microencapsulated bifidobacterium bifidum atcc 35914  (ATCC)

Journal: bioRxiv

Article Title: Epstein-Barr Virus Latent Membrane Protein 1 Subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1101/2024.11.07.622457

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. Double 7-AAD/annexin V positivity indicates apoptosis. (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 Milipore, 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 Middledorp).

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

Journal: bioRxiv

Article Title: Epstein-Barr Virus Latent Membrane Protein 1 Subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1101/2024.11.07.622457

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 expresing 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 Milipore, 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 Middledorp).

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

Journal: bioRxiv

Article Title: Epstein-Barr Virus Latent Membrane Protein 1 Subverts IMPDH pathways to drive B-cell oncometabolism

doi: 10.1101/2024.11.07.622457

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 apoptosis.

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 Milipore, 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 Middledorp).

Techniques: Inhibition, Expressing