Journal: Nature communications

Article Title: Transcriptional and epigenetic rewiring by the NUP98::KDM5A fusion oncoprotein directly activates CDK12.

doi: 10.1038/s41467-025-59930-9

Figure Lengend Snippet: Fig. 7 | CDK12-controlled DNA repair is a vulnerability in NUP98::KDM5A-driven leukemia. A Cell viability assay of primary human NUP98-rearranged AML cells, healthy donor BM MNC and CD34+ progenitors treated with indicated con- centrations of THZ531 for 3 days (n = 3 technical replicates). B GI50 values from cell viability assays of murine AML cells treated with THZ531 for 3 days. C RT-qPCR analysis of dTAG-NUP98::KDM5A cells treated with THZ531 (2 µM, 24 h) showing log2FC values (n = 3 technical replicates). D Gene set enrichment analysis of RNA- seq data from a doxycycline-controlled NUP98::KDM5A cell line (Tet-Off) after 5 days of doxycycline-induced NUP98::KDM5A downregulation compared to DMSO. E Time series plot of Tet-Off RNA-seq data showing gene expression levels of DNA double strand break repair genes at day 3 and day 5 after NUP98::KDM5A downregulation normalized to NUP98::KDM5A-expressing cells, highlighting selected genes (left), with a density plot of normalized expression of all genes

Article Snippet: For the generation of the CDK12 wild type overexpression construct pCR8/GW_TOPO_Cdk12_Nterm_FlagHa (Addgene plasmid #127177) expressing the mouse Cdk12 transgene (NM_001109626.1 isoform) was cloned into pLX303-Gw (Addgene plasmid #25897) using Gateway LR Clonase II EnzymeMix (Invitrogen, USA) according the manufacturer’s protocol.

Techniques: Viability Assay, Quantitative RT-PCR, RNA Sequencing, Gene Expression, Expressing

Journal: Journal of Virology

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF61 protein sequesters APOBEC3B in filamentous aggregates

doi: 10.1128/jvi.00789-25

Figure Lengend Snippet: Rhadinovirus R1 proteins accumulate in elongated cytoplasmic condensates. ( A ) Schematic representation of KSHV ORF61 containing the Induced Protein Aggregation Motif (IPAM) and an alignment of the IPAM sequences within selected herpesvirus R1 homologs, including the γ-herpesviruses EBV, KSHV, RRV (rhesus rhadinovirus), and MHV-68. Confocal microscopy images of ( B ) ARPE-19 cells infected with KSHV mNeon-ORF61 or mNeon-ORF61 mut IPAM and ( C ) 10.1 cells infected with MHV-68 ORF61- or ORF61 mut IPAM-FLAG. Cells were fixed at different times post-infection, and nuclei were counterstained with Hoechst 33342. 10.1 cells were immunostained with an anti-FLAG antibody. Fluorescence images were acquired by cLSM. Scale bar, 20 µm.

Article Snippet: To generate pcDNA3-KSHV ORF61 mut IPAM-3xFLAG, the IPAM within ORF61 (aa positions 697-701) was mutated by four alanine substitutions (PFVDQ to PAAAA). pmCherry-Nucleolin ( ) and pcDNA3-MCMV M45-HA ( ) have been described. pcDNA3.1-hRIPK1-Flag (#112487) and pCMVTAG-NEMO (#11970) were purchased from Addgene.

Techniques: Confocal Microscopy, Infection, Fluorescence

Journal: Journal of Virology

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF61 protein sequesters APOBEC3B in filamentous aggregates

doi: 10.1128/jvi.00789-25

Figure Lengend Snippet: Rhadinovirus R1 proteins become insoluble during infection. ( A ) ARPE-19 cells were infected with KSHV mNeon-ORF61 or mNeon-ORF61 mut IPAM (MOI 0.1) and harvested at different times post-infection. The detergent-soluble (S) and insoluble (I) fractions were analyzed by immunoblot. GAPDH was used as a fractionation control and β-Actin as a loading control. ( B ) Murine 10.1 fibroblasts were infected with MHV-68 ORF61-FLAG or ORF61 mut IPAM-FLAG (MOI 2) and harvested at different times post-infection. Total cell lysates and the detergent-soluble (S) and insoluble (I) fractions were analyzed by immunoblot. ( C ) ARPE-19 cells were infected with KSHV mNeon-ORF61 at MOI 0.1 and NIH/3T3 fibroblasts with either MHV-68 ORF61-FLAG or MCMV M45-HA at MOI 5. At 6 hpi, cells were treated with 5 mM 3-methyladenine (3-MA) until lysate collection to inhibit autophagy or left untreated. Cell lysates were collected at 24 hpi and separated into soluble (S) and insoluble (I) fractions.

Article Snippet: To generate pcDNA3-KSHV ORF61 mut IPAM-3xFLAG, the IPAM within ORF61 (aa positions 697-701) was mutated by four alanine substitutions (PFVDQ to PAAAA). pmCherry-Nucleolin ( ) and pcDNA3-MCMV M45-HA ( ) have been described. pcDNA3.1-hRIPK1-Flag (#112487) and pCMVTAG-NEMO (#11970) were purchased from Addgene.

Techniques: Infection, Western Blot, Fractionation, Control

Journal: Journal of Virology

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF61 protein sequesters APOBEC3B in filamentous aggregates

doi: 10.1128/jvi.00789-25

Figure Lengend Snippet: Rhadinovirus R1 protein condensates are solid aggregates composed of filamentous bundles. ( A ) Fluorescence recovery after photobleaching (FRAP) analysis of mCherry-tagged R1 proteins. KSHV and MHV-68 ORF61-pmCherry were expressed in U2OS cells by plasmid transfection. mCherry-positive structures were half-bleached, and fluorescence recovery was recorded at two frames per second for 6 min. pmCherry-Nucleolin was used as a control. Scale bar, 10 µm. (B and C) Relative fluorescence intensity (RFI) of bleached areas over time in at least 10 different cells per sample. The shaded areas are the mean values ± SEM. For correlative light and electron microscopy (CLEM) of ORF61 aggregates, ( D ) ARPE-19 cells were infected with KSHV mNeon-ORF61, and ( E ) MEF cells with MHV-68 ORF61-mNeon. At 40 hpi (KSHV) or 20 hpi (MHV-68), the cells were fixed, and nuclei were stained with Hoechst 33342. Fluorescent Z-stacks were acquired by cLSM. Ultrathin 50 nm serial sections of the samples were prepared and imaged by transmission electron microscopy (TEM). Representative overview images and magnified views of the indicated areas are shown.

Article Snippet: To generate pcDNA3-KSHV ORF61 mut IPAM-3xFLAG, the IPAM within ORF61 (aa positions 697-701) was mutated by four alanine substitutions (PFVDQ to PAAAA). pmCherry-Nucleolin ( ) and pcDNA3-MCMV M45-HA ( ) have been described. pcDNA3.1-hRIPK1-Flag (#112487) and pCMVTAG-NEMO (#11970) were purchased from Addgene.

Techniques: Fluorescence, Plasmid Preparation, Transfection, Control, Electron Microscopy, Infection, Staining, Transmission Assay

Journal: Journal of Virology

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF61 protein sequesters APOBEC3B in filamentous aggregates

doi: 10.1128/jvi.00789-25

Figure Lengend Snippet: KSHV ORF61 relocalizes APOBEC3B to aggregates, whereas MHV-68 ORF61 does not relocalize human or mouse APOBECs. U2OS cells were transfected with plasmids expressing ( A ) A3B-HA or A3G-HA and KSHV ORF61-FLAG or ORF61 mut IPAM-FLAG, ( B ) A3B-HA, A3G-HA, mA1-HA, or mA3-HA and MHV-68 ORF61-FLAG. Cells were fixed, immunostained with anti-HA and anti-FLAG antibodies, and counterstained with Hoechst 33342. Fluorescence images were acquired by cLSM. Scale bar, 20 µm.

Article Snippet: To generate pcDNA3-KSHV ORF61 mut IPAM-3xFLAG, the IPAM within ORF61 (aa positions 697-701) was mutated by four alanine substitutions (PFVDQ to PAAAA). pmCherry-Nucleolin ( ) and pcDNA3-MCMV M45-HA ( ) have been described. pcDNA3.1-hRIPK1-Flag (#112487) and pCMVTAG-NEMO (#11970) were purchased from Addgene.

Techniques: Transfection, Expressing, Fluorescence

Journal: Journal of Virology

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF61 protein sequesters APOBEC3B in filamentous aggregates

doi: 10.1128/jvi.00789-25

Figure Lengend Snippet: KSHV ORF61 binds and relocalizes A3B in an IPAM-dependent manner in infected cells. ( A ) The Tet-inducible expression of mScarlet-HA-tagged A3B or A3G in stably transduced RPE-1 cells was verified by treating the cells with increasing concentrations of doxycycline for 24 h and detecting the A3 proteins by immunoblot. ( B ) TetOn A3B and A3G RPE-1 cells were infected with either KSHV mNeon-ORF61, KSHV mNeon-ORF61 mut IPAM (MOI 0.1), or MHV-68 ORF61-mNeon (MOI 5). A3 expression was induced 16 hpi with 1 µg/mL doxycycline, and cell lysates were used for mNeon pulldown at 40 hpi. Co-precipitating proteins were detected by immunoblot. ( C ) TetOn A3B and A3G RPE-1 cells infected with KSHV mNeon-ORF61 or KSHV mNeon-ORF61 mut IPAM. A3 expression was induced 16 hpi with 1 µg/mL doxycycline. At 48 hpi, cells were fixed, nuclei were counterstained with Hoechst 33342, and fluorescence images were acquired by cLSM. Scale bar, 20 µm. ( D ) Colocalization was quantified by calculating Pearson’s correlation coefficient for ORF61 and A3 signals using Z-stacks acquired for each condition ( n = 10). Mean values ± SEM are shown. Significance was calculated using an unpaired Student’s t -test. ***, P < 0.001. ( E ) The percentage of nuclear A3B fluorescence intensity was determined by dividing the nuclear by the total A3B intensity for each infected cell ( n = 12). Mean values ± SEM are shown. Significance was calculated using an unpaired Student’s t -test. ***, P < 0.001.

Article Snippet: To generate pcDNA3-KSHV ORF61 mut IPAM-3xFLAG, the IPAM within ORF61 (aa positions 697-701) was mutated by four alanine substitutions (PFVDQ to PAAAA). pmCherry-Nucleolin ( ) and pcDNA3-MCMV M45-HA ( ) have been described. pcDNA3.1-hRIPK1-Flag (#112487) and pCMVTAG-NEMO (#11970) were purchased from Addgene.

Techniques: Infection, Expressing, Stable Transfection, Western Blot, Fluorescence

Journal: Journal of Virology

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF61 protein sequesters APOBEC3B in filamentous aggregates

doi: 10.1128/jvi.00789-25

Figure Lengend Snippet: Requirement of the KSHV ORF61 IPAM for efficient replication and protection from A3B-mediated genome editing. ( A ) Multistep MHV-68 replication kinetics. MEF cells were infected with MHV-68 ORF61-FLAG or ORF61 mut IPAM-FLAG at MOI 0.01. Viral titers in the supernatants were determined. Mean values ± SEM of triplicates are shown. DL, detection limit. ( B ) Multistep KSHV replication kinetics. TetOn A3B RPE-1 cells were infected with either KSHV mNeon-ORF61 or mNeon-ORF61 mut IPAM at MOI 0.025. At 4 hpi, the cells were treated with 1 µg/mL doxycycline to induce A3B expression or left untreated. Viral titers were determined in the supernatants. Mean values ± SEM of triplicates are shown. DL, detection limit. ( C ) Differential DNA denaturation PCR (3D-PCR) of KSHV mNeon-ORF61 and mNeon-ORF61 mut IPAM with (induced) or without (uninduced) A3B overexpression in TetOn A3B RPE-1 cells. DNA extracted from supernatants at 7 dpi was used to PCR-amplify a fragment of the viral ORF45. A PCR with a denaturation temperature gradient was used to determine the lowest denaturation temperature that allowed amplification. The dotted lines indicate the lowest denaturation temperature for the ORF45 fragment of KSHV mNeon-ORF61 without A3B induction. M, DNA size marker.

Article Snippet: To generate pcDNA3-KSHV ORF61 mut IPAM-3xFLAG, the IPAM within ORF61 (aa positions 697-701) was mutated by four alanine substitutions (PFVDQ to PAAAA). pmCherry-Nucleolin ( ) and pcDNA3-MCMV M45-HA ( ) have been described. pcDNA3.1-hRIPK1-Flag (#112487) and pCMVTAG-NEMO (#11970) were purchased from Addgene.

Techniques: Infection, Expressing, Over Expression, Amplification, Marker

Journal: Neuron

Article Title: Differing Strategies Despite Shared Lineages of Motor Neurons and Glia to Achieve Robust Development of an Adult Neuropil in Drosophila

doi: 10.1016/j.neuron.2018.01.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A SpeI-VGlut-Spe1 fragment containing the 5.9 kb sequence upstream of the VGlut translation start site was taken from a pCR8/GW/TOPO ( Enriquez et al., 2015 ) vector and clone in a pattb-QF-hsp70 vector (addgene).

Techniques: Recombinant, Plasmid Preparation, Software