Journal: Nature Communications

Article Title: Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells

doi: 10.1038/s41467-023-42689-2

Figure Lengend Snippet: a General scheme of GCE. RS designates aminoacyl-tRNA synthetase, ncAA - noncanonical amino acid. b Schematic representation of the PKR-dependent eIF2α phosphorylation pathway. c Schematic representation of the iRFP-GFP 39TAG reporter. The gray population represents untransfected cells; gray arrow – CMV promoter; gray box – polyA signal, FC denotes flow cytometry. d Heat map illustrating the effect of adding various stress remodelers on GCE efficiency in units of relative efficiency GFP (%). Percentage relative efficiency GFP is calculated as the median GFP signal for each particular case divided by the median GFP signal for control samples with the addition of mock plasmid. Median GFP signals were obtained after FC analysis of corresponding samples. The heat map shows the mean value for the relative efficiencies of three independent experiments. Source data are provided as a Source Data file. e FC analysis of the iRFP-GFP 39TAG reporter in the absence and presence of the tested stress remodelers. Concatenated data from three independent experiments (50 ng tRNA Pyl plasmid) are shown.

Article Snippet: Next day membranes were washed three times with 2% BSA in 1× TBST and incubated with secondary antibodies: IRDye® 800CW goat anti-rabbit antibody (LI-COR, 926-32211, 1:10,000 in 2% BSA in 1× TBST) and IRDye® 680RD goat anti-mouse antibody (LI-COR, 926-68070, 1:10000 in 2% BSA in 1× TBST)—for assessment of eIF2α phosphorylation level—and IRDye® 800CW goat anti-mouse antibody (LI-COR, 925-32210, 1:10,000 in 2% BSA in 1× TBST)—for puromycin incorporation assay.

Techniques: Flow Cytometry, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells

doi: 10.1038/s41467-023-42689-2

Figure Lengend Snippet: a Schematic representation of ncAA-modified trastuzumab and constructs used in the study. SP denotes secretion peptide, LC - light chain, HC - heavy chain, purple star represents ncAA, gray arrow - CMV promoter; gray box - polyA signal. b Bar plot illustrating the effect of adding various stress remodelers on concentration of SCO-K-modified trastuzumab expressed in HEK293T cells. WT designates trastuzumab WT (trastuzumab LC + HC, without amber stop codon). Data are presented as mean values +/− standard deviation (SD). Results from three independent experiments are shown. Ns denotes not significant ( p value > 0.05), * p value ≤ 0.05, p values were calculated using one-way ANOVA with Dunnett’s multiple comparison test to no stress remodeler addition (Mock). Exact p values for the samples are 0.7741 (eIF2Bγ + eIF2Bε), 0.0279 (PKR∆), 0.9974 (eIF2α S51A), 0.2585 (PKR∆ + eIF2α S51A v2). Source data are provided as a Source Data file.

Article Snippet: Next day membranes were washed three times with 2% BSA in 1× TBST and incubated with secondary antibodies: IRDye® 800CW goat anti-rabbit antibody (LI-COR, 926-32211, 1:10,000 in 2% BSA in 1× TBST) and IRDye® 680RD goat anti-mouse antibody (LI-COR, 926-68070, 1:10000 in 2% BSA in 1× TBST)—for assessment of eIF2α phosphorylation level—and IRDye® 800CW goat anti-mouse antibody (LI-COR, 925-32210, 1:10,000 in 2% BSA in 1× TBST)—for puromycin incorporation assay.

Techniques: Modification, Construct, Concentration Assay, Standard Deviation, Comparison

Journal: Aging Cell

Article Title: The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans

doi: 10.1111/acel.12101

Figure Lengend Snippet: Conservation in gene structure and function of Caenorhabditis elegans gcn-2 . (A) Gene structure and predicted protein domains of GCN-2, designed using the Prosite MyDomains ( http://prosite.expasy.org/mydomains/ ): Black boxes represent exons linked by lines corresponding to introns, and white boxes indicate the 5′ and 3′ UTR found in a second alternative isoform ( http://www.wormbase.org ). The graphic was created using the Exon-Intron Graphic Maker ( http://wormweb.org/exonintron ). Branches point to the sequences deleted in the two alleles ok871 and ok886 . Black arrowheads indicate the position of primers used in this study (Table ). (B) Reverse transcription (RT)–PCR analysis with primers G1/G2 (shown in A pane) and qRT-PCR analysis with primers G4/G5 (shown in A pane). (C) Western blot analysis showing the levels of phosphorylated (P-eIF2α) normalized by the total amount of eIF2α, in whole worm extracts. Basal levels of P-eIF2α under well-fed conditions in untreated or gcn-2(RNAi) -treated N2 and gcn-2 mutants (left pane). Induced levels of P-eIF2α under amino acid limitation in krs-1(RNAi) treated worms (right pane).

Article Snippet: After 2–3 washes to remove bacteria, they were frozen in ethanol dry ice and, before loading onto SDS-PAGE, worm pellets were boiled in 50 μL 2X SDS-sample buffer for 10 min. Primary antibodies were a polyclonal antibody raised against worm eIF2α, a kind gift from Dr. Shin Takagi (Nukazuka et al ., ), for total (T)-eIF2α and an anti-phospho-eIF2α antibody (Santa Cruz Biotechnology, sc-101670) for phosphorylated (P)-eIF2α levels.

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

doi: 10.3390/ijms20030580

Figure Lengend Snippet: Ribosome profiling of MG1-infected U343 cells highlights cellular targets of eIF2α phosphorylation and apoptosis. ( A ) Schematic of the ribosome profiling strategy used. Polysomes were fixed using cycloheximide prior to extraction. RPF; Ribosome protected fragments. ( B ) Cumulative frequency distributions of gene expression at the transcriptome (RNA) and translatome (RPF) levels in mock and MG1-infected cells. ( C ) Violin plots showing distribution of the translation efficiency (TE = RPF/RNA) for sequenced genes between mock and MG1-infected U343 cells. The median (central dot), interquartile range (black box), 95% confidence interval (vertical line) are shown, while the grey area represents a density plot wherein the width is proportional to the frequency of the TE values. ( D ) Radiograph (top) showing nascent peptide synthesis in mock-infected U343 cells (0 MOI) or increasing MOI of MG1 virus. Western blot (bottom) probing for phospho-eIF2α. ( E ) TE of ATF4 , a known translational target of phospho-eIF2α, in mock- vs. MG1-infected U343 cells. ( F ) Distribution of TE for genes classified as “apoptotic processes” by the Gene Ontology consortium. Two known translation substrates of phospho-eIF2α, CHOP and GADD34 demonstrated enhanced TE, while other apoptotic regulators such as Bcl-xL and XIAP exhibited antipodal TEs. **** p < 0.0001; determined using a non-parametric Kolmogorov-Smirnov statistical test.

Article Snippet: Protein samples were separated at the constant voltage of 150 V for 1.5 h and transferred onto the 0.2 µm PVDF membrane and the levels of following proteins were determined: rabbit anti-Phospho-eIF2α (Cell Signaling Technology #9721, Danvers, MA, USA), rabbit anti-eIF2α (Abcam# ab26197, Toronto, ON, Canada), rabbit anti-XIAP (Cell Signaling Technology #2045), rabbit anti-RIAP3 ([ ]; kindly provided by Dr. Robert Korneluk, CHEO, Ottawa, ON, Canada), rabbit anti-Bcl-xL (Cell Signaling Technology, Cat#2762S), rabbit anti-4EBP1 (Cell Signaling Technology #9644), mouse anti-EIF5B (Santa-Cruz #sc-393564), rabbit anti-Maraba viral proteins, mouse anti-α-Tubulin (Abcam #ab7291), mouse anti-GAPDH (Advanced Immunochemical #2-RGM2).

Techniques: Infection, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

doi: 10.3390/ijms20030580

Figure Lengend Snippet: The impact of MG1 infection on the host protein synthesis. ( A ) U2OS cells were infected with MG1 at MOI = 0.1 for 18-h time course. The cells were pulsed labeled with [ 35 S]-methionine for 20 min. Next, the obtained lysates were separated through 10% SDS-PAGE electrophoresis. Identity of viral proteins is indicated to the right of the image; Coomassie blue staining demonstrates the protein loading. ( B ) Western blot of U2OS cells infected with MG1 at MOI = 0.1 during an 18-h time course demonstrated phosphorylation of eIF2α, de-phosphorylation of 4E-BP1 and up-regulation of Bcl-xL protein. The levels of Bcl-xL and XIAP proteins were quantified relative to Tubulin used as a loading control. ( C ) Steady-state levels of Bcl-xL and XIAP mRNAs were determined in mock- and MG1-infected cells at 12 hpi. Ct values were normalized by the standard curve of each primer and represented relative to the geometric mean of RPL13A and RPL36. (n.s. = not significant)

Article Snippet: Protein samples were separated at the constant voltage of 150 V for 1.5 h and transferred onto the 0.2 µm PVDF membrane and the levels of following proteins were determined: rabbit anti-Phospho-eIF2α (Cell Signaling Technology #9721, Danvers, MA, USA), rabbit anti-eIF2α (Abcam# ab26197, Toronto, ON, Canada), rabbit anti-XIAP (Cell Signaling Technology #2045), rabbit anti-RIAP3 ([ ]; kindly provided by Dr. Robert Korneluk, CHEO, Ottawa, ON, Canada), rabbit anti-Bcl-xL (Cell Signaling Technology, Cat#2762S), rabbit anti-4EBP1 (Cell Signaling Technology #9644), mouse anti-EIF5B (Santa-Cruz #sc-393564), rabbit anti-Maraba viral proteins, mouse anti-α-Tubulin (Abcam #ab7291), mouse anti-GAPDH (Advanced Immunochemical #2-RGM2).

Techniques: Infection, Labeling, SDS Page, Electrophoresis, Staining, Western Blot, De-Phosphorylation Assay

Journal: International Journal of Molecular Sciences

Article Title: Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

doi: 10.3390/ijms20030580

Figure Lengend Snippet: The effect of eIF2α phosphorylation on the rate of host and MG1 protein synthesis. ( A ) WT and S51A MEFs were infected with MG1 at MOI = 0.1 for the indicated time points and pulse labeled with [ 35 S]-methionine for 20 min. Protein lysate were then separated through 10% SDS-PAGE gel. Coomassie blue stain was used as loading control. ( B ) Top: Densitometry of the relative radioactive signal of the area between the viral L and G bands was normalized to the entire related lane on Coomassie gel at 0, 9 and 12 hpi to determine the rate of host global translation. Bottom: the relative radioactive signal from the viral M protein to Coomassie stain was normalized to the signals from the WT MEFs at each time points to measure the rate of viral translation between the two cell lines.

Article Snippet: Protein samples were separated at the constant voltage of 150 V for 1.5 h and transferred onto the 0.2 µm PVDF membrane and the levels of following proteins were determined: rabbit anti-Phospho-eIF2α (Cell Signaling Technology #9721, Danvers, MA, USA), rabbit anti-eIF2α (Abcam# ab26197, Toronto, ON, Canada), rabbit anti-XIAP (Cell Signaling Technology #2045), rabbit anti-RIAP3 ([ ]; kindly provided by Dr. Robert Korneluk, CHEO, Ottawa, ON, Canada), rabbit anti-Bcl-xL (Cell Signaling Technology, Cat#2762S), rabbit anti-4EBP1 (Cell Signaling Technology #9644), mouse anti-EIF5B (Santa-Cruz #sc-393564), rabbit anti-Maraba viral proteins, mouse anti-α-Tubulin (Abcam #ab7291), mouse anti-GAPDH (Advanced Immunochemical #2-RGM2).

Techniques: Infection, Labeling, SDS Page, Staining

Journal: International Journal of Molecular Sciences

Article Title: Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

doi: 10.3390/ijms20030580

Figure Lengend Snippet: The levels of MG1 proteins and up-regulation of Bcl-xL protein are independent of eIF2α phosphorylation. ( A ) Western blot analysis from the time course infection of WT and S51A MEFs with MG1 at MOI = 0.1 demonstrates the levels of Bcl-xL, XIAP proteins and de-phosphorylation of 4E-BP1. GAPDH is shown as the loading control. Relative Bcl-xL and XIAP expression levels to GAPDH at 12 hpi were normalized to uninfected cells (Bottom). ( B ) Top: IncuCyte live cell imaging demonstrates the confluency of GFP signal in WT and S51A MEFs in real time. Y axis depicts the percentage of GFP-MG1 infected cells relative to total cell confluency. Middle: Fold GFP-MG1 confluency between the two cell lines was quantified at 12 and 16 hpi. Bottom: Cytotox Red reagent was used in cells mock-infected or infected with MG1 virus and the activity of the fluorescent red dye was monitored over a time course. Y axis represents the number of cells emitting fluorescent red (dead cells) relative to the cell confluency. ( C ) MG1 genomic RNA was measured relative to the geometric mean of RPL13A and PPIA mRNAs in WT and S51A MEFs at 12 and 15 hpi.

Article Snippet: Protein samples were separated at the constant voltage of 150 V for 1.5 h and transferred onto the 0.2 µm PVDF membrane and the levels of following proteins were determined: rabbit anti-Phospho-eIF2α (Cell Signaling Technology #9721, Danvers, MA, USA), rabbit anti-eIF2α (Abcam# ab26197, Toronto, ON, Canada), rabbit anti-XIAP (Cell Signaling Technology #2045), rabbit anti-RIAP3 ([ ]; kindly provided by Dr. Robert Korneluk, CHEO, Ottawa, ON, Canada), rabbit anti-Bcl-xL (Cell Signaling Technology, Cat#2762S), rabbit anti-4EBP1 (Cell Signaling Technology #9644), mouse anti-EIF5B (Santa-Cruz #sc-393564), rabbit anti-Maraba viral proteins, mouse anti-α-Tubulin (Abcam #ab7291), mouse anti-GAPDH (Advanced Immunochemical #2-RGM2).

Techniques: Western Blot, Infection, De-Phosphorylation Assay, Expressing, Live Cell Imaging, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure

doi: 10.1016/j.jbc.2021.100395

Figure Lengend Snippet: Comparing signaling pathways that regulate protein synthesis in gastrocnemius muscle of WT and MKO mice . WT and MKO mice were treated with or without DEX (10 mg/kg body weight) for 1 week. At the end of treatment, mice were IP injected with insulin (1 unit per kg body weight) or PBS for 10 min, and gastrocnemius muscle was isolated and processed for ELISA to monitor the phosphorylation status of ( A ) p70S6K at Thr389, ( B ) 4E-BP1 at Thr37/Ser46 and ( C ) eIF2α at Ser52. n = 3 to 6, significance was denoted as ∗ p ≤ 0.05. Error bars represent the SD. DEX, dexamethasone.

Article Snippet: Relative total eIF2α and phosphorylated eIF2α levels were measured using the PathScan Phospho-eIF2α (Ser51) and total eIF2α Sandwich ELISA kits (Cell Signaling Technology, Catalog #7286 and Catalog #7952.)

Techniques: Protein-Protein interactions, Injection, Isolation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics