Journal: Experimental & Molecular Medicine

Article Title: YAP promotes global mRNA translation to fuel oncogenic growth despite starvation

doi: 10.1038/s12276-024-01316-w

Figure Lengend Snippet: a Heatmap of Z scores for RNA-seq expression profiles of MCF10A cells stably expressing the control vector or YAP 5SA subjected (or not) to overnight serum starvation. The samples are labeled “I to IV” for easy reference. b Venn diagrams showing the number of overlapping genes for the samples in ( a ) after filtering according to a p value of <0.05 and an absolute fold change of >3. The left Venn diagram shows the overlap of genes upregulated by YAP 5SA under serum starvation conditions with those downregulated by serum starvation in control cells. The right Venn diagram shows genes that behave in the opposite manner. The overlapping genes from each Venn diagram were combined to identify a gene signature regulated by YAP 5SA in response to serum starvation. These genes were then filtered relative to the KEGG_MTOR signature to identify any genes that encode proteins related to mTOR signaling. Only DDIT4 was identified using this analysis. c Schematic model illustrating the signaling axis by which DDIT4 ultimately regulates mRNA translation. d Quantitative reverse transcription and polymerase chain reaction (qRT‒PCR) analysis of the mRNA abundance of DDIT4 and CYR61 in MCF10A cells stably expressing the control vector or YAP 5SA subjected to serum starvation for the indicated times. The bar graphs represent the means of technical replicates ( n = 3 independent replicates). Data are presented as the means ± s.e.m. ( n = 3). The symbols * and # indicate comparisons with CYR61 and DDIT4 , respectively. */ # p < 0.05, ** p < 0.005; n.s. not significant (unpaired Student’s t test). e Immunoblot analysis of the samples in ( d ). f MCF10A cells harboring a doxycycline (Dox)-inducible DDIT4 expression construct (Tet-ON DDIT4) and stably expressing the vector control or YAP 5SA were serum starved and treated with doxycycline (1 µg/ml) before analysis, as shown in Fig. . g Relative anchorage-independent colony formation quantification of Tet-ON DDIT4 MCF10A cells stably expressing the control vector or YAP 5SA and maintained in the absence or presence of doxycycline for 3 weeks. The data are presented as the means ± s.e.m. ( n = 4 independent replicates). *** p < 0.0005, **** p < 0.0001; n.s. not significant (unpaired Student’s t test).

Article Snippet: Antibodies against the following antigens were used for immunoblot analysis: puromycin (Kerafast, EQ0001), FLAG (Sigma-Aldrich, F3165), YAP/TAZ (Cell Signaling Technology, #8418), YAP (Cell Signaling Technology, #14074), TEAD4 (Abcam, ab58310), CYR61 (Santa Cruz Biotechnology, sc-13100), DDIT4 (Novus Biologicals, NBP1-22966), mTOR (Cell Signaling Technology, #2972), phospho-S6 (Cell Signaling Technology, #2211), S6 (Cell Signaling Technology, #2217), phospho-4E-BP1 (Cell Signaling Technology, #2855), 4E-BP1 (Cell Signaling Technology, #9644), phospho-ERK (Cell Signaling Technology, #9101), ERK1/2 (Cell Signaling Technology, #4695), phospho-S6K1 (Cell Signaling Technology, #9205), S6K1 (Cell Signaling Technology, #9202), phospho-AKT (Cell Signaling Technology, #4060 and #4056), AKT2 (Cell Signaling Technology, #3063), eIF4E (Santa Cruz Biotechnology, sc-9976), PABP-C1 (Abcam, ab21060), phospho-paxillin (Cell Signaling Technology, #2541), phospho-p38 (Cell Signaling Technology, #4511), and vinculin (Cell Signaling Technology, #13901).

Techniques: RNA Sequencing, Expressing, Stable Transfection, Control, Plasmid Preparation, Labeling, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Construct

Journal: Experimental & Molecular Medicine

Article Title: YAP promotes global mRNA translation to fuel oncogenic growth despite starvation

doi: 10.1038/s12276-024-01316-w

Figure Lengend Snippet: a GNAQ Q209L -mutant 92.1 and BRAF V600E -mutant OCM1 uveal melanoma cells were serum starved (or not) overnight and then restimulated (or not) with serum for 1 h. The samples were then subjected to analysis, as shown in Fig. , as well as to YAP Phos-tag gel analysis. b Tet-ON DDIT4 92.1 and OCM1 cells were incubated in the absence or presence of doxycycline (Dox, 1 µg/ml) for 24 h and then analyzed as described in ( a ). c Relative anchorage-independent colony formation of Tet-ON DDIT4 92.1 or OCM1 cells maintained in the absence or presence of doxycycline for 3 weeks. The data are presented as the means ± s.e.m. ( n = 5 independent replicates). **** p < 0.0001; n.s. not significant (unpaired Student’s t test). d Relative transwell migration quantification of Tet-ON DDIT4 92.1 and OCM1 cells in the absence or presence of doxycycline. The data are presented as the means ± s.e.m. ( n = 4 independent replicates). *** p < 0.0005; n.s. not significant (unpaired Student’s t test). e Time course of xenograft tumor volume in nude mice injected with Tet-ON DDIT4 92.1 or OCM1 cells and treated (or not) with doxycycline (0.5 mg/ml) in the drinking water. The data are presented as the means ± s.e.m. ( n = 4 mice per group). * p < 0.05, ** p < 0.005, *** p < 0.0005; n.s. not significant (unpaired Student’s t test). f Weights of excised xenograft tumors from the mice in ( e ) at the end points (25 days or 14 days after Tet-ON DDIT4 92.1 or OCM1 cell injection, respectively). The data are presented as the means ± s.e.m. ( n = 4 mice per group). * p < 0.05; n.s. not significant (unpaired Student’s t test).

Article Snippet: Antibodies against the following antigens were used for immunoblot analysis: puromycin (Kerafast, EQ0001), FLAG (Sigma-Aldrich, F3165), YAP/TAZ (Cell Signaling Technology, #8418), YAP (Cell Signaling Technology, #14074), TEAD4 (Abcam, ab58310), CYR61 (Santa Cruz Biotechnology, sc-13100), DDIT4 (Novus Biologicals, NBP1-22966), mTOR (Cell Signaling Technology, #2972), phospho-S6 (Cell Signaling Technology, #2211), S6 (Cell Signaling Technology, #2217), phospho-4E-BP1 (Cell Signaling Technology, #2855), 4E-BP1 (Cell Signaling Technology, #9644), phospho-ERK (Cell Signaling Technology, #9101), ERK1/2 (Cell Signaling Technology, #4695), phospho-S6K1 (Cell Signaling Technology, #9205), S6K1 (Cell Signaling Technology, #9202), phospho-AKT (Cell Signaling Technology, #4060 and #4056), AKT2 (Cell Signaling Technology, #3063), eIF4E (Santa Cruz Biotechnology, sc-9976), PABP-C1 (Abcam, ab21060), phospho-paxillin (Cell Signaling Technology, #2541), phospho-p38 (Cell Signaling Technology, #4511), and vinculin (Cell Signaling Technology, #13901).

Techniques: Mutagenesis, Incubation, Migration, Injection

Journal: Experimental & Molecular Medicine

Article Title: YAP promotes global mRNA translation to fuel oncogenic growth despite starvation

doi: 10.1038/s12276-024-01316-w

Figure Lengend Snippet: (Left) In cells with an adequate supply of nutrients and growth factors (serum), G proteins such as G q /G 11 become activated, resulting in LATS1/2 inactivation and the consequent dephosphorylation of YAP/TAZ, which then translocate to the nucleus and bind to cognate TEAD transcription factors to activate the transcription of downstream target genes. However, some genes, such as DDIT4 , are transcriptionally repressed by YAP/TAZ. Given that DDIT4 suppresses mTORC1 activity via TSC1/2, downregulation of DDIT4 by YAP/TAZ promotes mTORC1 activation, which ultimately leads to increased cap-dependent translation, especially of 5′TOP-containing mRNAs that encode components of the translational machinery. (Right) Conversely, YAP/TAZ are inactive under nutrient-poor conditions, resulting in high DDIT4 expression, suppression of mTORC1 activity, and inhibition of translation. Forced YAP activation in serum-starved cells is sufficient to restore translation to levels characteristic of serum-replete conditions.

Article Snippet: Antibodies against the following antigens were used for immunoblot analysis: puromycin (Kerafast, EQ0001), FLAG (Sigma-Aldrich, F3165), YAP/TAZ (Cell Signaling Technology, #8418), YAP (Cell Signaling Technology, #14074), TEAD4 (Abcam, ab58310), CYR61 (Santa Cruz Biotechnology, sc-13100), DDIT4 (Novus Biologicals, NBP1-22966), mTOR (Cell Signaling Technology, #2972), phospho-S6 (Cell Signaling Technology, #2211), S6 (Cell Signaling Technology, #2217), phospho-4E-BP1 (Cell Signaling Technology, #2855), 4E-BP1 (Cell Signaling Technology, #9644), phospho-ERK (Cell Signaling Technology, #9101), ERK1/2 (Cell Signaling Technology, #4695), phospho-S6K1 (Cell Signaling Technology, #9205), S6K1 (Cell Signaling Technology, #9202), phospho-AKT (Cell Signaling Technology, #4060 and #4056), AKT2 (Cell Signaling Technology, #3063), eIF4E (Santa Cruz Biotechnology, sc-9976), PABP-C1 (Abcam, ab21060), phospho-paxillin (Cell Signaling Technology, #2541), phospho-p38 (Cell Signaling Technology, #4511), and vinculin (Cell Signaling Technology, #13901).

Techniques: De-Phosphorylation Assay, Activity Assay, Activation Assay, Expressing, Inhibition

Journal: Experimental & Molecular Medicine

Article Title: YAP promotes global mRNA translation to fuel oncogenic growth despite starvation

doi: 10.1038/s12276-024-01316-w

Figure Lengend Snippet:

Article Snippet: Antibodies against the following antigens were used for immunoblot analysis: puromycin (Kerafast, EQ0001), FLAG (Sigma-Aldrich, F3165), YAP/TAZ (Cell Signaling Technology, #8418), YAP (Cell Signaling Technology, #14074), TEAD4 (Abcam, ab58310), CYR61 (Santa Cruz Biotechnology, sc-13100), DDIT4 (Novus Biologicals, NBP1-22966), mTOR (Cell Signaling Technology, #2972), phospho-S6 (Cell Signaling Technology, #2211), S6 (Cell Signaling Technology, #2217), phospho-4E-BP1 (Cell Signaling Technology, #2855), 4E-BP1 (Cell Signaling Technology, #9644), phospho-ERK (Cell Signaling Technology, #9101), ERK1/2 (Cell Signaling Technology, #4695), phospho-S6K1 (Cell Signaling Technology, #9205), S6K1 (Cell Signaling Technology, #9202), phospho-AKT (Cell Signaling Technology, #4060 and #4056), AKT2 (Cell Signaling Technology, #3063), eIF4E (Santa Cruz Biotechnology, sc-9976), PABP-C1 (Abcam, ab21060), phospho-paxillin (Cell Signaling Technology, #2541), phospho-p38 (Cell Signaling Technology, #4511), and vinculin (Cell Signaling Technology, #13901).

Techniques: Sequencing

Journal: Cell Reports

Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response

doi: 10.1016/j.celrep.2019.02.077

Figure Lengend Snippet:

Article Snippet: Rabbit Antibody against REDD1/DDIT4 , Novus Biologicals , Cat# NBP1-77321SS; RRID: AB_11036185.

Techniques: Recombinant, Cell Isolation, Reverse Transcription, SYBR Green Assay, Isolation, Sequencing, Software

Journal: PLoS Pathogens

Article Title: Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication

doi: 10.1371/journal.ppat.1006635

Figure Lengend Snippet: ( A-C ) A549 cells were infected with WSN at MOI of 2 PFU/cell for the indicated times. Cell extracts were subjected to ( A ) western blot analysis to detect the depicted proteins and quantified as shown in or ( B ) RNA was purified for qRT-PCR to determine REDD1 mRNA levels. Mean and standard deviation are shown for qRT-PCR, n = 4 independent experiments done in triplicates. ** p <0.000004, Student's t -test. ( C ) A549 cells were transfected with siRNAs (pool of three each) targeting viral mRNAs and then infected for 7 h at MOI of 2 PFU/cell. Immunoblot analysis was performed to detect the depicted proteins, n = 3. ( D ) A549 cells were transfected with plasmids encoding the indicated virus proteins. At 48 h post-transfection, total RNA was purified and REDD1 mRNA levels were determined by qRT-PCR as in B . The bottom panel in D shows viral polymerase activity upon tranfection of the depicted viral proteins and/or minigenome as control. Minigenome mRNA was measured by qRT-PCR. In cells transfected with the complete set of plasmids that encode the viral polymerase we detect the minigenome RNA transcribed by pol I directly from the plasmid in addition to the minigenme RNA amplified by the influenza proteins, indicating protein activity. In cells transfected with the same plasmids except for PA, we only detect the minigenome RNA transcribed by pol I directly from the plasmid, and the average values was set to 1. The minigenome RNA level is higher when all plasmids were transfected ( n = 3). ( E, F ) MDCK cells were transfected with control plasmid of plasmid enconding the M2 protein. In E, RNA was purified for qRT-PCR to determine REDD1 mRNA levels as in B , n = 3, ***p<0.001. In F, cell extracts were subjected to western blot analysis to detect the depicted proteins ( n = 3). ( G ) U2OS-REDD1 cells were treated with vehicle or 1μg/ml tetracycline for 2 h prior to and during infection to induce REDD1 expression. Cells were infected at MOI of 2 PFU/cell for 6 h. Immunoblot analyses were performed to detect the depicted proteins. Total S6K serves as the loading control. The upper band in the S6K/p-S6K blots is p85 S6K, whereas the lower band is p70 S6K ( n = 3).

Article Snippet: Additional antibodies used for western blot analysis were against Rictor (Millipore 05–1471), IFITM3 (R&D Systems AF3377), MAVS (generated by Z. Chen laboratory), β-actin (Sigma A5441), REDD1 (Novus Biologicals NBP1-22966), ATG5 (Novus Biologicals NB110-53818), ATG7 (Sigma A2856), and LC3 (Novus Biologicals NB100-2220).

Techniques: Infection, Western Blot, Purification, Quantitative RT-PCR, Standard Deviation, Transfection, Virus, Activity Assay, Control, Plasmid Preparation, Amplification, Expressing

Journal: PLoS Pathogens

Article Title: Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication

doi: 10.1371/journal.ppat.1006635

Figure Lengend Snippet: The viral protein HA and virus replication promote mTORC1 activation through PDPK1-mediated phosphorylation of AKT at T308. In addition, down-regulation of REDD1 by the viral M2 protein amplifies or support mTORC1 activation downstream of AKT. NS1 promotes AKT phosphorylation at S473 via mTORC2 and this process is known to regulate apoptosis. Differential AKT phosphorylation dictates downstream effects.

Article Snippet: Additional antibodies used for western blot analysis were against Rictor (Millipore 05–1471), IFITM3 (R&D Systems AF3377), MAVS (generated by Z. Chen laboratory), β-actin (Sigma A5441), REDD1 (Novus Biologicals NBP1-22966), ATG5 (Novus Biologicals NB110-53818), ATG7 (Sigma A2856), and LC3 (Novus Biologicals NB100-2220).

Techniques: Virus, Activation Assay, Phospho-proteomics

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. R TP801 can be degraded by both the ubiquitin proteasome system and by the lysosomal pathway. NGF-differentiated PC12 cells or cortical neurons were treated during 4 hours with 1 μM epoxomycin, 10 μM MG132 or 50 μM chloroquine, and cell lysates were subjected to Western Blot. Membranes were probed first for RTP801 and then with α-actin, as a loading control. All samples were immunoblotted in the same membrane, but one irrelevant lane was deleted in the second panel. Graphs represent densitometric values (mean ± SEM) normalized to α-actin of three independent experiments in triplicates. Student's t -test, *** P < 0.001 and * P < 0.05 versus controls. b. NEDD4 polyubiquitinates RTP801 in a cell free assay. Recombinant NEDD4 E3 ligase, recombinant GST-RTP801, UbcH5b E2 enzyme, E1 enzyme, biotinylated ubiquitin and ATP were mixed and incubated at 37°C for 90 min. RTP801 was immunoprecipitated and immunocomplexes were analyzed by Western Blot. The membrane was incubated with Avidin/Biotin and then with chemiluminiscent peroxidase substrate solution (upper panel) and reprobed for RTP801 (lower panel). A representative image of three independent experiments is shown. (HMW Ub-RTP801, high molecular weight ubiquitinated RTP801) c. . HEK293 cells were transfected with pHAGE or pHAGE-NEDD4 along with HA-ubiquitin and pCMS-eGFP-RTP801 constructs. Forty-eight hours post-transfection either RTP801 was immunoprecipitated or non-specific rabbit immunoglobulins (Rb IgG) were added. Whole cell lysates (inputs) and the immunocomplexes were analyzed by Western Blot with anti-HA, anti-RTP801, anti-NEDD4 and anti-GAPDH (loading control) antibodies. A representative image of two independent experiments is shown. HMW Ub-RTP801 = High molecular weight ubiquitinated RTP801; IP = immunoprecipitation; IB = immunoblot. d. NEDD4 polyubiquitinates RTP801 with Ub-K63 chains. HEK293 cells were transfected with pCMS-eGFP-RTP801, along with pRK5-HA-Ub-K48 or pRK5-HA-Ub-K63 and pHAGE or pHAGE-NEDD4 as indicated. Forty-eight hours later, cultures were harvested and RTP801 was immunoprecipitated. Non-specific rabbit immunoglobulins (Rb IgG) were used as a negative control. Whole cell lysates (inputs) and RTP801 immunocomplexes were resolved in a Western Blot. Membrane was probed for HA, and reprobed for RTP801, for NEDD4 and for AKT as loading control. All samples were immunoblotted in the same membrane, but some irrelevant bands were deleted. Note the high molecular weight smears corresponding to polyubiquitinated RTP801. A representative image of three independent experiments is shown. IP = immunoprecipitation; IB = immunoblot.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Membrane, Cell-Free Assay, Recombinant, Incubation, Immunoprecipitation, Avidin-Biotin Assay, High Molecular Weight, Transfection, Construct, Negative Control

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. NEDD4 co-immunoprecipitates with RTP801 in cells exposed to DSP . HEK293 cells were co-transfected with empty vectors pCMS-eGFP and pCI-HA or with pCMS-eGFP-RTP801 and pCI-HA-NEDD4. Twenty-four hours post-transfection cells were exposed to cross-linker DSP for 2 hours at 4°C prior harvesting. RTP801 was immunoprecipitated and the samples were analyzed by Western Blotting. Membranes were probed with anti- NEDD4 and anti-RTP801 antibodies. Representative images are shown of at least three independent experiments. IP = immunoprecipitation; IB = immunoblot. b. . NGF-differentiated PC12 cells were treated with 1 μM epoxomicin for 2 hours. Then, cultures were exposed to DSP at 4 °C for 2 hours prior harvesting. RTP801 immunocomplexes were resolved in a Western Blotting. The membrane was incubated with anti-NEDD4 and anti-RTP801 antibodies. A representative image is shown of at least two independent assays. IP = immunoprecipitation; IB = immunoblot. c. NEDD4 and RTP801 co-localize in neurons. DIV 19 primary rat cortical neurons were transfected with pCI-HA-NEDD4. Forty-eight hours post-transfection, neurons were fixed and stained with anti-RTP801 (in red) and anti-HA (in green). Scale bar, 5 μm.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Transfection, Immunoprecipitation, Western Blot, Membrane, Incubation, Staining

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. Ectopic NEDD4 decreases RTP801 protein levels in neuronal PC12 cells. NGF-differentiated PC12 cells were transfected with pCI-HA, pCI-HA-NEDD4 or pCI-HA-NEDD4-C867S constructs. Forty-eight hours post-transfection cultures were harvested and analyzed by Western Blot with anti-NEDD4, anti-RTP801 and anti-α-actin antibodies. Representative immunoblots are shown along with densitometric quantification from at least three independent experiments. ($ endogenous NEDD4, # ectopic NEDD4, * specific band for RTP801). One-way ANOVA with Bonferroni multiple comparison test, * P < 0.05 versus pCI-HA. b. . DIV 8 rat primary cortical neurons were infected with lentiviruses containing the empty vector pHAGE, pHAGE-NEDD4 or pHAGE-NEDD4-C867S inactive mutant. Cell lysates were harvested 4 days later and analyzed by Western Blot with antibodies against RTP801, NEDD4 and α-actin as loading control. The graph represents RTP801 densitometries of at least three independent experiments done in triplicate. One-way ANOVA with Bonferroni multiple comparison test, ** P < 0.01 versus pHAGE. c. Ectopic NEDD4 does not affect RTP801 mRNA levels. DIV 8 cortical neurons were infected with lentiviruses containing the constructs pHAGE, pHAGE-NEDD4 or pHAGE-NEDD4-C867S. RNA was extracted 4 days later, and reverse transcription-qPCR was performed to quantify RTP801 transcripts. Results are displayed as RTP801 mRNA fold change respect to α-actin mRNA levels. The graph shows values (mean ± SEM) of three independent experiments. d. NEDD4 knockdown increases RTP801 protein levels and is detrimental for neurons. DIV 4 cortical neurons were infected with lentiviruses containing a scrambled shRNA (ShCt) or a mix of three shRNA sequences against NEDD4 (ShNEDD4). Six days later, cells were harvested and analyzed by Western Blot. Membranes were incubated with NEDD4, RTP801, P-AKT (S473), AKT and α-spectrin antibodies. The antibody against α-actin was used as loading control. For Western blotting using the α-spectrin antibody the caspase-cleaved fragment (spectrin breakdown product 120, SBDP120) is shown. Representative immunoblots are shown along with RTP801 and P-AKT (S473) densitometries (mean ± SEM) of at least three independent experiments. Student's t -test, * P < 0.05 and ** P < 0.01 versus ShCt. e. NEDD4f/f ;Emx1Cre conditional knockout mice have elevated RTP801 protein levels in the cortex. Cortical lysates of 6-week old mice were analyzed by Western Blotting with anti-NEDD4 and anti-RTP801 antibodies, and then reprobed with anti-α-actin antibody as loading control. Representative immunoblots are shown along with RTP801 densitometries (mean ± SEM) of at least three independent gels. All samples were immunoblotted in the same membrane, but some irrelevant lanes were deleted. (* Specific band for RTP801) Student's t -test, * P < 0.05 versus WT.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Transfection, Construct, Western Blot, Comparison, Infection, Plasmid Preparation, Mutagenesis, Control, Reverse Transcription, Knockdown, shRNA, Incubation, Knock-Out, Membrane

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. Ectopic WT NEDD4 protects from RTP801-induced cell death . NGF-differentiated PC12 cells were co-transfected with pCI-HA, pCI-HA-NEDD4 or pCI-HA-NEDD4-C867S constructs, together with either pCMS-eGFP or pCMS-eGFP-RTP801. Cells were fixed 24 hours later and cell survival (eGFP+ cells) scored under fluorescence microscopy. The graph represents mean ± SEM of at least three independent experiments in quadruplicates. One-way ANOVA with Bonferroni multiple comparison test, *** P < 0.001 versus pCI-HA/pCMS-eGFP, ## P < 0.01 versus pCI-HA/pCMS-eGFP-RTP801. b. . NGF-differentiated PC12 cells were co-transfected with pCI-HA or pCI-HA-NEDD4 together with pCMS-eGFP, pCMS-eGFP-RTP801 or pCMS-eGFP-RTP801-KR. Twenty-four hours later, cells were fixed and eGFP+ surviving cells scored using fluorescence microscopy. The graph represents mean ± SEM of at least three independent experiments in quadruplicates. One-way ANOVA with Bonferroni multiple comparison test, *** P < 0.001 versus pCI-HA/pCMS-eGFP and # P < 0.05 versus pCI-HA/pCMS-eGFP-RTP801.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Transfection, Construct, Fluorescence, Microscopy, Comparison

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. NEDD4 protein levels are diminished in 6-OHDA-treated neuronal PC12 cells. NGF-differentiated PC12 cells were exposed to 100 μM 6-OHDA for 16 hours prior harvesting. Cell lysates were analyzed by Western Blot with antibodies against NEDD4, RTP801, as well as α-actin antibody as loading control. Representative immunoblots are shown along with densitometries represented as mean ± SEM of at least three independent experiments. Student's t -test, ** P < 0.01 and *** P < 0.001 versus Ut (Untreated). b. NEDD4 mRNA levels are not modified in 6-OHDA-treated neuronal PC12 cells. NGF-differentiated PC12 cells were exposed to 100 μM 6-OHDA for 8 hours. RNA was extracted and reverse transcription-qPCR was performed. The graphs show values (mean ± SEM) of three independent experiments. Student's t -test, *** P < 0.001 versus ut (untreated). c. NEDD4 is cleaved by caspases after 6-OHDA exposure. Neuronal PC12 cells were treated with 100 μM Z-VAD-FMK pan-caspase inhibitor for 1 hour prior to 6-OHDA exposure. Sixteen hours later, cultures were harvested and analyzed by Western Blot. Membranes were incubated with antibodies against NEDD4 and α-spectrin as well as an α-actin antibody as loading control. Graphs represent mean ± SEM of NEDD4 and caspase-cleaved α-spectrin fragment (spectrin breakdown product 120 KDa, SBDP 120) densitometric quantification of at least three independent experiments. One-way ANOVA with Bonferroni multiple comparison test, *** P < 0.001 versus Untreated/Ct and # P < 0.05, ### P < 0.001 versus 100 μM 6-OHDA/Ct. d. NEDD4 is cleaved by calpains after 6-OHDA exposure. Neuronal PC12 cells were treated with 1 μM ALLN calpain inhibitor for 1 hour prior to 6-OHDA exposure. Sixteen hours later, cultures were harvested and subjected to Western Blot. Membranes were incubated with anti-NEDD4 and anti-α-spectrin antibodies and with anti-α-actin as loading control. Graphs represent mean ± SEM of NEDD4 and calpain-cleaved α-spectrin fragment (spectrin breakdown product 145 KDa, SBDP 145) densitometric quantification of at least three independent experiments. One-way ANOVA with Newman-Keuls multiple comparison test, ** P < 0.01, *** P < 0.001 versus Untreated/Ct and # P < 0.05, ### P < 0.001 versus 100 μM 6-OHDA/Ct.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Western Blot, Control, Modification, Reverse Transcription, Incubation, Comparison

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: a. Ectopic NEDD4 WT partially prevents from 6-OHDA-induced cell death. NGF-differentiated PC12 cells were co-transfected with pCI-HA/pCMS-eGFP, pCI-HA-NEDD4/pCMS-eGFP or pCI-HA-NEDD4-C867S/pCMS-eGFP vectors with a 4:1 ratio. Thirty-two hours later, cell cultures were exposed to 100 μM 6-OHDA for 16 hours. Then, eGFP+ surviving cells were scored under fluorescence microscopy. The graph shows mean ± SEM of at least three independent experiments done in quadruplicate. One-way ANOVA with Bonferroni multiple comparison test, *** P < 0.001 versus Untreated/pCI-HA and ## P < 0.01 versus 100 μM 6-OHDA /pCI-HA. b. Ectopic NEDD4 reduces RTP801 elevation after 6-OHDA exposure. NGF-differentiated PC12 cells were transfected with pCI-HA, pCI-HA-NEDD4 or pCI-HA-NEDD4-C867S. Thrity-two hours later, cell cultures were exposed to 100 μM 6-OHDA for 16 hours and cell lysates were analyzed by Western Blot. Membranes were incubated with antibodies against NEDD4, RTP801 and α-actin as loading control (* endogenous NEDD4, # ectopic NEDD4). Low signal of ectopic NEDD4 maybe due low sensitivity of NEDD4 antibody towards the human NEDD4 protein, as compared to endogenous rat NEDD4. Representative immunoblots are shown along with RTP801 densitometric normalized quantification (mean ± SEM) from at least three independent experiments. One-way ANOVA with Bonferroni multiple comparison test, * P < 0.05 versus Untreated/pCI-HA, # P < 0.05 versus 100 μM 6-OHDA/pCI-HA.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Transfection, Fluorescence, Microscopy, Comparison, Western Blot, Incubation, Control

Journal: Oncotarget

Article Title: Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

doi: 10.18632/oncotarget.11020

Figure Lengend Snippet: DIV 5 primary rat cortical neurons were infected with lentiviruses containing a scrambled shRNA (ShCt) or a shRNA against RTP801 (ShRTP801). Two days later, neurons were transduced with a mix of three shRNA sequences against NEDD4 (ShNEDD4) or the corresponding control shRNA (ShCt). Cell lysates were analyzed 4 days later by Western Blot. Membranes were incubated with RTP801, NEDD4, P-AKT (S473), AKT, P-S6 (S235/236), ERK1/2 and α-spectrin antibodies, and with α-actin antibody as loading control. For α-spectrin the caspase-cleaved fragment (spectrin breakdown product 120, SBDP120) is shown. Representative immunoblots are shown along with densitometries (mean ± SEM) of at least two independent experiments done in triplicate. One-way ANOVA with Newman-Keuls multiple comparison test, * P < 0.05, ** P < 0.01 versus ShCt/ShCt and # P < 0.05, ## P < 0.01 versus ShCt/ShNEDD4.

Article Snippet: Shortly, 1 mM dithiothreitol in 20 mM Tris-HCl pH 7.5, 20 U/ml inorganic pyrophosphatase, 5 mM Mg-ATP, 2,5 μM biotinylated ubiquitin, 0,1 μM His6-tagged recombinant human ubiquitin-activating enzyme E1, His6-tagged recombinant human ubiquitin-conjugating enzyme UbcH5b E2 (Enzo Life Sciences), recombinant human NEDD4 protein (Sigma Aldrich) and N-terminal GST-tagged recombinant human RTP801 full-length protein (Novus Biologicals, Littleton, CO, USA) were added to the reaction buffer, as specified in each condition, and then incubated at 37°C for 90 min. Next, RTP801 was immunoprecipitated using an anti-RTP801 antibody and equal volumes of each sample were analyzed by WB.

Techniques: Infection, shRNA, Transduction, Control, Western Blot, Incubation, Comparison