Journal: Microbiology Spectrum

Article Title: Identification of IMP Dehydrogenase as a Potential Target for Anti-Mpox Virus Agents

doi: 10.1128/spectrum.00566-23

Figure Lengend Snippet: Essential role of IMPDH in the anti-MPXV activity of MPA. (A) Schematic representation of the guanosine nucleotide synthesis pathway. In the de novo pathway, an initial substrate, i.e., glucose, is converted to ribose-5-phosphate (R5P), phosphoribosyl diphosphate (PRPP), and then to IMP (IMP) in a stepwise manner. IMPDH catalyzes the conversion from IMP to xanthine monophosphate (XMP) as a rate-limiting step of the pathway. XMP is converted through GMP and GDP to GTP. In the salvage pathway, GMP is also produced from guanine. (B) Huh7 cells were transfected with or without (−) siRNA targeting IMPDH (IMPDH) or randomized control siRNA (control). At 48 h posttransfection, intracellular RNA for IMPDH1 and IMPDH2 and the protein expression for IMPDH1, IMPDH2, and β-actin were detected by real-time RT-PCR (i) and immunoblot analysis (ii), respectively. The y axis in panel Bi shows the value relative to that for the untransfected cells. Upper, middle, and lower panels in Bii show the protein production for IMPDH1, IMPDH2, and β-actin, respectively. The positions for the molecular weight markers (62, 49, and 38 kDa) are also shown. Intracellular MPXV DNA levels at 72 h posttransfection with siRNA were quantified by real-time PCR and are shown as the percentage relative to that of the untransfected cells (iii). (C) Huh7 cells infected with the same amount of MPXV as inoculum in Fig. 1B were treated with or without 5 μM MPA and supplemented with or without varying amounts of guanosine (12.5, 25, and 50 μM). After 24 h of treatment, intracellular viral DNA was measured by real-time PCR and is shown as the value relative to that for the DMSO-treated cells. Statistical significance is shown.

Article Snippet: Anti-IMPDH1 rabbit polyclonal antibody (Invitrogen), anti-IMPDH2 polyclonal antibody (Proteintech), and anti-beta actin monoclonal antibody (Cell Signaling Technology) were used as primary antibodies.

Techniques: Activity Assay, Produced, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Infection

Journal: Cell reports

Article Title: A Purine Nucleotide Biosynthesis Enzyme Guanosine Monophosphate Reductase is a Suppressor of Melanoma Invasion

doi: 10.1016/j.celrep.2013.09.015

Figure Lengend Snippet: (A) Schematic representation of the de novo purine biosynthesis and salvage pathway. Enzymes and their products are shown by ovals and open boxes, respectively. * IMPDH 1/2 are rate-limiting enzymes and MPA targets. (B) Total cellular extracts from independently isolated populations of normal human melanocytes (NHM) and indicated melanoma cell lines were probed in western blotting with indicated antibodies. (C) Expression of GMPR and IMPDH2 in thin, thick primary melanomas and melanoma metastases. The box plots represent the distribution of the IHC index. The median, first quartile, and third quartile are shown in the “box” with outlying samples represented by points. The dashed lines represent the interquartile range (IQR) times 1.5 added to the first and third quartiles. The number of patient samples (n) is indicated for each cohort. (D) Representative IHC images for GMPR and IMPDH2 from the data presented in C. See also Figure S1.

Article Snippet: For IMPDH2 antibodies (Atlas Antibodies, HPA001400) and GMPR antibodies (Sigma-Aldrich HPA021476), the Novocastra (Newcastle,UK) PowerVision kit was used for visualization, followed by Fast Red (Thermo Scientific, Wilmington, DE).

Techniques: Isolation, Western Blot, Expressing

Journal: eLife

Article Title: SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome

doi: 10.7554/eLife.71945

Figure Lengend Snippet:

Article Snippet: Membranes were incubated with the following primary antibodies: mouse anti-Actin (Cell Signaling, 3700), rabbit anti-IMPDH2 (Proteintec, 12948-1-AP), mouse anti-Strep-tag (Quiagen, 34850).

Techniques: Transformation Assay, Clone Assay, Transfection, Construct, Plasmid Preparation, Mutagenesis, Software

Journal: PLOS Pathogens

Article Title: Epstein-Barr virus latent membrane protein 1 subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1371/journal.ppat.1013092

Figure Lengend Snippet: (A) Metabolomic experiment workflow. Conditional LMP1 Daudi or Akata Burkitt cells were mock induced or induced by doxycycline (250ng/ml) for LMP1 expression for 24h. Polar metabolites were analyzed by targeted metabolomic analysis. (B) Volcano plot analysis of liquid chromatography mass spectrometry (LC-MS) analysis of n = 3 replicates of Daudi cells mock induced or induced for LMP1 expression. Positive fold changes indicate higher metabolite concentrations in LMP1 + vs LMP1- cells. Selected host cell metabolites induced vs. suppressed by LMP1 are indicated. (C) Volcano plot analysis of LC-MS analysis of n = 6 replicates of Akata cells mock induced or induced for LMP1 expression. (D) Metaboanalyst analysis of LMP1 driven Daudi cell metabolic pathway impact from the data presented in (B), using data from significantly changed metabolites in LMP1 + vs LMP1- cells ( p value > 0.05). Higher pathway impact values indicates stronger effects of conditional LMP1 expression on the indicated pathway. Purine metabolism was amongst the most highly LMP1 impacted pathways. (E) Metaboanalyst pathway impact analysis of LMP1 + vs LMP1- Akata cells from the data presented in (C). (F) Volcano plot analysis cross-comparing fold change of metabolite abundances in LMP1+ versus LMP1- Akata cells (x-axis) vs Daudi cells (Y-axis). Shown are metabolites whose abundances were LMP1 increased by ≥1.2 fold in both cell models. Selected metabolites are annotated, including xanthosine-5-phosphate, which was highly LMP1-induced under both conditions. (G) Purine metabolism pathways. The de novo pathway uses Ribose-5-phosphate and PRPP to generate inosine monophosphate (IMP), whereas the salvage pathway metabolizes hypoxanthine into IMP. IMP can be converted by IMPDH1/2 to xanthosine monophosphate (XMP) and subsequently to guanosine monophosphate (GMP). Alternatively, IMP can be converted to adenosine monophosphate (AMP). Created in BioRender. Burton, E. (2025) https://BioRender.com/x8l0ikn .

Article Snippet: Antibodies against the following proteins were used in this study: IMPDH1 (Cell Signaling Technology, #57068), IMPDH2 (Cell Signaling Technology, # 35914S), GAPDH (EMD Millipore, MAB374), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), Fas-APC (Biolegend, 305612), ICAM-1-PE (BD Bioscience, 555511), Caspase 3 (Cell Signaling Technology, #9662), EBNA1 (a gift from Jaap Middeldorp).

Techniques: Expressing, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: PLOS Pathogens

Article Title: Epstein-Barr virus latent membrane protein 1 subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1371/journal.ppat.1013092

Figure Lengend Snippet: (A) Schematic diagram of mycophenolic acid (MPA) inhibition of the guanylate biosynthesis pathway enzymes IMPDH1 and IMPDH2. IMP, inosine monophosphate. XMP, xanthosine monophosphate, GMP, guanosine monophosphate. GDP, guanosine diphosphate. GTP, guanosine triphosphate. (B) FACS analysis of dose-dependent MPA effects on proliferation of latency I P3HR-1 and MUTU I cells versus latency III GM12878 and GM12881 LCLs, as judged by CFSE dye-dilution analysis. CFSE-stained cells were incubated with DMSO vehicle vs the indicated MPA concentrations for 96 hours and CFSE mean fluorescence intensity (MFI) was analyzed by FACS. CFSE levels are reduced by half with each mitosis. Shown are mean ± SD CFSE levels from n = 3 independent replicates. (C) FACS analysis of dose-dependent effects of MPA treatment for 48 hours on cell death of latency I P3HR-1 and MUTU I cells versus latency III GM12878 and GM12881 LCLs, as judged by uptake of the vital dye 7-AAD. Shown are mean ± SD percentages of 7-AAD+ (non-viable) cells from n = 3 independent replicates. (D) FACS analysis of DMSO versus MPA effects on viability of isogenic MUTU I versus III cells that differ by EBV latency I versus III programs, respectively. Shown are mean ± SD percentages of 7-AAD+ cells following DMSO versus 1 μM MPA treatment for 48 hours. (E) FACS analysis of DMSO versus MPA effects on P3HR-1 versus Jijoye Burkitt cell viability following DMSO versus 1 μM MPA treatment for 48 hours. Mean ± SD 7-AAD+ cell percentages from n = 3 replicates are shown. (F) FACS analysis of mean ± SD percentages of 7-AAD + /Annexin V+ cells following treatment with DMSO, 1 μM MPA with or without 100 μM GTP rescue for 48 hours. (G) Relative caspase 3/7 activity levels of cells analyzed in panel (F), as judged by Caspase3/7 Glo assay. Mean ± SD values from n = 3 replicates are shown. *, P < 0.05; **, P < 0.05; ***, P < 0.005; ns, nonsignificant using Student’s t-test.

Article Snippet: Antibodies against the following proteins were used in this study: IMPDH1 (Cell Signaling Technology, #57068), IMPDH2 (Cell Signaling Technology, # 35914S), GAPDH (EMD Millipore, MAB374), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), Fas-APC (Biolegend, 305612), ICAM-1-PE (BD Bioscience, 555511), Caspase 3 (Cell Signaling Technology, #9662), EBNA1 (a gift from Jaap Middeldorp).

Techniques: Inhibition, Staining, Incubation, Fluorescence, Activity Assay, Glo Assay

Journal: PLOS Pathogens

Article Title: Epstein-Barr virus latent membrane protein 1 subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1371/journal.ppat.1013092

Figure Lengend Snippet: (A) Mean ± SD live cell numbers of Cas9 + MUTU I expressing control, IMPDH1 or IMPDH2 targeting single guide RNAs (sgRNA) from n = 3 replicates. Cells transduced with lentiviruses expressing the indicated sgRNAs were puromycin selected. Cell numbers immediately following puromycin selection (defined as day 0 of the graph) were set to 1. Live cell numbers were quantitated by CellTiter-Glo assay. (B) Mean ± SD live cell numbers of Cas9 + Daudi cells as in (A). (C) Mean ± SD live cell numbers of Cas9 + GM12878 LCLs as in (A). (D) Mean ± SD live cell numbers of Cas9 + GM13111 LCLs as in (A). (E) Mean ± SD live cell numbers of Cas9 + P3HR-1 or GM12878 cells transduced with lentivirus expressing control, IMPDH1, IMPDH2 or IMPDH1 and 2 sgRNAs at 8 days post-puromycin selection. (F) Immunoblot analysis of WCL from Cas9 + P3HR-1 or GM12878 expressing the indicated sgRNA. * = non-specific band present in analysis of GM12878 lysates. Immunoblots are representative of n = 3 independent replicates. (G) Mean ± SD MFI of Cas9 + P3HR-1 or GM12878 cells transduced with lentivirus expressing control, IMPDH1, IMPDH2 or IMPDH1 and 2 sgRNAs at 8 days post-puromycin selection, performed on cells from the same replicates shown in (E).

Article Snippet: Antibodies against the following proteins were used in this study: IMPDH1 (Cell Signaling Technology, #57068), IMPDH2 (Cell Signaling Technology, # 35914S), GAPDH (EMD Millipore, MAB374), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), Fas-APC (Biolegend, 305612), ICAM-1-PE (BD Bioscience, 555511), Caspase 3 (Cell Signaling Technology, #9662), EBNA1 (a gift from Jaap Middeldorp).

Techniques: Expressing, Control, Transduction, Selection, Glo Assay, Western Blot

Journal: PLOS Pathogens

Article Title: Epstein-Barr virus latent membrane protein 1 subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1371/journal.ppat.1013092

Figure Lengend Snippet: In Latency I, IMPDH1 and 2 each contribute to production of XMP and downstream guanylates to support demand. IMPDH1/2 inhibition by MPA triggers Burkitt growth arrest and de-represses EBV lytic antigens. In Latency III, LMP1 activated IMPDH2 predominantly supports XMP production and guanylate synthesis, sensitizing LMP1-expressing cells to MPA-driven killing. Created in BioRender. Burton, E. (2025) https://BioRender.com/76p9mp7 .

Article Snippet: Antibodies against the following proteins were used in this study: IMPDH1 (Cell Signaling Technology, #57068), IMPDH2 (Cell Signaling Technology, # 35914S), GAPDH (EMD Millipore, MAB374), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), Fas-APC (Biolegend, 305612), ICAM-1-PE (BD Bioscience, 555511), Caspase 3 (Cell Signaling Technology, #9662), EBNA1 (a gift from Jaap Middeldorp).

Techniques: Inhibition, Expressing

Journal: PLOS Pathogens

Article Title: Epstein-Barr virus latent membrane protein 1 subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1371/journal.ppat.1013092

Figure Lengend Snippet: (A) Schematic diagram of mycophenolic acid (MPA) inhibition of the guanylate biosynthesis pathway enzymes IMPDH1 and IMPDH2. IMP, inosine monophosphate. XMP, xanthosine monophosphate, GMP, guanosine monophosphate. GDP, guanosine diphosphate. GTP, guanosine triphosphate. (B) FACS analysis of dose-dependent MPA effects on proliferation of latency I P3HR-1 and MUTU I cells versus latency III GM12878 and GM12881 LCLs, as judged by CFSE dye-dilution analysis. CFSE-stained cells were incubated with DMSO vehicle vs the indicated MPA concentrations for 96 hours and CFSE mean fluorescence intensity (MFI) was analyzed by FACS. CFSE levels are reduced by half with each mitosis. Shown are mean ± SD CFSE levels from n = 3 independent replicates. (C) FACS analysis of dose-dependent effects of MPA treatment for 48 hours on cell death of latency I P3HR-1 and MUTU I cells versus latency III GM12878 and GM12881 LCLs, as judged by uptake of the vital dye 7-AAD. Shown are mean ± SD percentages of 7-AAD+ (non-viable) cells from n = 3 independent replicates. (D) FACS analysis of DMSO versus MPA effects on viability of isogenic MUTU I versus III cells that differ by EBV latency I versus III programs, respectively. Shown are mean ± SD percentages of 7-AAD+ cells following DMSO versus 1 μM MPA treatment for 48 hours. (E) FACS analysis of DMSO versus MPA effects on P3HR-1 versus Jijoye Burkitt cell viability following DMSO versus 1 μM MPA treatment for 48 hours. Mean ± SD 7-AAD+ cell percentages from n = 3 replicates are shown. (F) FACS analysis of mean ± SD percentages of 7-AAD + /Annexin V+ cells following treatment with DMSO, 1 μM MPA with or without 100 μM GTP rescue for 48 hours. (G) Relative caspase 3/7 activity levels of cells analyzed in panel (F), as judged by Caspase3/7 Glo assay. Mean ± SD values from n = 3 replicates are shown. *, P < 0.05; **, P < 0.05; ***, P < 0.005; ns, nonsignificant using Student’s t-test.

Article Snippet: Antibodies against the following proteins were used in this study: IMPDH1 (Cell Signaling Technology, #57068), IMPDH2 (Cell Signaling Technology, # 35914S), GAPDH (EMD Millipore, MAB374), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), Fas-APC (Biolegend, 305612), ICAM-1-PE (BD Bioscience, 555511), Caspase 3 (Cell Signaling Technology, #9662), EBNA1 (a gift from Jaap Middeldorp).

Techniques: Inhibition, Staining, Incubation, Fluorescence, Activity Assay, Glo Assay

Journal: PLOS Pathogens

Article Title: Epstein-Barr virus latent membrane protein 1 subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1371/journal.ppat.1013092

Figure Lengend Snippet: (A) Mean ± SD live cell numbers of Cas9 + MUTU I expressing control, IMPDH1 or IMPDH2 targeting single guide RNAs (sgRNA) from n = 3 replicates. Cells transduced with lentiviruses expressing the indicated sgRNAs were puromycin selected. Cell numbers immediately following puromycin selection (defined as day 0 of the graph) were set to 1. Live cell numbers were quantitated by CellTiter-Glo assay. (B) Mean ± SD live cell numbers of Cas9 + Daudi cells as in (A). (C) Mean ± SD live cell numbers of Cas9 + GM12878 LCLs as in (A). (D) Mean ± SD live cell numbers of Cas9 + GM13111 LCLs as in (A). (E) Mean ± SD live cell numbers of Cas9 + P3HR-1 or GM12878 cells transduced with lentivirus expressing control, IMPDH1, IMPDH2 or IMPDH1 and 2 sgRNAs at 8 days post-puromycin selection. (F) Immunoblot analysis of WCL from Cas9 + P3HR-1 or GM12878 expressing the indicated sgRNA. * = non-specific band present in analysis of GM12878 lysates. Immunoblots are representative of n = 3 independent replicates. (G) Mean ± SD MFI of Cas9 + P3HR-1 or GM12878 cells transduced with lentivirus expressing control, IMPDH1, IMPDH2 or IMPDH1 and 2 sgRNAs at 8 days post-puromycin selection, performed on cells from the same replicates shown in (E).

Article Snippet: Antibodies against the following proteins were used in this study: IMPDH1 (Cell Signaling Technology, #57068), IMPDH2 (Cell Signaling Technology, # 35914S), GAPDH (EMD Millipore, MAB374), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), Fas-APC (Biolegend, 305612), ICAM-1-PE (BD Bioscience, 555511), Caspase 3 (Cell Signaling Technology, #9662), EBNA1 (a gift from Jaap Middeldorp).

Techniques: Expressing, Control, Transduction, Selection, Glo Assay, Western Blot

Journal: PLOS Pathogens

Article Title: Epstein-Barr virus latent membrane protein 1 subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1371/journal.ppat.1013092

Figure Lengend Snippet: In Latency I, IMPDH1 and 2 each contribute to production of XMP and downstream guanylates to support demand. IMPDH1/2 inhibition by MPA triggers Burkitt growth arrest and de-represses EBV lytic antigens. In Latency III, LMP1 activated IMPDH2 predominantly supports XMP production and guanylate synthesis, sensitizing LMP1-expressing cells to MPA-driven killing. Created in BioRender. Burton, E. (2025) https://BioRender.com/76p9mp7 .

Article Snippet: Antibodies against the following proteins were used in this study: IMPDH1 (Cell Signaling Technology, #57068), IMPDH2 (Cell Signaling Technology, # 35914S), GAPDH (EMD Millipore, MAB374), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), Fas-APC (Biolegend, 305612), ICAM-1-PE (BD Bioscience, 555511), Caspase 3 (Cell Signaling Technology, #9662), EBNA1 (a gift from Jaap Middeldorp).

Techniques: Inhibition, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Glutamine deprivation initiates reversible assembly of mammalian rods and rings

doi: 10.1007/s00018-014-1567-6

Figure Lengend Snippet: Purine and pyrimidine biosynthesis pathways and rods and rings. a Purine and pyrimidine de novo biosynthesis pathways. Enzymes in these pathways that are known RR components are shown in red and steps requiring glutamine in blue. In the steps eventually leading to GTP formation, IMPDH2 catalyzes the conversion of IMP to XMP, which is converted to GMP in the glutamine-dependent GMP synthase reaction. CTPS1 is an enzyme involved in the CTP biosynthesis pathway and catalyzes the conversion of UTP to CTP. Arrows and numbers signify steps in the pathway. The pathway schematic is adapted from Hofer et al. [29]. b, c Antibodies from prototype anti-RR serum It2006 recognize typical rod (~3–10 μm in length) and ring (~2–5 μm in diameter) structures in the cytoplasm of HeLa cells. Nuclei are counterstained with DAPI

Article Snippet: Western blotting The expression levels of CTPS and IMPDH were analyzed using rabbit anti-CTPS (Abcam 1:1,000) and rabbit anti-IMPDH2 (Santa Cruz Biotechnology, Paso Robles, CA, USA 1:1,000; Proteintech, Chicago, IL, USA 12948-1-AP, 1:1,000) as described [ 28 ].

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Glutamine deprivation initiates reversible assembly of mammalian rods and rings

doi: 10.1007/s00018-014-1567-6

Figure Lengend Snippet: Glutamine deprivation induces rods and rings. HeLa cells maintained in DMEM medium with 10 % FBS without glutamine (a), DMEM medium with 1 % FBS without glutamine (b), DMEM medium with 10 % FBS (c), and DMEM medium with 1 % FBS (d) for 48 h were stained with rabbit anti-IMPDH2 (red). Nuclei were counterstained with DAPI (blue). The average percentage of cells with RR are displayed in the lower right corner, with total number of cells counted in parentheses. e mRNA levels of CTPS1 and IMPDH2 are significantly elevated in cells depleted of glutamine. mRNA levels were normalized to GAPDH. f Western blot of CTPS1 and IMPDH2 protein levels using GAPDH as a loading control in HeLa cells maintained in 10 % FBS medium with glutamine or without glutamine. There was an increase (56 %) in IMPDH2 protein levels after glutamine deprivation in 10 % FBS compared with cells maintained in DMEM medium with 10 % FBS. Data are from three independent experiments (a–d mean, e mean ± SD). * p < 0.05 (two-tailed unpaired t test). Scale bar 10 μm

Article Snippet: Western blotting The expression levels of CTPS and IMPDH were analyzed using rabbit anti-CTPS (Abcam 1:1,000) and rabbit anti-IMPDH2 (Santa Cruz Biotechnology, Paso Robles, CA, USA 1:1,000; Proteintech, Chicago, IL, USA 12948-1-AP, 1:1,000) as described [ 28 ].

Techniques: Staining, Western Blot, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Glutamine deprivation initiates reversible assembly of mammalian rods and rings

doi: 10.1007/s00018-014-1567-6

Figure Lengend Snippet: Time-course of RR formation during glutamine starvation. HeLa cells were maintained in normal DMEM medium with 10 % FBS (a–c), or in glutamine-depleted medium with 10 % FBS for 1 day (d–f), 2 days (g–i), or 3 days (j–l). Cells were co-stained with human anti-RR serum It2006 (green c, f, i, l) and rabbit anti-IMPDH2 (red b, e, h, k). Merged images are shown in a, d, g, and j. Nuclei were counterstained with DAPI (blue). m Summary of the percentage of cells with short RR alone, short and long RR, and long RR alone for each day of glutamine starvation and control. Total number of cells counted are shown in parentheses

Article Snippet: Western blotting The expression levels of CTPS and IMPDH were analyzed using rabbit anti-CTPS (Abcam 1:1,000) and rabbit anti-IMPDH2 (Santa Cruz Biotechnology, Paso Robles, CA, USA 1:1,000; Proteintech, Chicago, IL, USA 12948-1-AP, 1:1,000) as described [ 28 ].

Techniques: Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Glutamine deprivation initiates reversible assembly of mammalian rods and rings

doi: 10.1007/s00018-014-1567-6

Figure Lengend Snippet: Glutamine replenishment results in disassembly of RR previously formed in response to glutamine deprivation. a–c RR were not detected in HeLa cells maintained in normal DMEM medium with 10 % FBS. d–f RR formed in cells maintained in glutamine-deprived medium for 72 h. g–i Glutamine deprivation-induced RR disassembled when cells were supplemented with 2.5 mM glutamine for 15 min. Scale bar 10 μm. j mRNA levels of CTPS1 and IMPDH2 were elevated in glutamine-deprived cells and returned to apparently lower levels in cells supplemented with 2.5 mM glutamine after 24 h. mRNA levels were normalized to GAPDH

Article Snippet: Western blotting The expression levels of CTPS and IMPDH were analyzed using rabbit anti-CTPS (Abcam 1:1,000) and rabbit anti-IMPDH2 (Santa Cruz Biotechnology, Paso Robles, CA, USA 1:1,000; Proteintech, Chicago, IL, USA 12948-1-AP, 1:1,000) as described [ 28 ].

Techniques: