Figure S2. Annexin-containing EVs are... | Rockefeller University Press

Figure S2.

$Annexin-containing EVs are shed from the repair scab after plasma membrane damage. (A) Confocal micrographs of ANXA2-mScarlet recruitment in three laser ablation experiments are shown. Image times are relative to the first image taken after ablation. White arrows in the top panes indicate the sites of ablation. Arrows in the bottom panes indicate EVs. Scale bars: 5 μm. (B) Representative confocal micrographs of ANXA2-mScarlet shedding are shown. Image times are relative to the first image taken after ablation. White arrows in panel indicate the site of ablation. Scale bars: 5 μm. (C) Representative confocal micrographs of ANXA2-mScarlet and ANXA1-mNeonGreen–expressing cells after laser ablation. Arrows indicate the ablation site. Scale bars: 10 μm. (D) Representative confocal micrographs of ANXA2-mScarlet and ANXA6-mNeonGreen–expressing cells after laser ablation. Arrows indicate the ablation site. Scale bars: 10 μm. (E) Representative widefield micrographs of cells stained with 1 μM Sytox Green after a treatment period with the indicated SLO concentration and recovery period. Scale bars: 150 μm. (F) Immunoblots show expression of annexin A2-Nluc (A2-Nluc) using a low expression promoter. (G) Immunoblots show enrichment of EV markers after capture with immobilized annexin A5 from the conditioned medium 100k × g pellet fraction (FT—flow through; Elu—elution). (H) EV production index from ANXA2-Nluc cells expressing mCherry-VPS4a (dominant mutant) under control of a doxycycline-inducible promoter. Cells were pretreated with 200 ng/ml doxycycline (Dox) or DMSO for 6 h, followed by treatment with 200 ng/ml SLO. Error bars indicate three experimental replicates. Source data are available for this figure: SourceData FS2.$

Annexin-containing EVs are shed from the repair scab after plasma membrane damage. (A) Confocal micrographs of ANXA2-mScarlet recruitment in three laser ablation experiments are shown. Image times are relative to the first image taken after ablation. White arrows in the top panes indicate the sites of ablation. Arrows in the bottom panes indicate EVs. Scale bars: 5 μm. (B) Representative confocal micrographs of ANXA2-mScarlet shedding are shown. Image times are relative to the first image taken after ablation. White arrows in panel indicate the site of ablation. Scale bars: 5 μm. (C) Representative confocal micrographs of ANXA2-mScarlet and ANXA1-mNeonGreen–expressing cells after laser ablation. Arrows indicate the ablation site. Scale bars: 10 μm. (D) Representative confocal micrographs of ANXA2-mScarlet and ANXA6-mNeonGreen–expressing cells after laser ablation. Arrows indicate the ablation site. Scale bars: 10 μm. (E) Representative widefield micrographs of cells stained with 1 μM Sytox Green after a treatment period with the indicated SLO concentration and recovery period. Scale bars: 150 μm. (F) Immunoblots show expression of annexin A2-Nluc (A2-Nluc) using a low expression promoter. (G) Immunoblots show enrichment of EV markers after capture with immobilized annexin A5 from the conditioned medium 100k × g pellet fraction (FT—flow through; Elu—elution). (H) EV production index from ANXA2-Nluc cells expressing mCherry-VPS4a (dominant mutant) under control of a doxycycline-inducible promoter. Cells were pretreated with 200 ng/ml doxycycline (Dox) or DMSO for 6 h, followed by treatment with 200 ng/ml SLO. Error bars indicate three experimental replicates. Source data are available for this figure: SourceData FS2.

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