Journal: bioRxiv

Article Title: Macropinocytosis of aggregated amyloid-beta and tau requires Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641073

Figure Lengend Snippet: A) IPSC-derived neurons transduced with LAMP1-mCh (red) were treated with DMSO (vehicle control), NAV-2729 (Arf6 inhibitor), EHT 1864 (Rac1 inhibitor), ML 141 (Cdc42 inhibitor) or Rhosin (RhoA inhibitor). After treatment, neurons were incubated with Aβ42o (red) for 30 min the imaged live. Colocalization between Aβ42o and LAMP1 was quantitated with Imaris 10.1 (Bitplane) software to determine uptake of oligomers to lysosomes (white). B) Quantification of the mean percent of Aβ42o signal colocalized with LAMP1 from three replicate experiments (n=3), each containing at least 6 individual neuron cell body images at each time point for each treatment. Differences in colocalization between DMSO, NAV-2729, EHT 1864, ML 141, and Rhosin were assessed using a one-way ANOVA with Tukey’s test. C) Neurons treated with DMSO, NAV-2729, EHT 1864, ML 141, and Rhosin were incubated with tauPFFs (green) for 30 min, then imaged lived. Colocalization between tauPFFs and LAMP1-labeled lysosomes (white pixels) was measured. D) Quantification of the mean percent of tauPFF colocalized with LAMP1 from three replicate experiments (n=3), each containing at least 6 individual neuron cell body images at each time point for each treatment. Differences between each treatment were quantitated using a one-way ANOVA with Tukey’s test. Data is presented as mean ± SEM. * p<0.05. Scale bar = 10μm .

Article Snippet: The RhoA selective inhibitor Rhosin hydrochloride (5003) and Ethylisopropyl amiloride (EIPA; 3378) were purchased from Tocris (5003; Bristol, UK).

Techniques: Derivative Assay, Transduction, Control, Incubation, Software, Labeling

Journal: bioRxiv

Article Title: Macropinocytosis of aggregated amyloid-beta and tau requires Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641073

Figure Lengend Snippet: A) IPSC-derived neurons transduced with LAMP1-mCh (magenta) were treated with DMSO (vehicle control), Pitstop2, NAV-2729, EHT 1864, ML 141 or Rhosin. After treatment, neurons were incubated with Aβ42o (red) and 70kDa dextran (Dex70; green) for 30 min then imaged live. To quantify the uptake of Aβ42o and Dex70, signal from each were used to create spots demarking puncta using Imaris. The number of puncta of each within the cell body of each neuron was counted. Puncta containing both Aβ42o and Dex70 (Aβ42o+/Dex70+) were also counted. Number of B) Aβ42o puncta, C) Dex70 puncta, and D) Aβ42o+/Dex70+ was quantified from three replicate experiments (n=3), each containing at least 6 individual neuron cell body images at each time point for each treatment. Differences in the number of puncta between DMSO, Pitstop2 NAV- 2729, EHT 1864, ML 141, and Rhosin were assessed using a one-way ANOVA with Tukey’s test. D) Image of a neuron transduced with PLC8PH (magenta) marking membrane ruffles and stained with fluorescently tagged phalloidin (red) marking F-actin. After 10 min incubation with Aβ42o (green), oligomers can be observed within membrane ruffles containing actively polymerizing actin. Data are presented as mean ± SEM. * p<0.05. Scale bar = 5μm .

Article Snippet: The RhoA selective inhibitor Rhosin hydrochloride (5003) and Ethylisopropyl amiloride (EIPA; 3378) were purchased from Tocris (5003; Bristol, UK).

Techniques: Derivative Assay, Transduction, Control, Incubation, Membrane, Staining

Journal: bioRxiv

Article Title: Macropinocytosis of aggregated amyloid-beta and tau requires Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641073

Figure Lengend Snippet: A) IPSC-derived neurons transduced with LAMP1-mCh (magenta) were treated with DMSO (vehicle control), Pitstop2, NAV-2729, EHT 1864, ML 141 or Rhosin. After treatment, neurons were incubated with tauPFFs (red) and 70kDa dextran (Dex70; green) for 30 min the imaged live. To quantify the uptake of tauPFFs and Dex70, signal from each were used to create spots demarking puncta using Imaris. Puncta containing both tauPFFs and Dex70 (TauPFF+/Dex70+) were also counted. The number of B) TauPFF puncta, C) Dex70 puncta, and D) TauPFF+/Dex70+ puncta within neuron cell bodies were quantified from from three replicate experiments (n=3), each containing at least 6 individual neuron cell body images at each time point for each treatment. Differences in the number of puncta between DMSO, Pitstop2 NAV-2729, EHT 1864, ML 141, and Rhosin were assessed using a one-way ANOVA with Tukey’s test. E) Image of a neuron transduced with PLC8PH (magenta) marking membrane ruffles and stained with fluorescently tagged phalloidin (red) marking F-actin. After 10 min incubation with tauPFFs (green), they can be observed within membrane ruffles containing actively polymerizing actin. Data are presented as mean ± SEM. * p<0.05. Scale bar = 5μm .

Article Snippet: The RhoA selective inhibitor Rhosin hydrochloride (5003) and Ethylisopropyl amiloride (EIPA; 3378) were purchased from Tocris (5003; Bristol, UK).

Techniques: Derivative Assay, Transduction, Control, Incubation, Membrane, Staining

Journal: bioRxiv

Article Title: Macropinocytosis of aggregated amyloid-beta and tau requires Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641073

Figure Lengend Snippet: AB42o or tauPFF incubation increases Rac1, Cdc42 and RhoA activity in Neuro2a cells. Neuro2a (N2a) mouse neuroblastoma cells were incubated with Aβ42o, tauPFFs or EGF (positive control) while on ice, then incubated for 5 minutes at 37°C and 5% CO 2 to allow for the initiation of macropinocytosis. Cells were moved back to ice following this incubation and lysates were immediately collected and used for Rac1, Cdc42 or RhoA G-LISA activation assays. To measure the relative amount of active GTP-bound levels of each protein, the absolute optical density (OD) was measured in lysate loaded into G-LISA assay wells excited with a 490nm laser. Absolute OD was measured in lysates from untreated cells, or cells treated with Aβ42o, tauPFFs, and EGF and levels of A) GTP-bound Rac1, B) GTP-bound Cdc42, and C) GTP-bound RhoA were compared. Each of these experiments was ran in technical triplicates, and three biological replicates were performed (n=3). The difference in absolute OD was compared to untreated control by a one-way ANOVA with Tukey’s test. Data are presented as mean ± SEM. * p<0.05 .

Article Snippet: The RhoA selective inhibitor Rhosin hydrochloride (5003) and Ethylisopropyl amiloride (EIPA; 3378) were purchased from Tocris (5003; Bristol, UK).

Techniques: Incubation, Activity Assay, Positive Control, Activation Assay, Control

Journal: Cancer

Article Title: Generation and external validation of a tumor-derived 5-gene prognostic signature for recurrence of lymph node-negative, invasive colorectal carcinoma

doi: 10.1002/cncr.27628

Figure Lengend Snippet: Genes Used to Generate Final Candidate Rules

Article Snippet: ARHGDIB , Rho GDP dissociation inhibitor beta , 81 , Hs00171288_m1 , 12.

Techniques: Gene Expression, Variant Assay, Ubiquitin Proteomics