rtp801  (Novus Biologicals)

Journal: Journal of Extracellular Vesicles

Article Title: RTP801 mediates transneuronal toxicity in culture via extracellular vesicles

doi: 10.1002/jev2.12378

Figure Lengend Snippet: RTP801 is present in EVs and its overexpression increases EVs release. For EVs characterization, EVs were isolated from the culture media of cortical neurons at DIV13 following a sequential ultracentrifugation protocol. (a) Size distribution of nanoparticles by NanoSight particle tracking analysis (NTA). (b) Transmission electron micrographs of the vesicles show particles with the characteristic morphology and size of EVs. (c) WB analysis of total cell lysates, large EVs (P10K or lEVs) and small EVs (P100K or sEVs). Membranes were probed against common EVs markers (Alix, TSG‐101 and Flotillin‐1), EVs negative marker (Calnexin), and RTP801. Actin is used as a loading control for the lysates. (d) Cultured neurons were transfected at DIV12 with an eGFP plasmid (in green). EVs were isolated from sister neuronal cultures and labeled with the fluorogenic cell membrane probe MemGlow‐560 (in red). EVs particles were administered to eGFP‐cultured neurons with a ratio of 400 EVs:cell. MemGlow‐560 specifically binds to EVs, as no fluorescent signal was detected in 1X PBS samples. Orthogonal views show EVs internalization into neurons. Scale bar of 5 µm. (e) Cortical primary neurons (DIV11) were transduced with lentiviral particles containing eGFP empty vector (as control) or eGFP‐RTP801 constructs. 48 h after, EVs were isolated and total protein content was analysed by WB. Membranes were probed against RTP801 and eGFP, TSG‐101, and flotillin‐1 as EVs markers and actin as a loading control for the lysates. Representative immunoblots are shown. (f) Graphs show values obtained by densitometric analysis of WB data relative to the total protein content of the cell lysate. Values represent culture replicates of at least three independent neuronal cultures (mean ± SEM). Data were analysed by unpaired T ‐test with Welch's correction in the cell lysate and in the EVs fraction (* P < 0.05 vs. control EVs).

Article Snippet: Although RTP801 antibody (Proteintech, #10638‐1‐AP) show several non‐specific bands, the specific 34 KDa band has been widely validated using shRNAs against RTP801 (Canal et al., ; Malagelada et al., , , ; Martín‐Flores et al., , ; Pérez‐Sisqués, Martín‐Flores et al., ; Pérez‐Sisqués, Sancho‐Balsells et al., ; Pérez‐Sisqués, Solana‐Balaguer et al., ) or with a RTP801 KO model (Pérez‐Sisqués, Martín‐Flores et al., ).

Techniques: Over Expression, Isolation, Transmission Assay, Marker, Control, Cell Culture, Transfection, Plasmid Preparation, Labeling, Membrane, Transduction, Construct, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: RTP801 mediates transneuronal toxicity in culture via extracellular vesicles

doi: 10.1002/jev2.12378

Figure Lengend Snippet: Modulation of RTP801 expression alters protein cargo of neuronal‐derived EVs in vitro. (a) Heatmap showing the differentially expressed proteins in GFP and RTP801‐derived EVs. Significantly overexpressed proteins in the RTP801 group in comparison to the GFP group are depicted in red, whereas proteins that are underrepresented in the RTP801 group are shown in blue. In the right annotation the log2 fold change (FC) is displayed as a bar plot for each of the proteins. (b) The volcano plot shows the whole set of proteins detected in the comparison between GFP and RTP801 experimental groups. The most overexpressed proteins being significant (‐log10 p‐value > = 1.2 and log2 fold change < −2.5) are plotted in red and the most downregulated proteins (‐log10 p‐value > = 1.2 and log2 fold change > 2.5) in blue. (c) Heatmap showing the differentially expressed proteins in shCT and shRTP801‐derived EVs. Significantly overexpressed proteins in the shRTP801 group in comparison to the shCT group are depicted in red, whereas proteins that are underrepresented in the shRTP801 group are shown in blue. In the right annotation, the log2 fold change (FC) is displayed as a barplot for each of the proteins. (d) The volcano plot shows the whole set of proteins detected in the comparison between shCT and shRTP801 experimental groups. The most overexpressed proteins being significant (‐log10 p‐value > = 1.2 and log2 fold change < −2.5) are plotted in red and the most downregulated proteins (‐log10 p‐value > = 1.2 and log2 fold change > 2.5) in blue. In both cases, these proteins are also tagged with the gene symbol. In both cases these proteins are also tagged with the gene symbol. Note that the heatmaps (a) and (c) only show the differentially expressed proteins between groups, while the whole set of proteins is used for the representation in volcano plots (b) and (d).

Article Snippet: Although RTP801 antibody (Proteintech, #10638‐1‐AP) show several non‐specific bands, the specific 34 KDa band has been widely validated using shRNAs against RTP801 (Canal et al., ; Malagelada et al., , , ; Martín‐Flores et al., , ; Pérez‐Sisqués, Martín‐Flores et al., ; Pérez‐Sisqués, Sancho‐Balsells et al., ; Pérez‐Sisqués, Solana‐Balaguer et al., ) or with a RTP801 KO model (Pérez‐Sisqués, Martín‐Flores et al., ).

Techniques: Expressing, Derivative Assay, In Vitro, Comparison

Journal: Journal of Extracellular Vesicles

Article Title: RTP801 mediates transneuronal toxicity in culture via extracellular vesicles

doi: 10.1002/jev2.12378

Figure Lengend Snippet: Significantly altered proteins involved in apoptosis found in EVs derived from RTP801‐overexpressing neurons compared to the control. Pro‐apoptotic proteins are shown in red, anti‐apoptotic proteins in green, and proteins with a dual role as pro‐ and anti‐apoptotic in orange. Up‐arrows indicate that the protein is upregulated and down‐arrows that the protein is downregulated (Cas, abbreviated form os caspase).

Article Snippet: Although RTP801 antibody (Proteintech, #10638‐1‐AP) show several non‐specific bands, the specific 34 KDa band has been widely validated using shRNAs against RTP801 (Canal et al., ; Malagelada et al., , , ; Martín‐Flores et al., , ; Pérez‐Sisqués, Martín‐Flores et al., ; Pérez‐Sisqués, Sancho‐Balsells et al., ; Pérez‐Sisqués, Solana‐Balaguer et al., ) or with a RTP801 KO model (Pérez‐Sisqués, Martín‐Flores et al., ).

Techniques: Derivative Assay, Control, Activation Assay, Blocking Assay, Activity Assay, Knockdown, Inhibition, Migration, In Vivo, Over Expression, Knock-Out, In Vitro

Journal: Journal of Extracellular Vesicles

Article Title: RTP801 mediates transneuronal toxicity in culture via extracellular vesicles

doi: 10.1002/jev2.12378

Figure Lengend Snippet: Significantly altered proteins involved in apoptosis in EVs derived from neurons where RTP801 was silenced with an shRNA compared to the control. Pro‐apoptotic proteins are shown in red, anti‐apoptotic proteins in green, and proteins with a dual role as pro‐ and anti‐apoptotic in orange. Up‐arrows indicate that the protein is upregulated and down‐arrows that the protein is downregulated (Cas, abbreviated form of caspase).

Article Snippet: Although RTP801 antibody (Proteintech, #10638‐1‐AP) show several non‐specific bands, the specific 34 KDa band has been widely validated using shRNAs against RTP801 (Canal et al., ; Malagelada et al., , , ; Martín‐Flores et al., , ; Pérez‐Sisqués, Martín‐Flores et al., ; Pérez‐Sisqués, Sancho‐Balsells et al., ; Pérez‐Sisqués, Solana‐Balaguer et al., ) or with a RTP801 KO model (Pérez‐Sisqués, Martín‐Flores et al., ).

Techniques: Derivative Assay, shRNA, Control, Activation Assay, Knockdown, Inhibition, Histone Deacetylase Assay, Over Expression

Journal: Journal of Extracellular Vesicles

Article Title: RTP801 mediates transneuronal toxicity in culture via extracellular vesicles

doi: 10.1002/jev2.12378

Figure Lengend Snippet: EVs derived from RTP801 overexpressing neurons induce neuron cell death via apoptosis. (a) Schematic representation of the experimental procedure. Cultured cortical neurons were transduced with lentiviral particles containing eGFP, eGFP‐RTP801, shCT and shRTP801 constructs. EVs were isolated from the culture medium and used to treat sister cortical neurons at three different doses: 400, 800 and 1,600 EVs:cell. After 24 h, neurons were fixed, and immunofluorescence against active caspase‐3 was performed. (b) Representative images of neuronal cultures at DIV14. MAP2 (in green) is used to identify neurons and ClvCas3 (in red) to identify cells expressing cleaved caspase‐3. Scale bar of 50 µm. (c) Neuronal nuclei were classified by phenotype: ClvCas3(+) (red outline) represents neurons expressing ClvCas3 and ClvCas3(‐) (blue outline) represents neurons with no active caspase‐3. Scale bar of 50 μm. Neuronal nuclei were then classified by machine learning into viable or apoptotic. Apoptotic nuclei were subclassified as condensed and fragmented. (d) Confusion matrix obtained with the classification training with CellProfiler Analyst. (e) ClvCas3 mean intensity in neurons. Data of the 3 doses of EVs‐treatment. (f) Data of ClvCas3 mean intensity only at the highest EVs dose (1,600 EVs:cell). (g) Quantification of neurons undergoing the last step of the apoptotic process (Fragmented ClvCas(‐) neuronal nuclei). At least neurons of 5 different fields were analysed per replicate. Values represent culture replicates of at least three independent neuronal cultures (mean ± SEM). Data was analysed by One‐way ANOVA (*P < 0.05, **P < 0.01, ****P < 0.0001).

Article Snippet: Although RTP801 antibody (Proteintech, #10638‐1‐AP) show several non‐specific bands, the specific 34 KDa band has been widely validated using shRNAs against RTP801 (Canal et al., ; Malagelada et al., , , ; Martín‐Flores et al., , ; Pérez‐Sisqués, Martín‐Flores et al., ; Pérez‐Sisqués, Sancho‐Balsells et al., ; Pérez‐Sisqués, Solana‐Balaguer et al., ) or with a RTP801 KO model (Pérez‐Sisqués, Martín‐Flores et al., ).

Techniques: Derivative Assay, Cell Culture, Transduction, Construct, Isolation, Immunofluorescence, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: RTP801 mediates transneuronal toxicity in culture via extracellular vesicles

doi: 10.1002/jev2.12378

Figure Lengend Snippet: Treatment with EVs derived from RTP801 overexpressing neurons reduces neuron arborization while EVs derived from shRTP801 increase neuron morphology's complexity. (a) Neurons were untreated (UT) or treated with EVs derived from eGFP, eGFP‐RTP801, shCT or shRTP801 transduced neurons, at a dose of 1,600 EVs:cell. Immunocytochemistry of MAP2 was performed to visualize cultured neurons. Using CellProfiler software, neurons not touching the borders of the image were identified as independent objects, and neuron skeleton was obtained. (b) Representative images of neuron arborization in UT neurons and neurons treated with EVs derived from eGFP, RTP801, shCT or shRTP801 transduced neurons. (c) Morphological assessment of neurons. Primary dendrites, intermediate branches, termini branches (endpoints), and the total tree length were analysed. Scale bar of 50 μm. At least neurons of 5 different fields were analysed per replicate. Values represent culture replicates of at least three independent neuronal cultures (mean ± SEM). Data was analysed by One‐way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Although RTP801 antibody (Proteintech, #10638‐1‐AP) show several non‐specific bands, the specific 34 KDa band has been widely validated using shRNAs against RTP801 (Canal et al., ; Malagelada et al., , , ; Martín‐Flores et al., , ; Pérez‐Sisqués, Martín‐Flores et al., ; Pérez‐Sisqués, Sancho‐Balsells et al., ; Pérez‐Sisqués, Solana‐Balaguer et al., ) or with a RTP801 KO model (Pérez‐Sisqués, Martín‐Flores et al., ).

Techniques: Derivative Assay, Immunocytochemistry, Cell Culture, Software

Journal: Journal of Extracellular Vesicles

Article Title: RTP801 mediates transneuronal toxicity in culture via extracellular vesicles

doi: 10.1002/jev2.12378

Figure Lengend Snippet: 6‐OHDA exposure increases RTP801 protein levels in EVs and elevates their secretion in cortical primary cultures. (a) Rat cortical neurons at DIV14 were treated with the toxin 6‐OHDA 50 µM for 16 h or untreated (UT) as control. Afterward, cell media was collected, and EVs were isolated by sequential centrifugations. EVs suspension was negatively stained and observed by TEM. Electron micrographs show the presence of vesicles between 30 and 110 nm. Scale bars are 500 nm and 200 nm in magnification micrographs (b) Sister cultures were treated with 6‐OHDA 50 μM for 16 h or not (∅) and total protein content was analysed by WB. Membranes were probed against RTP801 and NEDD4, TSG101, and Flotillin‐1 as EVs markers and actin as a loading control. Graphs show values obtained by densitometric analysis of WB data relative to the total protein content of the cell lysate. Values represent culture replicates of at least three independent neuronal cultures (mean ± SEM). Representative immunoblots are shown. Data were analysed by Student's T ‐test in the cell lysate and the EVs fraction (* P < 0.05, ** P < 0.01).

Article Snippet: Although RTP801 antibody (Proteintech, #10638‐1‐AP) show several non‐specific bands, the specific 34 KDa band has been widely validated using shRNAs against RTP801 (Canal et al., ; Malagelada et al., , , ; Martín‐Flores et al., , ; Pérez‐Sisqués, Martín‐Flores et al., ; Pérez‐Sisqués, Sancho‐Balsells et al., ; Pérez‐Sisqués, Solana‐Balaguer et al., ) or with a RTP801 KO model (Pérez‐Sisqués, Martín‐Flores et al., ).

Techniques: Control, Isolation, Suspension, Staining, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: RTP801 mediates transneuronal toxicity in culture via extracellular vesicles

doi: 10.1002/jev2.12378

Figure Lengend Snippet: 6‐OHDA‐induced RTP801 counteract EVs trophic effect in recipient neurons. (a) Rat cortical neurons at DIV10 were untransduced (CT) or transduced with shCT or shRTP801. Seventy‐hours later, cells were exposed to 6‐OHDA 50 µM or not (∅) for 16 h. After that, EVs were isolated from the culture media by sequential centrifugations and EVs were administrated to cortical cultures (400 EVs:cell), and a control condition without EVs treatment was performed as a negative control (UT). Twenty‐four hours later, cells were harvested, and protein extracts were subjected to western blotting. (b) Membranes were probed against phosho‐Ser473‐Akt, Phospho‐Ser235/236‐RPS6, total Akt, total RPS6 and actin as a loading control. Representative immunoblots are shown. (c) Densitometric analysis of phospho‐Ser473‐Akt levels and phospho‐Ser235/246‐RPS6 levels. Values represent culture replicates of at least three independent neuronal cultures (mean ± SEM). Data were analysed by One‐way ANOVA followed by Bonferroni's posthoc test. (*** P < 0.005, ** P < 0.01 , *P < 0.05).

Article Snippet: Although RTP801 antibody (Proteintech, #10638‐1‐AP) show several non‐specific bands, the specific 34 KDa band has been widely validated using shRNAs against RTP801 (Canal et al., ; Malagelada et al., , , ; Martín‐Flores et al., , ; Pérez‐Sisqués, Martín‐Flores et al., ; Pérez‐Sisqués, Sancho‐Balsells et al., ; Pérez‐Sisqués, Solana‐Balaguer et al., ) or with a RTP801 KO model (Pérez‐Sisqués, Martín‐Flores et al., ).

Techniques: Transduction, Isolation, Control, Negative Control, Western Blot