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

Journal: bioRxiv

Article Title: Axonal transport of Hrs is activity-dependent and rate limiting for synaptic vesicle protein degradation

doi: 10.1101/2020.04.16.044818

Figure Lengend Snippet: (A) Schematic diagram of transport assay utilizing FKBP-tagged BicD2 motor domain, FRB- tagged Kinesin cargo-binding tail domain, and fluorescently-tagged cargo of interest. Upon addition of the rapamycin analogue, FKBP and FRB interact and are co-transported to the centriole. If the protein/vesicle of interest interacts with the Kinesin tail, it is also transported. (B) Representative images of N2a cells expressing RFP-Hrs (red), BicD2-GFP-FKBP (green), and KIF13A-FRB-Myc (blue), +/- rapamycin analogue to induce FKBP/FRB interaction (linker). Size bar, 10 µm. (C) Line scan graphs of the images in panel A , showing that addition of the linker induces colocalization of Hrs with BicD2 and KIF13A as depicted by a single peak in the lower graph. (D-E) Representative images of N2a cells expressing RFP-Hrs (red), BicD2-GFP-FKBP (green), and KIF13B-FRB-Myc (blue), +/- linker ( D ), and corresponding line scan graphs ( E ) showing that addition of linker does not induce Hrs colocalization with BicD2/KIF13B. (F) Immunoblot of lysates from N2a cells coexpressing mCherry or RFP-Hrs together with KIF13A- FRB-Myc or KIF13B-FRB-Myc, immunoprecipitated (IP) with mCherry antibody and probed with Myc or mCherry antibodies. (G) Quantitative analysis of co-immunoprecipitated KIF13A/B, normalized to immunoprecipitated RFP-Hrs and expressed as intensity normalized to KIF13B (****P<0.0001, unpaired t-test, n= 5 independent experiments). Bars show mean ± SEM.

Article Snippet: To create pFU-EGFP-R183A Hrs-W, an 868 nucleotides fragment of Hrs containing the R183A mutation was synthesized at Genewiz and subcloned into the BsrGI/PshAI sites. shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (catalog #TL706020).

Techniques: Transport Assay, Binding Assay, Expressing, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Axonal transport of Hrs is activity-dependent and rate limiting for synaptic vesicle protein degradation

doi: 10.1101/2020.04.16.044818

Figure Lengend Snippet: (A-B) Representative images for proximity ligation assay (PLA) in 13-15 DIV hippocampal neurons expressing EGFP ( A ) or EGFP-Hrs ( B ) under control or 2hrs Bic/4AP conditions. PLA puncta (red), representing EGFP-Hrs/KIF13A interactions, were quantified in the soma (white rectangles). Size bar, 10 μm. (C) PLA puncta number per soma, showing a significant increase in Hrs/KIF13A interactions following 2hrs Bic/4AP treatment (*P=0.0129, ****P<0.0001, one-way ANOVA with Sidak’s multiple comparisons test; n=13 for each EGFP control conditions, n=9 for each EGFP-Hrs conditions, ≥3 independent experiments). (D) Immunoblot of lysates from N2a cells coexpressing RFP-Hrs and KIF13A-FRB-Myc, treated with control or high K+ Tyrodes solution for 2hrs, immunoprecipitated (IP) with mCherry antibody and probed with Myc or mCherry antibodies. (E) Quantitative analysis of co-immunoprecipitated KIF13A, normalized to immunoprecipitated RFP-Hrs and expressed as intensity normalized to control condition (*P=0.0249, unpaired t-test, 4 independent experiments). Bars show mean ± SEM.

Article Snippet: To create pFU-EGFP-R183A Hrs-W, an 868 nucleotides fragment of Hrs containing the R183A mutation was synthesized at Genewiz and subcloned into the BsrGI/PshAI sites. shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (catalog #TL706020).

Techniques: Proximity Ligation Assay, Expressing, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Axonal transport of Hrs is activity-dependent and rate limiting for synaptic vesicle protein degradation

doi: 10.1101/2020.04.16.044818

Figure Lengend Snippet: (A) Representative images and corresponding kymographs of EGFP-Hrs in microfluidically isolated axons expressing shCtrl under control conditions (upper panels) and after 2hrs treatment with Bic/4AP (lower panels)(see Video 8&9). (B) Representative images and corresponding kymographs of EGFP-Hrs in microfluidically isolated axons expressing shKIF13A1 under control conditions (upper panels) and after 2hrs treatment with Bic/4AP (lower panels)(see Video 10&11). Horizontal size bar =10 µm, vertical size bar =25 s. 1 frame taken every 5 s (0.2 fps). Dashed yellow lines highlight axons. (C) Percentage of motile Hrs puncta in axons, showing that shKIF13A1 prevents the increase in motility induced by 2hrs Bic/4AP treatment (***P=0.0004, ns P=0.9985, one-way ANOVA with Sidak’s multiple comparisons test; ≥3 separate experiments, n=14 (shCtrl control), 16 (shCtrl 2hrs Bic/4AP), 16 (shKIF13A1 control), 15 (shKIF13A1 2hrs Bic/4AP) videos/condition). (D) Percentage of motile Hrs puncta in axons expressing shCtrl, shKIF13A2 or shKIF13B under control or 2hrs Bic/4AP treatment. Expression of shKIF13A2, but not shKIF13B, prevents the increase in motility induced by 2hrs Bic/4AP treatment (****P<0.0001, ns P=0.0823, one-way ANOVA with Sidak’s multiple comparisons test; 3 separate experiments, n≥20 videos/condition). Bars show mean ± SEM.

Article Snippet: To create pFU-EGFP-R183A Hrs-W, an 868 nucleotides fragment of Hrs containing the R183A mutation was synthesized at Genewiz and subcloned into the BsrGI/PshAI sites. shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (catalog #TL706020).

Techniques: Isolation, Expressing

Journal: bioRxiv

Article Title: Axonal transport of Hrs is activity-dependent and rate limiting for synaptic vesicle protein degradation

doi: 10.1101/2020.04.16.044818

Figure Lengend Snippet: (A-C) Representative images of axons expressing mCh-Hrs/shCtrl ( A ), mCh-Hrs/shKIF13A1 ( B ), or mCh-Hrs/shKIF13A2 ( C ) together with EGFP-Synapsin, under control conditions (left panels) or after 20hrs treatment with Bic/4AP (right panels). Size bar, 10 µm. (D) Percentage of Hrs puncta colocalized with Synapsin under the conditions shown in A-C, demonstrating that KIF13A knockdown prevents activity-dependent recruitment of Hrs to SV pools (****P<0.0001, ns P=0.0823, one-way ANOVA with Sidak’s multiple comparisons test; 3 separate experiments, n≥20 videos/condition). (E) Representative immunoblots of SV2 and VAMP2, with corresponding tubulin loading controls, from lysates of 14 DIV hippocampal neurons expressing shCtrl or shKIF13A1 and treated for 24hrs with DMSO control (CON) or cycloheximide (CHX). (F-G) Quantification of the fold-change in SV2 ( F ) and VAMP2 ( G ) degradation under these conditions, calculated as previously described . Degradation of both proteins is significantly decreased in the presence of shKIF13A1 (*P=0.0191 (SV2), *P=0.0161 (VAMP2), unpaired t-test; ≥3 separate experiments, n = 5 (SV2), 11 (VAMP2) cell lysates/condition). All scatter plots show mean ± SEM.

Article Snippet: To create pFU-EGFP-R183A Hrs-W, an 868 nucleotides fragment of Hrs containing the R183A mutation was synthesized at Genewiz and subcloned into the BsrGI/PshAI sites. shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (catalog #TL706020).

Techniques: Expressing, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Axonal transport of Hrs is activity-dependent and rate limiting for synaptic vesicle protein degradation

doi: 10.1101/2020.04.16.044818

Figure Lengend Snippet: Neuronal firing increases the association of Hrs with plus-end directed kinesin motor protein KIF13A, stimulating the anterograde transport of these vesicles and their delivery to SV pools. Hrs+ vesicles also transport STAM1 and are a subset of Rab5+ early endosomes.

Article Snippet: To create pFU-EGFP-R183A Hrs-W, an 868 nucleotides fragment of Hrs containing the R183A mutation was synthesized at Genewiz and subcloned into the BsrGI/PshAI sites. shRNA scrambled control and KIF13A knockdown constructs (shCtrl, shKIF13A1 and A2, KIF13B) were purchased from Origene (catalog #TL706020).

Techniques: