Figure 3. ESCRT depletion resulted in... | Rockefeller University Press

Figure 3.

$ESCRT depletion resulted in enlargement of tauRD (P301L) aggregates and an increased population of ubiquitylated insoluble tauRD (P301L). (A) Schematic of tauRD (P301L)-Venus aggregate detection and quantification by a high-content image analyzer. Raw data consisting of DAPI-stained nuclei and GFP (Venus) fluorescence were obtained. The area of DAPI fluorescence was used to define the nucleus (circle), and a region of set width around the nucleus (ring) was used as the cytoplasm. Bright spots made by Venus within the circle and ring were defined as nuclear (red) and cytoplasmic (yellow) tauRD (P301L)-Venus aggregates, respectively. Ring spot total intensity was used to measure the size of cytoplasmic tauRD (P301L)-Venus aggregates. Scale bar: 10 μm. (B–D) Ring spot total intensity of tauRD (P301L)-Venus aggregates was quantified by a high-content image analyzer, and cells were imaged by a confocal microscope. TauRD (P301L)-Venus aggregate–positive cells were treated with 500 nM BTZ or 1 µM BafA1 (B), transfected with control, TSG101, CHMP4A/B/C, or VPS4A/B siRNA (C), or transfected with control or PTPN23 sgRNA, and successfully transfected cells were selected by puromycin treatment (D). Ring spot total intensity and images were acquired 16 h after drug treatment (B), 72 h (C), or 1 wk (D) after transfection. Data were normalized to the ring spot total intensity of DMSO (B)-, siControl (C)-, or sgControl (D)-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test (B and C) or two-tailed Student’s t test (D) with *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant. Scale bar: 10 μm. (E) TauRD (P301L)-Venus aggregate–positive cells were transfected with control or TSG101 siRNA. Cells were lysed 72 h after transfection, and Triton X-100–soluble and Triton X-100–insoluble fractions were immunoprecipitated with a GST-tagged anti-GFP nanobody. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of tauRD (P301L)-Venus or ubiquitin in lane 5 and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test with *P < 0.05, **P < 0.01. n.s., not significant. (F) UbiCRest analysis of tauRD (P301L)-Venus ubiquitylation. TauRD (P301L)-Venus aggregate–positive cells were lysed, and the insoluble fraction was immunoprecipitated with a GST-tagged anti-GFP nanobody. 15 µM K48-specific deubiquitinase OTUB1, 15 µM K63-specific deubiquitinase AMSH-LP, or both were added to the immunoprecipitated samples and rotated at 37°C for 30 min and then immunoblotted with the indicated antibodies. Data were normalized to the band intensity of ubiquitin in the control sample and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F3.$

ESCRT depletion resulted in enlargement of tauRD (P301L) aggregates and an increased population of ubiquitylated insoluble tauRD (P301L). (A) Schematic of tauRD (P301L)-Venus aggregate detection and quantification by a high-content image analyzer. Raw data consisting of DAPI-stained nuclei and GFP (Venus) fluorescence were obtained. The area of DAPI fluorescence was used to define the nucleus (circle), and a region of set width around the nucleus (ring) was used as the cytoplasm. Bright spots made by Venus within the circle and ring were defined as nuclear (red) and cytoplasmic (yellow) tauRD (P301L)-Venus aggregates, respectively. Ring spot total intensity was used to measure the size of cytoplasmic tauRD (P301L)-Venus aggregates. Scale bar: 10 μm. (B–D) Ring spot total intensity of tauRD (P301L)-Venus aggregates was quantified by a high-content image analyzer, and cells were imaged by a confocal microscope. TauRD (P301L)-Venus aggregate–positive cells were treated with 500 nM BTZ or 1 µM BafA1 (B), transfected with control, TSG101, CHMP4A/B/C, or VPS4A/B siRNA (C), or transfected with control or PTPN23 sgRNA, and successfully transfected cells were selected by puromycin treatment (D). Ring spot total intensity and images were acquired 16 h after drug treatment (B), 72 h (C), or 1 wk (D) after transfection. Data were normalized to the ring spot total intensity of DMSO (B)-, siControl (C)-, or sgControl (D)-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test (B and C) or two-tailed Student’s t test (D) with *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant. Scale bar: 10 μm. (E) TauRD (P301L)-Venus aggregate–positive cells were transfected with control or TSG101 siRNA. Cells were lysed 72 h after transfection, and Triton X-100–soluble and Triton X-100–insoluble fractions were immunoprecipitated with a GST-tagged anti-GFP nanobody. Input and IP samples were immunoblotted with the indicated antibodies. Data were normalized to the band intensity of tauRD (P301L)-Venus or ubiquitin in lane 5 and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test with *P < 0.05, **P < 0.01. n.s., not significant. (F) UbiCRest analysis of tauRD (P301L)-Venus ubiquitylation. TauRD (P301L)-Venus aggregate–positive cells were lysed, and the insoluble fraction was immunoprecipitated with a GST-tagged anti-GFP nanobody. 15 µM K48-specific deubiquitinase OTUB1, 15 µM K63-specific deubiquitinase AMSH-LP, or both were added to the immunoprecipitated samples and rotated at 37°C for 30 min and then immunoblotted with the indicated antibodies. Data were normalized to the band intensity of ubiquitin in the control sample and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F3.

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