Journal: The Journal of cell biology

Article Title: Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes

doi: 10.1083/jcb.202407013

Figure Lengend Snippet: (A) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against Snx15 following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm; inset bar, 2 μm. (B) Representative images of natively expressed Ist1-HaloTag in control cells and cells lacking Snx15 following labeling with the JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under each condition is also shown (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm. (C) Quantification of CHMP1B levels in control cells and cells depleted of CHMP1A, CHMP1B, or both CHMP1A and CHMP1B, based on immunoblot analysis ( n = 3). Error bars represent the mean ± SEM. ****P < 0.0001 and ***P < 0.001, as calculated using a one-way ANOVA and Tukey’s post hoc test. (D) Representative images of natively expressed Ist1-HaloTag in control cells treated with a scrambled siRNA (Mock) or siRNAs targeting CHMP1 isoforms following labeling with JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under the conditions shown is also provided (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm. (E) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against CHMP4B following labeling with JF650-HaloTag ligand and treatment with either a scrambled siRNA (Mock) or siRNAs targeting CHMP1A and CHMP1B. Representative images are shown (left) with quantification highlighting CHMP4B fluorescence intensity under each condition (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm; inset bar, 2 μm.

Article Snippet: Immunofluorescence studies were conducted as described previously using the following antibodies (1 μg/ml final concentration each): CHMP1A (rabbit polyclonal; Proteintech 15761-AP), CHMP1B (rabbit polyclonal; Proteintech 14639-1-AP), CHMP4B (rabbit polyclonal; Proteintech 13683-1-AP), Snx15 (rabbit polyclonal; Proteintech 16049-1-AP), spastin (mouse monoclonal; Santa Cruz Biotechnology SC-81624), GAPDH (mouse monoclonal; Proteintech 60004-1-Ig), and Ist1 (rabbit polyclonal; a gift of Dr. Wesley Sundquist, University of Utah, Salt Lake City, UT, USA).

Techniques: Expressing, Staining, Labeling, Marker, Control, Western Blot, Fluorescence

Journal: Nature cell biology

Article Title: Endosomal Membrane Tension Regulates Escrt-III-Dependent Intra-Lumenal Vesicle Formation

doi: 10.1038/s41556-020-0546-4

Figure Lengend Snippet: a) Confocal immunofluorescence images showing colocalization between CHMP4B-GFP and indicated ESCRT subunits after 10min hypertonic shock. Image representative of 4 experiments. Bars: 4 μm. b) Quantification of punctae number per cell from automated confocal images of immunofluorescence before (black) and after 30min hypertonic shock (red) for various markers. Bars are mean±SEM. One dot corresponds to the average number of punctae per cell in one field of view (N values are from 3 independent experiments: STAM, STAM hyper and VPS4B: 60 fields; VPS4B hyper: 61 fields; TSG101 hyper, CHMP4B and CHMP3: 71 fields; others: 72 fields; 10-40 cells per field) normalized to the average number before shock (black). c) Confocal convolved images of CHMP4B-GFP HeLa cells before and after 10min hypertonic shock, pre-treated with anti-ALIX siRNAs or control siRNAs against VSV-G. Image representative of 3 experiments. Bars: 15 μm. d) Mean number of CHMP4B punctae per cell, for cells treated with control siRNAs or siRNAs against ALIX, TSG101 or both, before and after hypertonic shocks of various duration. (N=3 independent replicates represented by dots on the graph with mean±SEM. Analysis by automated microscopy with a total of 72 fields per condition and10-40 cells per field.)

Article Snippet: ESCRT were then immunolabeled in this blocking solution for 1h at room temperature with antibodies against the following proteins:, CHMP1B, CHMP3, CHMP4B, Alix, VPS4B, TSG101, STAM (All were rabbit polyclonal antibodies obtained from ProteinTech Group, Rosemont, IL, USA, with the following reference numbers, respectively: 14639-1-AP, 15472-1-AP, 13683-1-AP, 12422-1-AP, 17673-1-AP, 14497-1-AP, 12434-1-AP) all at a 300-fold dilution.

Techniques: Immunofluorescence, Control, Microscopy

Journal: Nature cell biology

Article Title: Endosomal Membrane Tension Regulates Escrt-III-Dependent Intra-Lumenal Vesicle Formation

doi: 10.1038/s41556-020-0546-4

Figure Lengend Snippet: a-b) Maximum projection of confocal images of live HeLa-CHMP4B-GFP cells labelled with LysoTracker before and after a 10min incubation with 0.5mM LLOMe. Image representative of 2 experiments. Bars: 10 μm. In (b) cells were pre-treated with control siRNAs (VSV, blue) or siRNAs against TSG101 (green). Average number of LysoTracker punctae per cell. N= 3 independent replicates, mean±SD. Analysis by automated microscopy with a total of 56 fields per condition and 10-40 cells per field. c) Average number of CHMP4B-GFP punctae per cell with time, during and after LLOMe treatment. Shaded area: SEM (N=51 cells from 3 independent replicates). d) Recovery after photobleaching of individual CHMP4B-GFP punctae induced by LLOMe treatment. Shaded area: SEM. (N=18 endosomes from 3 independent replicates). e) Confocal images of immuno-fluorescence against several markers after 10min incubation with LLOMe treatment. Image representative of 3 experiments. Bars: 4 μm. f) Number of punctae per cell for different ESCRT subunits before (black) and after LLOMe treatment (red). Each dot represents the mean number of ESCRT punctae/cell in one field of view. Bars are mean±SEM (N values for 3 independent experiments are respectively: STAM, STAM LLOMe and VPS4B: 60 fields; VPS4B LLOMe: 61 fields; CHMP1B LLOMe: 68 fields; TSG101 LLOMe: 70 fields; CHMP4B and CHMP3: 71 fields; others: 72 fields. In all cases: 10-40 cells per field. g) FLIM images of endosomes in Hela MZ cells stained by Lyso Flipper before and after 0.5 mM LLOMe treatment. Image representative of 6 experiments. Bars: 10 μm. h) Lyso Flipper lifetime measurements before and 5min after LLOMe addition (quantification as in ). Thin coloured lines: 6 independent experiments; thick red line: mean with SEM (two-tailed paired t-test: P=0.00454651). For each experiment, average fluorescent lifetimes were calculated from >500 endosomes taken from at least 3 different cells. i) FLIM images of RAB5Q79L endosomes in Hela MZ cells stained after a 2h incubation with FliptR before and after 0.5 mM LLOMe treatment. Images representative of 4 experiments. Bars: 10 μm. j) FliptR lifetime measurements of RAB5Q79L endosomes before and 10min after LLOMe treatment (lifetime was measured as in ). Thin coloured lines: 4 independents experiments; thick red line: mean with SEM (two-tailed paired t-test: P=0.89443). k) Average number of CHMP4B-GFP punctae per cell before (control) and after treatment with LLOMe in cells transfected with control siRNAs (siVSV) or siRNAs against ALIX, TSG101 or both ALIX and TSG101. Error bars are SEM. N=3 independent replicates represented by dots on the graph with mean±SEM. Analysis by automated microscopy with a total of 72 fields per condition, with 10-40 cells per field.

Article Snippet: ESCRT were then immunolabeled in this blocking solution for 1h at room temperature with antibodies against the following proteins:, CHMP1B, CHMP3, CHMP4B, Alix, VPS4B, TSG101, STAM (All were rabbit polyclonal antibodies obtained from ProteinTech Group, Rosemont, IL, USA, with the following reference numbers, respectively: 14639-1-AP, 15472-1-AP, 13683-1-AP, 12422-1-AP, 17673-1-AP, 14497-1-AP, 12434-1-AP) all at a 300-fold dilution.

Techniques: Incubation, Control, Microscopy, Fluorescence, Staining, Two Tailed Test, Transfection