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. Author manuscript; available in PMC: 2023 Jul 17.
Published in final edited form as: Nat Cell Biol. 2023 Feb 16;25(3):415–424. doi: 10.1038/s41556-023-01092-1

Fig. 2. Akt promotes exosome secretion from cells grown on stiff matrix.

Fig. 2.

a, Heatmap of RPPA data showing the levels of phospho-Akt and its downstream signaling proteins p-4E-BP1 and pGSK-3 in Huh7 cells grown on soft vs. stiff matrix. b, Exosomes from Huh7 cells expressing GFP control or myr-Akt on soft or stiff matrix were analyzed by NTA. Exosomes from cells expressing GFP on soft matrix was normalized to 1. Relative values are presented as Mean ± S.D., n=3.c, Huh7 cells grown on soft or stiff matrix were treated with DMSO or Akt inhibitor MK-2206. Exosome concentration from the cells treated with DMSO on soft matrix was normalized to 1. Values are presented as Mean ± S.D., n=3. d, Exosomes were purified from the media of equal amounts of Huh7 cells treated with DMSO or MK-2206. The levels of HRS, Syntenin-1, and CD63 in whole cell lysates (“WCL”) and purified exosomes were examined by immunoblotting. Total Akt (t-Akt) and p-Akt in WCL was also examined. GAPDH was used as WCL loading control. e, Quantification of the levels of HRS, Syntenin-1, and CD63. Values are presented as Mean ± S.D. n=3. f, Huh7 cells were treated with MK-2206 and immunostained for CD63 and LAMP1. Scale Bar: 10 μm. Cell peripherals were indicated with dashed lines and the perinuclear region was indicated with solid lines. g, Quantification of the percentage of perinuclear CD63 or LAMP1. Values are presented as Mean ± S.D. n=3. h, An equal amount of Huh7 cells grown on stiff matrix were treated with FAK inhibitor PND-1168, and exosomes in the conditioned media were purified. Exosome markers (HRS, Syntenin-1 and CD63) were examined. The levels of t-Akt and p-Akt in cell lysates were also examined. i, Quantification of the levels of HRS, Syntenin-1 and CD63. Mean ± S.D. n=3. j, Huh7 cells growing on soft or stiff matrix were treated with FAK inhibitor PND-1168. Exosome concentration from cells treated with DMSO on soft matrix was normalized to 1. Values are presented as Mean ± S.D. n=3. Source numerical data and unprocessed blots are available in source data. n represents the number of independent experiments.