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: Cell Reports

Article Title: Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion

doi: 10.1016/j.celrep.2021.110187

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CHMP4B , Novus Biologicals , Cat#NBP1-91782; RRID: AB_11020392.

Techniques: Isolation, Virus, Recombinant, Electron Microscopy, Staining, Western Blot, Control, Plasmid Preparation, Software

Journal: The Journal of Cell Biology

Article Title: The ESCRT protein Chmp4c regulates mitotic spindle checkpoint signaling

doi: 10.1083/jcb.201709005

Figure Lengend Snippet: Chmp4c-depletion reduces cold-stable microtubule polymers. (A–D) Cells transfected with negative siRNA (control), Chmp4c siRNA (siChmp4c), Chmp4a siRNA (siChmp4a), or Chmp4b siRNA (siChmp4b) were treated with ice-cold medium for the indicated times. (E) Centromere length in metaphase-like spindles. Insets show magnified centromeres, and values indicate the centromere length. Bars: (inset) 0.5 µm. (F) Quantification of centromere length from cells as in E or incubated with nocodazole (nocod) for 4 h. Control: n = 291 centromeres; 20 cells; siChmp4c: n = 697 centromeres, 26 cells; nocodazole: n = 200 centromeres, 20 cells from three independent experiments. (G) Cells expressing GFP, WT or S210A mutant Chmp4c:GFP resistant to degradation by siChmp4c-2, or GFP:Vps4-K173Q were treated with ice-cold medium for 15 min. Mean tubulin intensity is shown, and values in the controls were set to one. n = 90 cells (B, D, and G) from three independent experiments. Error bars show the SD. ***, P < 0.001 compared with the control. Statistically significant differences were determined by ANOVA and Student’s t test. Bars, 5 µm.

Article Snippet: Human Chmp4c located in the open reading frame (5′-GCUUGGGCUACCUAAACUA-3′), Chmp4c-2 located in the 3′-untranslated region (5′-GUAGAGGAGUCUUAUAUGA-3′), ZW10 (a pool of three different siRNAs: 5′-GCAUGGAGCUCACAAUACA-3′, 5′-CCAAGUAUUUGCACCUUUA-3′, and 5′-CAAGCCAGCUUGCAAGAAA-3′), Chmp4a (a pool of three different siRNAs: 5′-CAAACUGACGGGACAUUAU-3′, 5′-CUGAGUGGGUAUCCUGAUA-3′, and 5′-GAAGGAUCUUGCUACAGAA-3′), and Chmp4b (a pool of three different siRNAs: 5′-CAACAUGGACAUCGAUAAA-3′, 5′-CAGUCCCUCUACCAAAUGU-3′, and 5′-GCUGAAUCCUCAAUGGAAA-3′) siRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Incubation, Expressing, Mutagenesis

Journal: Advanced Materials Interfaces

Article Title: ESCRT III Regulates Lysosomal Perinuclear Clustering by Inhibiting Kinesin 1 Leading to Agnps Cytotoxicity in Human Cells

doi: 10.1002/admi.202400944

Figure Lengend Snippet: Figure 2. AgNPs induced LPC and biochemical markers expression in HS27 cells. a) Upper panel, no AgNPs treatment; middle panel, AgNPs treated HS27 cells for 4 h; lower panel, AgNPs treated HS27 cells for 24 h. Blue color, Hoechst33342 stained nucleus; Red color, Lyso-Tracker Red tracked lyso- some. Images were taken in a confocal microscope, scale bar = 100 μm(Phase-contract, Nucleus, Lysosome), scale bar = 50 μm(Merge). b) Comparison of survival rates in AgNPs alone or combined with other chemicals; c) CHMP4B and Kinesin 1 protein expression in AgNPs treated HS27 cells by western blot (Figure S1, Supporting Information). AgNPs, Silver Nanoparticles; LPC, lysosomal perinuclear clustering; CHMP4B, charged multivesicular body protein 4B.

Article Snippet: For CHMP4B and Kinesin 1 over-expression, Myc-DDK-tagged human CHMP4B (Catalog#: RC207637, OriGene, Rockville, MD, USA) and Kinesin 1 full-length cDNA was cloned into pCDNA 3.1 plasmid.

Techniques: Expressing, Staining, Microscopy, Comparison, Western Blot

Journal: Advanced Materials Interfaces

Article Title: ESCRT III Regulates Lysosomal Perinuclear Clustering by Inhibiting Kinesin 1 Leading to Agnps Cytotoxicity in Human Cells

doi: 10.1002/admi.202400944

Figure Lengend Snippet: Figure 4. CHMP4B and Kinesin 1 inhibition enhanced LPC in AgNPs treated HS27 cells. a) Lysosome images in different conditions. Upper panel, AgNPs treatment; middle panel, AgNPs with CHMP4B siRNA treatment; lower panel, AgNPs with Kinesin 1 siRNA treatment. Red color, Lyso-Tracker Red tracked lysosome. Images were taken in a confocal microscope, scale bar = 100 μm. b) HS27 cell survival rate in AgNps or CHMP4B and Kinesin 1 siRNA. c) CHMP4B and Kinesin 1 protein expression in AgNps, CHMP4B siRNA, and Kinesin 1 siRNA treated HS27 cells by western blot. AgNps, Silver Nanoparticles; LPC, lysosomal perinuclear clustering; CHMP4B, charged multivesicular body protein 4B.

Article Snippet: For CHMP4B and Kinesin 1 over-expression, Myc-DDK-tagged human CHMP4B (Catalog#: RC207637, OriGene, Rockville, MD, USA) and Kinesin 1 full-length cDNA was cloned into pCDNA 3.1 plasmid.

Techniques: Inhibition, Microscopy, Expressing, Western Blot

Journal: Advanced Materials Interfaces

Article Title: ESCRT III Regulates Lysosomal Perinuclear Clustering by Inhibiting Kinesin 1 Leading to Agnps Cytotoxicity in Human Cells

doi: 10.1002/admi.202400944

Figure Lengend Snippet: Figure 5. CHMP4B and Kinesin 1 overexpression rescued AgNPs induced the LPC model and cell survival. a) LPC cell images in AgNPs and other reagent treatments (scale bar = 50 μm). b) HS27 cell viability in AgNPs and other reagent treatments. c) CHMP4B and Kinesin 1 protein expression by western blot in AgNPs and other reagent treatments. AgNPs, Silver Nanoparticles; LPC, lysosomal perinuclear clustering; CHMP4B, charged multivesicular body protein 4B.

Article Snippet: For CHMP4B and Kinesin 1 over-expression, Myc-DDK-tagged human CHMP4B (Catalog#: RC207637, OriGene, Rockville, MD, USA) and Kinesin 1 full-length cDNA was cloned into pCDNA 3.1 plasmid.

Techniques: Over Expression, Expressing, Western Blot