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: PloS one

Article Title: Lactate activates HIF-1 in oxidative but not in Warburg-phenotype human tumor cells.

doi: 10.1371/journal.pone.0046571

Figure Lengend Snippet: Figure 2. Lactate inhibits PHD2 activity in oxidative tumor cells. (A) HIF-1a and b-actin protein expression was detected using Western blotting in the lysates of SiHa TCs incubated during 24-h with 10 mM lactate or not and increasing doses of 2-oxoglutarate. The upper panels show representative experiments and the graph HIF-1a protein expression normalized to b-actin levels. Data are expressed as % of lactate induction. **p,0.01, ***p,0.005 versus 10 mM lactate without 2-oxoglutarate; n = 8. (B) ODD-driven luciferase activity was measured in SiHa TCs treated during 24-h with 10 mM lactate or not. *p = 0.0206; n = 6. (C) HIF-1a and b-actin protein expression was detected using Western blotting in the lysates of SiHa TCs transfected with a specific siRNA against PHD2 (siPHD2) or with a control siRNA (siCTR) and incubated during 24-h with 10 mM lactate or not. The upper panels show representative experiments and the graph HIF-1a protein expression normalized to b-actin levels. ns, p.0.05, *p,0.05; n = 3. doi:10.1371/journal.pone.0046571.g002

Article Snippet: Control shRNA (shCTR) was Addgene plasmid 1864.

Techniques: Activity Assay, Expressing, Western Blot, Incubation, Luciferase, Transfection, Control

Journal: PloS one

Article Title: Lactate activates HIF-1 in oxidative but not in Warburg-phenotype human tumor cells.

doi: 10.1371/journal.pone.0046571

Figure Lengend Snippet: Figure 5. Targeting MCT1 inhibits lactate-induced but not basal HIF-1 activity in tumor cells. (A) SiHa TCs were cultured during 24-h in fresh medium containing 10 mM lactate, lactate +5 mM a-cyano-4-hydroxycinnamate (CHC), or none of the drugs. HIF-1a and b-actin were detected using Western blotting. The upper panels show a representative experiment and the graph shows HIF-1a protein expression normalized to b-actin. ***p,0.005 versus control; ###p,0.005 versus lactate alone; n = 3–8. (B) As in (A) but with WiDr TCs. ns, p.0.05 versus control; n = 3–8. (C–F) TCs were infected with a control shRNA (shCTR, left panels) or with a specific shRNA targeting MCT1 (shMCT1-1, right panels). The cells were then cultured during 24-h in the presence of 10 mM lactate or not (control), after which HIF-1 activity was quantified using a dual reporter luciferase assay. The assay was performed using (C) SiHa (n = 5–7), (D) HeLa (n = 3–4), (E) FaDu (n = 5–8), and (F) WiDr (n = 4–5) TCs. ns, p.0.05, *p,0.05, ** p,0.01, ***p,0.005 versus control. doi:10.1371/journal.pone.0046571.g005

Article Snippet: Control shRNA (shCTR) was Addgene plasmid 1864.

Techniques: Activity Assay, Cell Culture, Western Blot, Expressing, Control, Infection, shRNA, Luciferase