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: 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: Fig. 5 DDIT4 suppresses the tumorigenicity of YAP-dependent uveal melanoma cells. a GNAQQ209L-mutant 92.1 and BRAFV600E-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. 1a, 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), phosphoERK (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: Fig. 6 Proposed model for the regulation of translation by serum in a manner dependent on the YAP/TAZ–TEAD–DDIT4–mTORC1 axis. (Left) In cells with an adequate supply of nutrients and growth factors (serum), G proteins such as Gq/G11 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), phosphoERK (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: Cancer letters

Article Title: REDD1 is a determinant of the sensitivity of renal cell carcinoma cells to autophagy inhibition that can be therapeutically exploited by targeting PIM activity.

doi: 10.1016/j.canlet.2025.217496

Figure Lengend Snippet: Fig. 1. TP-3654 has selective anti-RCC effects and potently induces REDD1 expression. (A) TP-3654 selectively antagonizes RCC cell viability. Normal RPTEC renal cells and a panel of 6 RCC cell lines (Achn, A498, 786-O, RCC4, Caki-1 and Caki-2) were treated with the indicated concentrations of TP-3654 for 72 h. Cell viability was determined by MTT assay. Mean ± SD, n = 3. (B) TP-3654 diminishes ATP levels in RCC cells. 786-O, A498, Achn, and Caki-2 cells were treated with the indicated concentrations of TP-3654 for 72 h. Cellular ATP levels were quantified using the ATPLite assay according to the manufacturer’s directions. Mean ± SD, n = 3. (C) Effects of TP-3654 treatment on the transcriptome of RCC cells. 786-O cells were treated with 3 μM TP-3654 for 24 h. Cells were subjected to RNASeq analyses. Heatmap depicts the most significantly induced genes following TP-3654 treatment. DDIT4 (REDD1) emerged as a highly upregulated gene in response to treatment with TP-3654. (D) Validation of the induction of DDIT4 by TP-3654.786-O, A498, Caki-1, Caki-2, Achn, and RCC4 cells were treated with the indicated concentrations of TP-3654 for 24 h. Gene expression levels were quantified using qRT-PCR and normalized to GAPDH. Mean ± SD, n = 3, * indicates significant difference from Control, p < 0.05. (E) Pharmacodynamic effects of TP-3654 on the AMPK/mTOR signaling cascade. 786-O and A498 cells were treated with the indicated concentrations of TP-3654 for 24 h. Protein lysates were subjected to immunoblotting to assess the effects of drug treatment on the expression of the following factors: phospho-AMPK (Thr172), total AMPK, REDD1, TXNIP, phospho-mTOR (Ser2448), total mTOR, phospho-p70S6K (Thr389), total p70S6K, phospho- 4E-BP1 (Thr37/46), total 4E-BP1, p62/SQSTM1, and LC3B. Tubulin documented equal protein loading.

Article Snippet: 786-O cells were infected with lentiviral particles containing GFP and a puromycin resistance gene (cat. # PS100093V) or a DDIT4 cDNA construct (cat. # RC202847L4V) according to the manufacturer’s protocol (Origene, Rockville, MD).

Techniques: Expressing, MTT Assay, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Control, Western Blot

Journal: Cancer letters

Article Title: REDD1 is a determinant of the sensitivity of renal cell carcinoma cells to autophagy inhibition that can be therapeutically exploited by targeting PIM activity.

doi: 10.1016/j.canlet.2025.217496

Figure Lengend Snippet: Fig. 4. DDIT4/REDD1 status determines sensitivity to autophagy inhibition. (A) DDIT4 was knocked down in 786-O RCC cells using lentiviral shRNA and knocked out in HAP1 cells using CRISPR. Knockdown/knockout efficiency was assessed for both cell lines by immunoblotting. (B) DDIT4 knockdown significantly reduces CQ- induced LC3B punctae formation. 786-O cells infected with control or DDIT4-targeted shRNA were treated with 25 μM CQ for 24 h LC3B punctae were visualized and quantified by immunocytochemistry. Mean ± SD, n = 10. * indicates a significant difference from shRNA controls, p < 0.05. (C) Genetic impairment of DDIT4 reduces cellular sensitivity to CQ. 786-O cells infected with control or DDIT4-targeted shRNA (left) and isogenic HAP1 cells with and without DDIT4 knockout (right) were treated with the indicated concentrations of CQ for 72 h. The impact of drug treatment on cell viability was determined by MTT assay. Mean ± SD, n = 3. * indicates a significant difference from shRNA controls or DDIT4 +/+ cells, p < 0.05. (D) Targeting DDIT4 blunts the pro-apoptotic effects of CQ. 786-O cells infected with control or DDIT4-targeted shRNA (left) and isogenic HAP1 cells with and without DDIT4 knockout (right) were treated with the indicated concentrations of CQ for 48 h. The impact of drug treatment on apoptosis induction was determined by staining with a FITC-tagged active caspase-3 antibody followed by flow cytometry. Mean ± SD, n = 3. * indicates a significant difference from shRNA controls or DDIT4 +/+ cells, p < 0.05. (E) REDD1 (DDIT4) overexpression synergistically enhances the sensitivity of RCC cells to autophagy inhibition. REDD1 was overexpressed in 786-O cells using a GFP-tagged lentiviral construct. Overexpression efficiency was assessed by immunoblotting (left). GFP and REDD1-GFP overexpressing cells were treated with the indicated concentrations of CQ for 72 h (middle, MTT) or 48 h (right, active caspse-3). The effects of drug treatment on cell viability (middle) and apoptosis (right) were quantified for each experimental condition. Mean ± SD, n = 3. * indicates a significant difference from GFP control cells, p < 0.05.

Article Snippet: 786-O cells were infected with lentiviral particles containing GFP and a puromycin resistance gene (cat. # PS100093V) or a DDIT4 cDNA construct (cat. # RC202847L4V) according to the manufacturer’s protocol (Origene, Rockville, MD).

Techniques: Inhibition, shRNA, CRISPR, Knockdown, Knock-Out, Western Blot, Infection, Control, Immunocytochemistry, MTT Assay, Staining, Flow Cytometry, Over Expression, Construct