Figure S7.
Latruculin B (LatB) control experiments and knockdowns of ESCRT proteins. (A) moDC preincubated in the presence of 100 μM LatB and cultured for 1 h in the absence of LatB. The cell is immunolabeled for IST1 (magenta), phalloidin (green), and DAPI (blue). No F-actin staining was observed in the presence of LatB, and phalloidin showed weak nuclear staining. Scale bar: 10 μm. (B) Left: Scheme of workflow of adhesion isolation. Right: Volcano plot of mass spectrometry hits. Key consensus adhesion proteins (Horton et al., 2015) were depleted from adhesions in response to LatB treatment. (C.I) Western blot of moDCs treated with non-targeting (NT) siRNA, IST1 siRNA, or TSG101 siRNA. Total protein levels were measured using Ponceau S and the blot was probed for TSG101 and IST1. (C.II) Quantification of C.I. Protein levels were corrected for loading using Ponceau S intensities and subsequently normalized to the NT condition. Graphs show mean ± SD (n ≥ 5 donors). (D) Effect of IST1 knockdown and TSG101 knockdown on the average number of IST1-positive structures per cell, the average size of the structures and the average total area covered by the structures per cell. Effects are shown both in untreated cells and in LatB treated cells. Each dot-line-dot pair represents 1 donor. *: P < 0.05; NS: not significant (paired two-sided t test). (E) Fold change in the number of adherent cells upon LatB treatment in moDCs with IST1 or TSG101 knockdown compared to cells treated with NT siRNA. (paired two-sided t test). (F) Quantification of integrin β2 signal in the ESCRT structures, based on immunofluorescence labeling of moDCs for integrin β2 and IST1. Knockdown of IST1 and TSG101 significantly reduced the level of integrin β2 (n = 3 donors; paired two-sided t test; ***: P < 0.001). (G.I) Western blot and quantification of knockdown of TSG101 and ALIX. Note unsuccessful knockdown of ALIX. (G.II) Flow cytometry of surface levels of integrin αM and integrin β2 in moDCs. TSG101 knockdown significantly lowered surface levels of integrin β2 (n = 3 donors; paired two-sided t test). A representative histogram is shown. (H) DAPI influx in moDCs was not affected by knockdown of TSG101. Scale bars, 10 μm. For all statistical analysis, data distribution was assumed to be normal, but this was not formally tested. Source data are available for this figure: SourceData FS7.

Latruculin B (LatB) control experiments and knockdowns of ESCRT proteins. (A) moDC preincubated in the presence of 100 μM LatB and cultured for 1 h in the absence of LatB. The cell is immunolabeled for IST1 (magenta), phalloidin (green), and DAPI (blue). No F-actin staining was observed in the presence of LatB, and phalloidin showed weak nuclear staining. Scale bar: 10 μm. (B) Left: Scheme of workflow of adhesion isolation. Right: Volcano plot of mass spectrometry hits. Key consensus adhesion proteins (Horton et al., 2015) were depleted from adhesions in response to LatB treatment. (C.I) Western blot of moDCs treated with non-targeting (NT) siRNA, IST1 siRNA, or TSG101 siRNA. Total protein levels were measured using Ponceau S and the blot was probed for TSG101 and IST1. (C.II) Quantification of C.I. Protein levels were corrected for loading using Ponceau S intensities and subsequently normalized to the NT condition. Graphs show mean ± SD (n ≥ 5 donors). (D) Effect of IST1 knockdown and TSG101 knockdown on the average number of IST1-positive structures per cell, the average size of the structures and the average total area covered by the structures per cell. Effects are shown both in untreated cells and in LatB treated cells. Each dot-line-dot pair represents 1 donor. *: P < 0.05; NS: not significant (paired two-sided t test). (E) Fold change in the number of adherent cells upon LatB treatment in moDCs with IST1 or TSG101 knockdown compared to cells treated with NT siRNA. (paired two-sided t test). (F) Quantification of integrin β2 signal in the ESCRT structures, based on immunofluorescence labeling of moDCs for integrin β2 and IST1. Knockdown of IST1 and TSG101 significantly reduced the level of integrin β2 (n = 3 donors; paired two-sided t test; ***: P < 0.001). (G.I) Western blot and quantification of knockdown of TSG101 and ALIX. Note unsuccessful knockdown of ALIX. (G.II) Flow cytometry of surface levels of integrin αM and integrin β2 in moDCs. TSG101 knockdown significantly lowered surface levels of integrin β2 (n = 3 donors; paired two-sided t test). A representative histogram is shown. (H) DAPI influx in moDCs was not affected by knockdown of TSG101. Scale bars, 10 μm. For all statistical analysis, data distribution was assumed to be normal, but this was not formally tested. Source data are available for this figure: SourceData FS7.

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