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