Figure EV5. Analysis of EV function and proteins secreted by control and knockdown HCT116 colorectal cancer cells.

• A
  Number of DAPI‐stained cells after 120‐h culture in serum‐depleted conditions (1% serum), following pre‐incubation either with EVs from HCT116 cells cultured in glutamine‐replete or glutamine‐depleted conditions for 24 h, or with vehicle (PBS). Bar charts derived from three independent experiments.
• B
  Overall levels of apoptosis, measured in arbitrary units (AU), of fluorescence, and levels of apoptosis per cell (right‐hand graph) detected in HCT116 cells by analysis of caspase‐3 and caspase‐7 activities, following treatments with EVs isolated as in (A) or with vehicle (PBS).
• C
  Growth curves for HCT116 recipient cells in 1% serum conditions following 30‐min pre‐incubation with 0.5 × 10³, 1.0 × 10³ and 2.0 × 10³ EVs per cell, isolated by SEC from glutamine‐replete and glutamine‐depleted HCT116 cells or PBS.
• D–F
  Western blot analysis of cell lysate proteins, and Nanosight Tracking Analysis (NTA) of EV size and number in D′ and E′. Bar charts indicate putative exosome protein levels normalised to Tubulin on Western blots. (D) With relevance to EVs shown in Fig 6C, Western blot analysis of lysates from EV‐secreting cells with and without knockdown of Rab11a in glutamine‐replete and glutamine‐depleted conditions. Total protein levels were increased by 1 ± 3% following knockdown. Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates and relative levels of phosphorylation of mTORC1 downstream readouts, S6 and 4E‐BP1 (measured as a ratio of p‐S65-4E‐BP1 to pan 4E‐BP1). Significantly decreased levels are in blue and increased levels are in red.(D′) NTA of EVs isolated from HCT116 cells with and without knockdown of Rab11a. (E) With relevance to EVs shown in Fig 6F, Western blot analysis of proteins isolated from EV‐secreting HCT116 cells with and without knockdown of Rab7. Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates and relative levels of phosphorylation of mTORC1 downstream readouts, S6 and 4E‐BP1 (measured as a ratio of p‐S65-4E‐BP1 to pan 4E‐BP1). Significantly decreased levels are in blue and increased levels are in red. (E′) NTA of EVs isolated from HCT116 cells with and without knockdown of Rab7. (F) With relevance to EVs shown in Fig 7C, Western blot analysis of cell lysates from HCT116 cells cultured in glutamine‐replete (2.00 mM) and glutamine‐depleted (0.15 mM) medium for 24 h. Gel was loaded with equal protein amounts. The activity of mTORC1 was assessed via phosphorylation of S6 and 4E‐BP1. Bar chart shows the abundance of selected exosome/EV proteins relative to tubulin in these lysates. Significantly decreased levels are in blue and increased levels are in red.

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 the Kruskal–Wallis test: **P < 0.01, *P < 0.05 Bars and error bars denote mean ± SD.