Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) RPE-1 cells were arrested in G0 by contact inhibition and 48 h serum starvation (-FCS 48 h), in G1 by 24 h serum starvation (-FCS 24 h), in S phase by 24 h treatment with 1 mM hydroxyurea (HU) and in G2/M by 24 h treatment with 200 ng/ml nocodazole. Asynchronous (asynchr.) cells are shown for comparison. The cells were fixed and stained with propidium iodide. The DNA content was analyzed by flow cytometry to verify synchronization. Cells in G0 and G1 have a DNA content of 2n, cells in S phase between 2n and 4n, and cells in G2/M a 4n DNA content. The percentages of cells in each cell cycle phase are indicated. (B) FAM111A and FAM111B expression in cell-cycle arrested cells was analyzed by immunoblot. Cyclin E and cyclin B1 were analyzed as markers for S and G2/M phases, respectively. GAPDH was used as a loading control. ( C) Subconfluent RPE-1 cells were treated with an E2F inhibitor (HLM0006474, 40 μM) or vehicle (DMSO). Cell lysates were harvested at the indicated times (hours post-treatment, hpt) and analyzed by immunoblot. (D to F) WT RPE-1 cell, E2F1 KO, and E2F3 KO cells were synchronized by serum starvation for 24 h and infected with MCMVs expressing full-length or mutant M117 (MOI 5). Cell lysates were harvested at the indicated times post-infection and analyzed by immunoblot. The MCMV immediate-early 1 (IE1) protein and β-actin were used as infection and loading controls, respectively. Representative blots out of multiple experiments are shown. hpt: hours post treatment.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Inhibition, Comparison, Staining, Flow Cytometry, Expressing, Western Blot, Control, Infection, Mutagenesis

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) RPE-1 cells were infected (MOI 5 TCID 50 /cell) with recombinant MCMV-GFP encoding FAM111A-specific (A sh1, A sh2), FAM111B-specific (B sh1, B sh2), or scrambled (scr) control shRNA. Cell lysates were harvested 24 and 28 hpi and analyzed by immunoblot. (B) RPE-1 cells (MOI 0.2), (C) ARPE-19 (MOI 1) and (D) RPTEC (MOI 1) were infected with the same viruses as above. Viral replication kinetics were determined by titration of virus released into the supernatant. (C) Rhesus fibroblasts were infected (MOI 1.5 TCID 50 /cell) with the same viruses as above and analyzed by immunoblot or infected at MOI 1 TCID 50 /cell (D) with the same viruses as above. Viral replication kinetics were determined by titration of virus released into the supernatant. Mean ±SEM of triplicates are shown. DL, detection limit. (E, F) Cell lysates of WT RPE-1 cells, empty vector (EV)-transduced RPE-1 cells, two FAM111B KO RPE-1 clones (cl 1-9 and 3-18), and one FAM111A KO RPE-1 (cl 3-10) clone were analyzed by immunoblot. (G) Multistep replication kinetic of WT MCMV in the same RPE-1 as in E. Supernatants of infected cells were harvested at the indicated times post infection and titrated. Mean ±SEM of triplicates are shown. DL, detection limit.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Infection, Recombinant, Control, shRNA, Western Blot, Titration, Virus, Plasmid Preparation, Clone Assay

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) 10.1 mouse fibroblasts were infected (MOI 1 TCID 50 /cell) with recombinant MCMVs encoding HA-tagged WT or mutant FAM111B. Cell lysates were analyzed by immunoblot. (B) 10.1 cells were infected (MOI 0.02) with the same viruses as above to analyze multistep replication kinetics. Mean ±SEM of triplicates are shown. DL, detection limit. (C) NIH-3T3 fibroblasts transduced with a lentiviral vector encoding tet-inducible FAM111B were induced with 2 µg/ml doxycyline or left untreated. Cell lysates were harvested and analyzed by immunoblot. (D) Transduced NIH-3T3 cells were treated with doxycycline 1h before (−1 hpi) or 4 h after (+4 hpi) infection with WT MCMV (MOI 0.02) to analyze multistep replication kinetics. Mean ±SEM of triplicates are shown.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Infection, Recombinant, Mutagenesis, Western Blot, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) Immunofluorescence of 10.1 fibroblasts mock-infected or infected with MCMV-eGFP-FAM111B (MOI 1 TCID 50 /cell). Cells were fixed 24 hpi and stained with an antibody recognizing the MCMV E1 protein (red), a marker for vRCs. Nuclei were stained with DAPI. Scale bar, 25 µm. (B) Immunofluorescence of FAM111B KO or WT RPE-1 cells mock-infected or infected with WT MCMV (MOI 1 TCID 50 /cell). Cells were fixed 40 hpi and stained with antibodies recognizing FAM111B (green) or MCMV E1 (red). Nuclei were stained with DAPI or Hoechst 33342. Arrow heads indicate FAM111B in vRCs. Scale bar, 25 µm.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Immunofluorescence, Infection, Staining, Marker