Journal: The Journal of Biological Chemistry

Article Title: Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF–ERK1/2 signaling pathways

doi: 10.1016/j.jbc.2022.101675

Figure Lengend Snippet: High-content imaging analysis of glucosomes from Hs578T cells. A , the percentages (%) of EGF-treated Hs578T cells displaying each size of PFKL-mEGFP assemblies were quantified from at least three independent imaging sessions in the presence of SCH772984 ( gray ), shERK1 ( yellow ), shERK2 ( blue ), or shControl Scrambled ( green ). Note that the previously reported distributions of glucosomes in various sizes in the absence and presence of 30 ng/ml EGF ( purple and red , respectively) are also graphed together for direct comparison. B , a table shows the average percentages (%) of Hs578T cells displaying the given sized glucosomes at each condition along with their SDs (±). Statistical analyses were performed using Tukey’s multiple comparison tests for two-way ANOVA analysis. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.0001. N.S.; not significant. EGF, epidermal growth factor; mEGFP, monomeric enhanced GFP; PFKL, liver-type phosphofructokinase 1.

Article Snippet: Hs578T cells were transfected with shRNAs targeting ERK1/2 (shERK1 and shERK2) and a control shRNA with a scrambled sequence (shControl Scrambled , CGCGAAGTCTGTACTCTTG, Addgene, Cat# 65232) ( ) using Lipofectamine 2000.

Techniques: Imaging

Journal: The Journal of Biological Chemistry

Article Title: Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF–ERK1/2 signaling pathways

doi: 10.1016/j.jbc.2022.101675

Figure Lengend Snippet: Western blot analysis of ERK1/2 knockdown. Epidermal growth factor-treated Hs578T cells were transfected with shERK1, shERK2, or shControl Scrambled and subsequently selected in the presence of puromycin (1 μg/ml). A , Western blot analysis showed the knockdown of total ERK1/2, but no change was detected for pERK1/2 in the presence of shERK1/2. B and C , expression levels of total ERK1/2 and phosphorylated ERK1/2 (pERK1/2) were normalized based on load controls (β-actin), respectively. The error bars represent the SDs of at least three independent experiments. Statistical analyses were performed using two-sample two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01. N.S.; not significant. ERK, extracellular signal-regulated kinase.

Article Snippet: Hs578T cells were transfected with shRNAs targeting ERK1/2 (shERK1 and shERK2) and a control shRNA with a scrambled sequence (shControl Scrambled , CGCGAAGTCTGTACTCTTG, Addgene, Cat# 65232) ( ) using Lipofectamine 2000.

Techniques: Western Blot, Transfection, Expressing, Two Tailed Test

Journal: Molecular cell

Article Title: Glutaminolysis activates Rag-mTORC1 signaling.

doi: 10.1016/j.molcel.2012.05.043

Figure Lengend Snippet: Figure 3. Inhibition of Glutaminolysis Inhibits the Translocation of mTOR to the Lysosome (A) Lysosomal localization of mTORC1 in leucine and glutamine (LQ)- stimulated U2OS cells transfected with either nontargeting siRNA (siCtrl) or with siRNA against GLS or GDH.

Article Snippet: Plasmid expressing FLAG-Rheb-N153T (RhebGTP) (Addgene plasmid 19997), plasmid containing nontargeting shRNA (shCtrl, Addgene plasmid 1864), pRK5-HA GST RagB WT plasmid expressing wild-type RagB (Addgene plasmid 19301), and pRK5-HA GST RagB 99L plasmid expressing GTP-bound mutant of RagB (Addgene plasmid 19303) were obtained from Addgene.

Techniques: Inhibition, Translocation Assay, Transfection

Journal: Molecular cell

Article Title: Glutaminolysis activates Rag-mTORC1 signaling.

doi: 10.1016/j.molcel.2012.05.043

Figure Lengend Snippet: Figure 5. Glutaminolysis Increases GTP Loading of RagB (A and B) U2OS cells were cotransfected with a GST-RagB-expressing plasmid plus either nontargeting siRNA (siCtrl) or siRNAs targeting GLS and GDH. Forty-eight hours after transfection, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ), as indicated. After GST-RagB pull-down, radiolabeled GTP and GDP were analyzed by TLC (A) and quantified using ImageJ software (B). (C) HeLa cells were cotransfected with either empty vector or GST-RagB-GTP and GST-RagC-GDP-expressing plasmid plus either nontargeting siRNA (siCtrl) or siRNAs targeting GLS and GDH. Forty-eight hours after transfection, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ), as indicated. Phosphorylation of S6K was measured by immunoblotting. (D) HeLa cells were transfected with either empty vector or GST-RagB-GTP and GST-RagC-GDP. Forty-eight hours later, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ) either in the presence or absence of DON, as indicated. Phosphorylation of S6K was then measured by immunoblotting. The mean is shown; error bars represent SEM (n > 3, *p < 0.05).

Article Snippet: Plasmid expressing FLAG-Rheb-N153T (RhebGTP) (Addgene plasmid 19997), plasmid containing nontargeting shRNA (shCtrl, Addgene plasmid 1864), pRK5-HA GST RagB WT plasmid expressing wild-type RagB (Addgene plasmid 19301), and pRK5-HA GST RagB 99L plasmid expressing GTP-bound mutant of RagB (Addgene plasmid 19303) were obtained from Addgene.

Techniques: Expressing, Plasmid Preparation, Transfection, Software, Phospho-proteomics, Western Blot

Journal: PLoS Pathogens

Article Title: The herpesvirus accessory protein γ 1 34.5 facilitates viral replication by disabling mitochondrial translocation of RIG-I

doi: 10.1371/journal.ppat.1009446

Figure Lengend Snippet: (A) Validation of RIG-I knockdown in HEL cells. Cell lysates from HELs expressing control shRNA (shCtrl) or RIG-I target shRNA (shRIG-I) were subjected to western blot analysis with anti-RIG-I and β-actin antibodies. (B) Effects of γ 1 34.5 on antiviral gene expression in control or RIG-I knockdown HEL cells. Cells infected with wild type HSV-1 or Δγ 1 34.5 (5 pfu/cell) for 8 h were analyzed for transcript levels of IFN-β, Ifit1, Ifit2, and Ccl5 by quantitative PCR analysis. The data were statistically analyzed by one-way ANOVA (**, P < 0.01) with SD (n = 3). (C) Effects of γ 1 34.5 on IRF3 phosphorylation in shCtrl-transfected HEL or RIG-I knockdown HEL. Cells were infected as described in panel B and processed for Western blot analysis with antibodies against p-IRF3, IRF3, ICP27, γ 1 34.5 and β-actin. The experimental data are representative of results from three independent experiments.

Article Snippet: HEL stably expressed Non-Target shRNA (shCtrl) or RIG-I target shRNA (shRIG-I) were selected with puromycin (sc-205821, Santa Cruz Biotechnology) at the concentration 3μg/ml.

Techniques: Biomarker Discovery, Knockdown, Expressing, Control, shRNA, Western Blot, Gene Expression, Infection, Real-time Polymerase Chain Reaction, Phospho-proteomics, Transfection

Journal: PLoS Pathogens

Article Title: The herpesvirus accessory protein γ 1 34.5 facilitates viral replication by disabling mitochondrial translocation of RIG-I

doi: 10.1371/journal.ppat.1009446

Figure Lengend Snippet: (A) Viral replication in Rig-I +/+ or Rig-I -/- MEFs. Cells were infected with wild-type HSV-1 or the γ 1 34.5 deletion virus (Δγ 1 34.5) at a MOI 0.01. At 48 h postinfection, virus yields were determined on Vero cells by plaque assay. (B) Kinetics of viral growth in Rig-I +/+ or Rig-I -/- MEFs. Viral infection was performed as described for panel (A) and viral yields were measured at the indicated time points. (C) Viral replication in control and RIG-I knockdown human lung fibroblasts cells. shCtrl (control) or shRIG-I (RIG-I knockdown) HEL cells were infected with wild type HSV-1 or Δγ 1 34.5 (0.01 pfu/cell). At 48 h postinfection, virus yields were determined by plaque assay. (D) Kinetics of viral growth in control and RIG-I knockdown cells. Viral infection was performed as described in panel (C) and viral yields were measured at the indicated time points. The data are representative of results from three experiments with triplicate samples. Differences between the selected groups were statistically assessed by one-way ANOVA (A and C) or a two-tailed Student’s t test (B and D) (**, P < 0.01).

Article Snippet: HEL stably expressed Non-Target shRNA (shCtrl) or RIG-I target shRNA (shRIG-I) were selected with puromycin (sc-205821, Santa Cruz Biotechnology) at the concentration 3μg/ml.

Techniques: Infection, Virus, Plaque Assay, Control, Knockdown, Two Tailed Test