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 Assay, 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: The Journal of experimental biology

Article Title: Different fuel regulation in two types of myofiber results in different antioxidant strategies in Daurian ground squirrels ( Spermophilus dauricus ) during hibernation.

doi: 10.1242/jeb.231639

Figure Lengend Snippet: Fig. 1. Expression of REDD1 in SOL and EDL muscles of Daurian ground squirrels during hibernation. Protein expression levels were visualized at four sampling points: pre-hibernation (PRE), hibernation (HIB), interbout arousal (IBA) and post-hibernation (POST). See Materials and Methods for more extensive definitions. Representative immunoblots and Stain-Free total protein loading controls are shown below for selected pairs of sampling points and muscles that are labeled on the top of the blot. Data were analyzed using an ANOVA with a post hoc Tukey’s test (P<0.05); values that are not statistically different from each other share the same letter notation.

Article Snippet: Membranes were then blocked with 4% Mowiol® PVA-203 (Aladdin, China) in Tris-buffered saline (TBS) for 10 min (Gholap et al., 2005; Rodda and Yamazaki, 1994), and incubated with REDD1 (Proteintech, 10638-1-AP), atrogin-1 (Proteintech, 12866-1-AP), PGC-1α (Novus, NBP1-04676SS), FOXO1 (C29H4) rabbit mAb (CST#2880), phospho-FOXO1 (Ser256) antibody (CST#9461), FOXO4 (CST#9472) and FOXO4 (phospho-Thr451) (Signalway antibody, 12053) in 0.1% TBST (TBS with 0.1% Tween 20) containing 2% polyvinylpyrrolidone (PVP-40; Amresco, USA, 0507-500G) at 4°C overnight.

Techniques: Expressing, Muscles, Sampling, Western Blot, Staining, Labeling

Journal: Scientific Reports

Article Title: High expression of DNA damage-inducible transcript 4 (DDIT4) is associated with advanced pathological features in the patients with colorectal cancer

doi: 10.1038/s41598-021-92720-z

Figure Lengend Snippet: Hub genes involved in pathways with P value < 0.05 based on KEGG pathway analysis.

Article Snippet: Subsequently, tissue sections with primary antibody specific for DDIT4 (1:80 dilution, Biorbyt, Cambridge, UK) were incubated overnight at 4 °C.

Techniques:

Journal: Scientific Reports

Article Title: High expression of DNA damage-inducible transcript 4 (DDIT4) is associated with advanced pathological features in the patients with colorectal cancer

doi: 10.1038/s41598-021-92720-z

Figure Lengend Snippet: Immunohistochemical analysis of DDIT4 expression in colorectal cancer (CRC) samples. Nuclear expression of DDIT4 in CRC: ( A , A-1 ) low expression and ( B , B-1 ) high expression. Cytoplasmic expression of DDIT4 in CRC: ( C , C-1 ) low expression and ( D , D-1 ) high expression. Membranous expression of DDIT4 in CRC: ( E , E1 ) low expression and ( F , F1 ) high expression. ( G , G1 ) DDIT4 expression in adjacent normal tissue. ( H ) DDIT4 expression in normal human liver as a positive control, ( I ) DDIT4 expression in normal human liver as a negative control, and ( J ) Isotype control. (Figures shown with magnification of 100 × and 200 ×).

Article Snippet: Subsequently, tissue sections with primary antibody specific for DDIT4 (1:80 dilution, Biorbyt, Cambridge, UK) were incubated overnight at 4 °C.

Techniques: Immunohistochemical staining, Expressing, Positive Control, Negative Control, Control

Journal: Scientific Reports

Article Title: High expression of DNA damage-inducible transcript 4 (DDIT4) is associated with advanced pathological features in the patients with colorectal cancer

doi: 10.1038/s41598-021-92720-z

Figure Lengend Snippet: Nuclear, cytoplasmic, and membranous DDIT4 expression in colorectal cancer (CRC) tissues and their adjacent normal tissue samples (Intensity of staining, percentage of positive tumor cells, and H-score).

Article Snippet: Subsequently, tissue sections with primary antibody specific for DDIT4 (1:80 dilution, Biorbyt, Cambridge, UK) were incubated overnight at 4 °C.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: High expression of DNA damage-inducible transcript 4 (DDIT4) is associated with advanced pathological features in the patients with colorectal cancer

doi: 10.1038/s41598-021-92720-z

Figure Lengend Snippet: The association between nuclear DDIT4 expression and clinicopathological features of colorectal cancer (CRC) samples (Intensity of staining and H-score).

Article Snippet: Subsequently, tissue sections with primary antibody specific for DDIT4 (1:80 dilution, Biorbyt, Cambridge, UK) were incubated overnight at 4 °C.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: High expression of DNA damage-inducible transcript 4 (DDIT4) is associated with advanced pathological features in the patients with colorectal cancer

doi: 10.1038/s41598-021-92720-z

Figure Lengend Snippet: The association between cytoplasmic DDIT4 expression and clinicopathological features of colorectal cancer (CRC) samples (Intensity of staining and H-score).

Article Snippet: Subsequently, tissue sections with primary antibody specific for DDIT4 (1:80 dilution, Biorbyt, Cambridge, UK) were incubated overnight at 4 °C.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: High expression of DNA damage-inducible transcript 4 (DDIT4) is associated with advanced pathological features in the patients with colorectal cancer

doi: 10.1038/s41598-021-92720-z

Figure Lengend Snippet: The association between membranous DDIT4 expression and clinicopathological features of colorectal cancer (CRC) samples (Intensity of staining and H-score).

Article Snippet: Subsequently, tissue sections with primary antibody specific for DDIT4 (1:80 dilution, Biorbyt, Cambridge, UK) were incubated overnight at 4 °C.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: High expression of DNA damage-inducible transcript 4 (DDIT4) is associated with advanced pathological features in the patients with colorectal cancer

doi: 10.1038/s41598-021-92720-z

Figure Lengend Snippet: Box plot analysis of DDIT4 expression levels in tumor differentiation groups and TNM stages. The bold line precisely represents median expression levels of DDIT4. ( A ) The result of data analysis showed a statistically significant association in the median nuclear expression of DDIT4 between well and moderately tumor differentiation ( P = 0.037). ( B ) A statistically significant association was observed in the median nuclear expression of DDIT4 between stages II and III ( P = 0.026).

Article Snippet: Subsequently, tissue sections with primary antibody specific for DDIT4 (1:80 dilution, Biorbyt, Cambridge, UK) were incubated overnight at 4 °C.

Techniques: Expressing

Journal: Scientific Reports

Article Title: High expression of DNA damage-inducible transcript 4 (DDIT4) is associated with advanced pathological features in the patients with colorectal cancer

doi: 10.1038/s41598-021-92720-z

Figure Lengend Snippet: Kaplan–Meier survival analysis according to nuclear expression levels of DDIT4 protein in colorectal cancer (CRC). Log-rank test did not show any significant difference in ( A ) DSS and ( B ) PFS between two groups of CRC patients (high versus low nuclear expression of DDIT4 protein). DSS : Disease-specific survival and PFS : progression-free survival.

Article Snippet: Subsequently, tissue sections with primary antibody specific for DDIT4 (1:80 dilution, Biorbyt, Cambridge, UK) were incubated overnight at 4 °C.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: DDIT4 overexpression associates with poor prognosis in lung adenocarcinoma

doi: 10.7150/jca.60118

Figure Lengend Snippet: Expression of DDIT4 is upregulated in LUAD cell lines and tissues. A. DDIT4 expression is upregulated in LUAD cell lines. B. DDIT4 expression is upregulated in LUAD tissues.

Article Snippet: Antibodies against DDIT4 were purchased from Atlas Antibodies AB (Sweden).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: DDIT4 overexpression associates with poor prognosis in lung adenocarcinoma

doi: 10.7150/jca.60118

Figure Lengend Snippet: Representative images of immunohistochemical staining of DDIT4 in lung adenocarcinoma tissue. A. Strong positive staining for DDIT4 in LUAD. B. Medium positive staining for DDIT4 in LUAD. C. Negative staining for DDIT4 in LUAD (A, B, C: 100× magnification).

Article Snippet: Antibodies against DDIT4 were purchased from Atlas Antibodies AB (Sweden).

Techniques: Immunohistochemical staining, Staining, Negative Staining

Journal: Journal of Cancer

Article Title: DDIT4 overexpression associates with poor prognosis in lung adenocarcinoma

doi: 10.7150/jca.60118

Figure Lengend Snippet: Relationship between expression levels of DDIT4 and clinicopathological features of 75 LUAD specimens

Article Snippet: Antibodies against DDIT4 were purchased from Atlas Antibodies AB (Sweden).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: DDIT4 overexpression associates with poor prognosis in lung adenocarcinoma

doi: 10.7150/jca.60118

Figure Lengend Snippet: Kaplan-Meier survival curves for 75 LUAD patients according to DDIT4 expression status (log-rank test, P = 0.013).

Article Snippet: Antibodies against DDIT4 were purchased from Atlas Antibodies AB (Sweden).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: DDIT4 overexpression associates with poor prognosis in lung adenocarcinoma

doi: 10.7150/jca.60118

Figure Lengend Snippet: Univariate and multivariate analysis of prognostic factors in 75 LUAD patients

Article Snippet: Antibodies against DDIT4 were purchased from Atlas Antibodies AB (Sweden).

Techniques:

Journal: Journal of Cancer

Article Title: DDIT4 overexpression associates with poor prognosis in lung adenocarcinoma

doi: 10.7150/jca.60118

Figure Lengend Snippet: Analysis of the DDIT4 gene in the TCGA database. A. Expression of DDIT4 in NSCLC in the TCGA database. B. Expression of DDIT4 in LUAD in the TCGA database. C. Expression of DDIT4 in LUSC in the TCGA database. D. The relationship between the expression levels of DDIT4 and OS in NSCLC. E. The relationship between the expression levels of DDIT4 and OS in LUAD. F. The relationship between the expression levels of DDIT4 and OS in LUSC.

Article Snippet: Antibodies against DDIT4 were purchased from Atlas Antibodies AB (Sweden).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: DDIT4 overexpression associates with poor prognosis in lung adenocarcinoma

doi: 10.7150/jca.60118

Figure Lengend Snippet: DDIT4 expression is unregulated in LUAD under hypoxia.

Article Snippet: Antibodies against DDIT4 were purchased from Atlas Antibodies AB (Sweden).

Techniques: Expressing

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: Frontiers in Microbiology

Article Title: Streptococcus pneumoniae promotes migration and invasion of A549 cells in vitro by activating mTORC2/AKT through up-regulation of DDIT4 expression

doi: 10.3389/fmicb.2022.1046226

Figure Lengend Snippet: Up-regulated DDIT4 regulates the mTORC2/AKT signaling pathway. (A) Differential expression of the DDIT4 gene in the co-culture and A549 control groups was analyzed using qPCR, ** P < 0.01. (B) The differential expression of DDIT4 protein in the co-culture and A549 control groups was analyzed by WB. (C) KEGG enrichment analysis based on significantly differentially expressed genes between the co-culture group and the A549 control group. (D) Compared with the control group, the expression of Rictor and phosphorylated AKT (p-AKT) in A549 cells in the co-culture group was up-regulated. At the same time, the remaining proteins had no significant change. (E,F) qPCR similarly confirmed that the Rictor gene but not the Raptor gene was up-regulated in the co-culture group, * P < 0.05. (G) Silencing of the DDIT4 gene resulted in reduced expression of Rictor and p-AKT without significant effects on other proteins. (H,I) Overexpression of DDIT4 up-regulated the expression of Rictor and p-AKT1, which was inhibited by the addition of AKT inhibitor perifosine.

Article Snippet: The primary antibodies used in this study are as follows: DDIT4 (1:500; ER1706-76, HUABIO, Hangzhou, China), AKT1 (1:500; ER1609-47, HUABIO, Hangzhou, China), phosphorylated AKT1 (Ser473) (p-AKT1; 1:500; ER1607-73, HUABIO, Hangzhou, China), RP70S6KB1 (1:1,000; #2708, Cell Signaling Technology, Boston, United States), p-RP70S6KB1 (Thr389) (p-S6K; 1:1,000; #9234, Cell Signaling Technology, Boston, United States), Raptor (1:500; ER1802-57, HUABIO, Hangzhou, China), Rictor (1:500; EM1709-50, HUABIO, Hangzhou, China), and β-actin (1:1,000; R1207-1, HUABIO, Hangzhou, China).

Techniques: Expressing, Co-Culture Assay, Over Expression

Journal: Frontiers in Microbiology

Article Title: Streptococcus pneumoniae promotes migration and invasion of A549 cells in vitro by activating mTORC2/AKT through up-regulation of DDIT4 expression

doi: 10.3389/fmicb.2022.1046226

Figure Lengend Snippet: High expression of DDIT4 enhances the migratory and invasive abilities of A549 cells and impairs the prognosis of LUAD patients. (A) Transwell migration assays of A549 cells with silencing of DDIT4, overexpression of DDIT4, overexpression of DDIT4 followed by addition of AKT inhibitor perifosine, and blank control. (B) Cell scratch assays of A549 cells with silencing of DDIT4, overexpression of DDIT4, overexpression of DDIT4 followed by addition of AKT inhibitor perifosine, and blank control at 0 and 24 h, respectively. (C) Expression and enrichment of DDIT4 and S. pneumoniae in lung cancer tissues. The white arrows refer to visible S. pneumoniae . (D) Expression of DDIT4 protein detected by immunohistochemistry in LUAD patients with different prognoses.

Article Snippet: The primary antibodies used in this study are as follows: DDIT4 (1:500; ER1706-76, HUABIO, Hangzhou, China), AKT1 (1:500; ER1609-47, HUABIO, Hangzhou, China), phosphorylated AKT1 (Ser473) (p-AKT1; 1:500; ER1607-73, HUABIO, Hangzhou, China), RP70S6KB1 (1:1,000; #2708, Cell Signaling Technology, Boston, United States), p-RP70S6KB1 (Thr389) (p-S6K; 1:1,000; #9234, Cell Signaling Technology, Boston, United States), Raptor (1:500; ER1802-57, HUABIO, Hangzhou, China), Rictor (1:500; EM1709-50, HUABIO, Hangzhou, China), and β-actin (1:1,000; R1207-1, HUABIO, Hangzhou, China).

Techniques: Expressing, Migration, Over Expression, Immunohistochemistry

Journal: Frontiers in Microbiology

Article Title: Streptococcus pneumoniae promotes migration and invasion of A549 cells in vitro by activating mTORC2/AKT through up-regulation of DDIT4 expression

doi: 10.3389/fmicb.2022.1046226

Figure Lengend Snippet: Model of S. pneumoniae up-regulates DDIT4 expression in A549 cells, which activates the mTORC2/AKT signaling pathway.

Article Snippet: The primary antibodies used in this study are as follows: DDIT4 (1:500; ER1706-76, HUABIO, Hangzhou, China), AKT1 (1:500; ER1609-47, HUABIO, Hangzhou, China), phosphorylated AKT1 (Ser473) (p-AKT1; 1:500; ER1607-73, HUABIO, Hangzhou, China), RP70S6KB1 (1:1,000; #2708, Cell Signaling Technology, Boston, United States), p-RP70S6KB1 (Thr389) (p-S6K; 1:1,000; #9234, Cell Signaling Technology, Boston, United States), Raptor (1:500; ER1802-57, HUABIO, Hangzhou, China), Rictor (1:500; EM1709-50, HUABIO, Hangzhou, China), and β-actin (1:1,000; R1207-1, HUABIO, Hangzhou, China).

Techniques: Expressing