without anti‐AREG neutralising antibody or with a control IgG. Solid red line shows growth‐promoting effect of EVs isolated under glutamine depletion, which is blocked by anti‐AREG antibody (red dashed line). ***P colour denotes significant increase relative to EVs from glutamine‐replete cells (black) and after anti‐AREG treatment of EVs (red). (C″) Western blot analysis of ERK phosphorylation (p‐ERK) in recipient serum‐deprived HCT116 cells pre‐treated with EVs isolated by SEC from glutamine‐replete (2.00 mM) or glutamine‐depleted (0.15 mM) HCT116 cells or vehicle (PBS), which were pre‐treated with anti‐AREG (AREGab) or a control immunoglobulin (Ig). Note increase in ERK phosphorylation using EVs from glutamine‐depleted cells, which is blunted by the addition of the anti‐AREG antibody.
Data information; Growth curves were reproduced in three independent experiments and analysed by two‐way ANOVA. Bar charts derived from three independent experiments and analysed by ANOVA or the Kruskal–Wallis test: ***P < 0.001, **P < 0.01, *P < 0.05. Bars and error bars denote mean ± SD.Source data are available online for this figure.