frt cells  (World Precision Instruments)

Journal: Nucleic Acids Research

Article Title: Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions

doi: 10.1093/nar/gkae006

Figure Lengend Snippet: Activation of GCN2 by agents that invoke translation inhibition. ( A ) ZAK was deleted by genome editing and CRISPR and WT and ZAK KO cells, designated + and -, respectively, were cultured and ZAK and actin proteins were measured in the prepared lysates by immunoblot analyses with antibodies that specifically recognize each protein. Molecular weight (MW) markers are indicated in kDal. ( B ) WT HEK293T cells (+) and those deleted for ZAK (−) were treated with 1 μM anisomycin (ANS) for 1 h (+) or not treated (−), followed by immunoblot measurements of the phosphorylated and total p38 proteins, along with ZAK. ( C ) WT (+, lanes 1–6) and ZAK (−, lanes 7–12) cells were treated with up to 200 nM ANS for 3 h. Levels of the indicated phosphorylated and total proteins were measured by immunoblot analyses. ANS treatment for lanes: 1 and 7 (0 nM, not treated -NT), 2 and 8 (5 nM), 3 and 9 (25 nM), 4 and 10 (50 nM), 5 and 11 (100 nM), and 6 and 12 (200 nM). ( D ) WT (+, lanes 1–5) and ZAK KO (−, lanes 6–10) HEK293T cells were treated with increasing amounts of HF for 3 h, followed by immunoblot measurements of the indicated phosphorylated and total proteins (left panel). HF treatment for lanes: 1 and 6 (0 nM, not treated -NT), 2 and 7 (5 nM), 3 and 8 (25 nM), 4 and 9 (50 nM), and 5 and 10 (100 nM). Relative levels of P-GCN2 and P-eIF2α normalized for the respectively total protein (P-total) are shown in the bar graph (right panel). The P/total for GCN2 and eIF2α proteins was normalized to the respective NT-WT group and presented as fold change. ( E ) WT (+) and ZAK KO (−) HEK293T cells were treated with 25 nM HF, 4 mM histidinol (HIS), or 2 μM borrelidin (BOR) for 3 h, followed by immunoblot measurements of the indicated phosphorylated and total proteins (left panel). Relative levels of P-GCN2 and P-eIF2α normalized for total levels of the corresponding proteins are shown in the bar graph (right panel). The P/total ratio for GCN2 and eIF2α proteins was normalized to the respective NT-WT group and presented as fold change. For panels D and E, statistical significance, denoted by *, was determined using a two-way analysis of variance (ANOVA); * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars indicate mean with SD ( n = 3).

Article Snippet: Deletion of GCN2 was achieved in the HEK293T-FRT cells using the CRISPR/Cas9 Human Gene Knockout Kit (Origene, # KN412459).

Techniques: Activation Assay, Inhibition, CRISPR, Cell Culture, Western Blot, Molecular Weight

Journal: Nucleic Acids Research

Article Title: Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions

doi: 10.1093/nar/gkae006

Figure Lengend Snippet: Role of ribosome collisions in the activation of GCN2. ( A ) WT HEK293T cells were either not treated (NT) or treated with either 25 nM anisomycin (ANS) alone or in combination with 1μM puromycin (Puro) for 3 h. Lysates were prepared, treated with RNase I, and subjected to sucrose gradient centrifugation and fractionation. Ribosomes were visualized by measuring the absorbance at 254nm. The profile indicates the free 40S and 60S ribosomal subunits, 80S monosomes and colliding translating ribosomes, which are represented by disomes and trisomes. M- monosomes, D-disomes. ( B ) WT HEK293T cells were not treated (−) or treated with 25 nM ANS in presence or absence of 1 μM Puro for 3 hours, as indicated (+). The designated phosphorylated and total proteins were measured by immunoblot analyses. ( C ) GCN2 KO HEK293T cells were not treated (−) or treated with 25 nM ANS in presence or absence of Puro for 3 h, as indicated (+), followed by immunoblot analyses with the indicated antibodies. ( D ) WT HEK293T cells were not treated (−) or treated with 25 nM HF in presence or absence of Puro for 3 h, as indicated (+), followed by immunoblot measurements of the specified phosphorylated and total proteins. ( E ) WT HEK293T cells were treated with 25 nM HF in the presence or absence of 1 μM Puro for 3 h or not treated (NT), as indicated. The percentage of uncharged tRNA Pro (top panel) and levels of total tRNA Pro relative to NT were measured by RT-qPCR and represented in the bar graph (bottom panel). Statistical significance was determined using a one-way analysis of variance (ANOVA); Significance is indicated by ** P ≤ 0.01 and **** P ≤ 0.0001 and ns indicates not significant difference. Error bars indicate mean with SD ( N = 3). ( F ) WT HEK293T cells were treated with 25 nM HF in presence or absence of 50 μg/ml cycloheximide (CHX) for 3 h or not treated (NT). The percentage of uncharged tRNA Pro (top panel) and relative levels of total tRNA Pro normalized to the NT group (bottom panel) were measured by RT-qPCR and represented in the bar graph. Statistical significance was determined using a one-way analysis of variance (ANOVA); Significant is indicated by ** P ≤ 0.01 and * P ≤ 0.05, and ns indicates no significant differences. Error bars indicate mean with SD ( n = 3). ( G ) WT HEK293T cells were not treated (−) or treated with 25 nM HF in presence or absence of 50 μg/ml cyclohexmide (CHX) for 3 h, as indicated (+). Lysates were prepared and the specified phosphorylated and total proteins were measured by immunoblot analyses. ( H ) HEK293T cells stably expressing only FLAG-WT-GCN2 or FLAG-GCN2-m2 were treated with 25 nM ANS (+) or no treatment (−) for 3 h, followed by measurements of phosphorylated or total GCN2 and eIF2α by immunoblot analyses.

Article Snippet: Deletion of GCN2 was achieved in the HEK293T-FRT cells using the CRISPR/Cas9 Human Gene Knockout Kit (Origene, # KN412459).

Techniques: Activation Assay, Gradient Centrifugation, Fractionation, Western Blot, Quantitative RT-PCR, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions

doi: 10.1093/nar/gkae006

Figure Lengend Snippet: GCN2 binds tRNA Pro in response to HF treatment. (A and B) To measure charging of tRNAs genome-wide, MEF cells were either treated with a 100 nM HF for 3 hours or not treated (NT). Only the tRNA isoacceptors measured in the MEF cells by the genome-wide assay are shown. The percentage of charged tRNAs is shown as a heatmap ( A ) and combined tRNA isoacceptors for each cognate amino acid as a bar graph ( B ). The error bars represent the SD of the mean ( N = 3). *Indicates P ≤ 0.05 determined using a two-sided Welch's t -test followed by correction for multiple hypothesis testing using Benjamini–Hochberg FDR method. ( C ) HEK293T cells stably expressing only FLAG-WT-GCN2 or FLAG-GCN2-m2, or GCN2 KO HEK293T cells, were treated with 25 nM HF (+) or no treatment (–) for 3 hours, followed by measurements of phosphorylated or total GCN2 and eIF2α by immunoblot analyses. (D and E) HEK293T cells expressing FLAG-WT-GCN2 or FLAG-GCN2-m2, or GCN2 KO HEK293T cells, were treated with 25 nM HF (+) for 3 hours or not treated (–). Cell lysates were prepared and FLAG-WT-GCN2 and FLAG-GCN2-m2 were pulled down using anti-FLAG antibody linked with magnetic beads. The associated RNA was purified and bound tRNA Pro ( D ) and tRNA Asn ( E ) were measured by qRT-PCR. The bar graph shows fold enrichment for tRNA bound to GCN2 (WT/m 2 ) in response to HF (+) or no treatment (–) and is normalized to FLAG-WT-GCN2-no treatment group. Statistical significance was determined using a two-way analysis of variance (ANOVA); ** P ≤ 0.01 and ns indicates no significant difference. Error bars indicate mean with SD ( n = 3). ns-not significant. ( F ) Model for activation of GCN2 by accumulating tRNA Pro in response to HF treatment.

Article Snippet: Deletion of GCN2 was achieved in the HEK293T-FRT cells using the CRISPR/Cas9 Human Gene Knockout Kit (Origene, # KN412459).

Techniques: Genome Wide, Stable Transfection, Expressing, Western Blot, Magnetic Beads, Purification, Quantitative RT-PCR, Activation Assay

Journal: Nucleic Acids Research

Article Title: Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions

doi: 10.1093/nar/gkae006

Figure Lengend Snippet: ZAK is not required for UV activation of GCN2 in human keratinocytes. ( A ) Human keratinocyte NTERT cells expressing ZAK (+, lanes 1–6) or deleted for ZAK (-, lanes 7–12) were treated with up to 1000 J/m 2 of UV-B ( A ) or not treated (NT), followed by 3 h of culture. Protein lysates were prepared and the indicated phosphorylated and total proteins were measured by immunoblot analyses. UV-B treatment for lanes: 1 and 7 (0 J/m 2 ), 2 and 8 (200 J/m 2 ), 3 and 9 (400 J/m 2 ), 4 and 10 (600 J/m 2 ). 5 and 11 (800 J/m 2 ) and 6 and 12 (1000 J/m 2 ). Relative levels of P-GCN2 and P-eIF2α are shown in the bar graph (right panels). P/total ratio for GCN2 and eIF2α proteins was normalized to the respective not treated (NT) WT group and presented as fold change. Statistical significance was determined using a two-way analysis of variance (ANOVA); Significance is indicated as ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars indicate mean with SD ( n = 3). ( B ) NTERT cells expressing ZAK (+, lanes 1–7) or deleted for ZAK (−, lanes 8–14) were treated with up to 1200 J/M 2 of UV-C or left untreated (NT), followed by 3 h of culture. The indicated phosphorylated and total proteins were measured by determined by immunoblot. UV-C treatment for lanes: 1 and 8 (0 J/m 2 ), 2 and 9 (20 J/m 2 ), 3 and 10 (75 J/m 2 ), 4 and 11 (150 J/m 2 ). 5 and 12 (300 J/m 2 ), 6 and 13 (600 J/m 2 ), and 7 and 14 (1200 J/m 2 ). Relative levels of P-GCN2 and P-eIF2α are shown in the bar graphs (right panel). P/total ratio for GCN2 and eIF2α proteins was normalized to the respective not treated (NT) WT group and presented as fold change. Statistical significance was determined using a two-way analysis of variance (ANOVA); Statistical significance is indicated as *** P ≤ 0.001, **** P ≤ 0.0001. Error bars indicate Mean with SD ( N = 3). ( C ) WT (+) and ZAK KO (−) NTERT cells were treated with a 400 J/m 2 UV-B or left untreated, followed by recovery for 3 h. Cells were collected, lysed, and analyzed by centrifugation in sucrose gradients. Gradients were fractionated and ribosomes monitored at A 254 nm. Free 40S and 60S ribosomal subunits, 80S monosomes, and polysomes are indicated in the figures. Polysome to monosome ratios were determined for each group and presented in a bar graph (bottom panel). Statistical significance was determined using a two-way analysis of variance (ANOVA); Statistical significance is indicated as * P ≤ 0.05, ** P ≤ 0.01. Error bars indicate mean with SD ( N = 3). ( D ) WT NTERT cells were either left untreated or treated with 400 J/m 2 UV-B in presence or absence of 1 μM puromycin (Puro) for 3 h, as indicated, followed by immunoblot measurements of the indicated phosphorylated and total proteins. Phosphorylated GCN2 or eIF2α normalized to total protein are presented for each treatment arrangement, with a value of 1 indicated for the sample not treated with either UV-B or Puro. ( E ) HEK293T cells stably expressing only FLAG-WT-GCN2 or FLAG-GCN2-m2 were treated with 400 J/m 2 UV-B or not treated (−) for 3 h, followed by measurements of phosphorylated or total GCN2 and eIF2α by immunoblot analyses.

Article Snippet: Deletion of GCN2 was achieved in the HEK293T-FRT cells using the CRISPR/Cas9 Human Gene Knockout Kit (Origene, # KN412459).

Techniques: Activation Assay, Expressing, Western Blot, Centrifugation, Stable Transfection