human immortalized retinal pigment epithelial cells  (ATCC)

Journal: bioRxiv

Article Title: Human cytomegalovirus antagonizes SMC5/6 driven genome silencing via UL35 instituted proteasomal degradation of SLF2

doi: 10.64898/2026.05.26.727865

Figure Lengend Snippet: ARPE-19 cells were left uninfected or were infected with HCMV TB40/E (MOI of 1) as indicated. After 8 and 24 h, cells were harvested and SLF2 protein expression was analysed through western blotting. In parallel, expression of SMC6, IE1 and UL44 were analysed; β-actin served as a loading control. Three independent biological replicates were performed.

Article Snippet: Primary human foreskin fibroblasts (HFFs), isolated from human foreskin tissue as described previously, and human retinal pigment epithelial cells ARPE-19 (ATCC-CRL-2302) were cultivated in Dulbeccós modified eagle medium (DMEM) (Gibco) supplemented with 5% fetal bovine serum (FBS) (Capricorn) and penicillin-streptomycin (Sigma-Aldrich) [ ].

Techniques: Infection, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Human cytomegalovirus antagonizes SMC5/6 driven genome silencing via UL35 instituted proteasomal degradation of SLF2

doi: 10.64898/2026.05.26.727865

Figure Lengend Snippet: (A) Requirement of viral entry for SLF2 degradation. ARPE-19 cells were infected for 4 hours (MOI 1) either with an entry proficient strain of TB40/E (TB40/E BAC KL17 UL32-EGFP UL100-mCherry) or with the entry-deficient strain TB40/E UL128insA332 (TB40/E BAC KL17 UL128insA332 UL32-EGFP UL100-mCherry) or were left uninfected (mock). Whole cell lysates were analysed by immunoblotting, and the SLF2 protein levels compared to mock cells. Viral UL83 was detected as control for viral entry and β-actin as loading control. Three biological replicates were performed. (B-D) Evidence for SLF2 degradation by a viral structural protein. (B) HFF cells were either left uninfected (mock) or were infected for 8 hours (MOI 1) with TB40/E IE2-EYFP. Where indicated, supernatants were UV-inactivated and added to the respective condition. The expression levels of SLF2 compared by western blotting. UV inactivation of the viral inoculum was controlled via the detection of IE1 expression. The experiment was performed in three biological replicates. (C) Indirect immunofluorescence analysis to detect the colocalization of PML and NSMCE3 either after mock infection or after infection of HFF with TB40/E IE2-EYFP. UV: UV treatment of supernatants. Scale bar: 2 µm. (D) Colocalization analysis of NSMCE3 and PML via quantification of PCCs. The analysis was restricted to the PML signals in the cell nuclei. More than 90 cells were analysed and the violin plot shows results of two independent biological replicates for the TB40/E IE2-EYFP sample and of three biological replicates for the other tested conditions. The median, the first and the third quartile are indicated. One-way ANOVA (Kruskal-Wallis test): n.s.: not significant, **** p< 0.0001. (E-F) SLF2 is degraded in a proteasome-dependent manner. (E) Western blot analysis of HFFs that were either mock infected or infected with TB40/E (MOI 1) for 8 hours. As indicated, cells were treated with 10 µM of MG132, 5 µM of PYR-41, 10 µM of MLN4924 or DMSO. SLF2, UL83, IE1 and Daxx were detected using specific antibodies; β-actin served as a loading control. (F) Indirect immunofluorescence analysis to detect the colocalization of NSMCE3 and PML. Infection and inhibitor treatment were performed as described for panel E. Scale bar: 2 µm. (F) Colocalization analysis of PML and NSMCE3 via quantification of PCCs. The violin plot summarizes the results of three independent biological replicates. The median, the first and the third quartile are indicated.

Article Snippet: Primary human foreskin fibroblasts (HFFs), isolated from human foreskin tissue as described previously, and human retinal pigment epithelial cells ARPE-19 (ATCC-CRL-2302) were cultivated in Dulbeccós modified eagle medium (DMEM) (Gibco) supplemented with 5% fetal bovine serum (FBS) (Capricorn) and penicillin-streptomycin (Sigma-Aldrich) [ ].

Techniques: Infection, Western Blot, Control, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Human cytomegalovirus antagonizes SMC5/6 driven genome silencing via UL35 instituted proteasomal degradation of SLF2

doi: 10.64898/2026.05.26.727865

Figure Lengend Snippet: (A-B) Targeted screening for SLF2 degradation in ARPE-19 cells. (A) ARPE-19 cells were co-transfected with expression vectors for SLF2 and candidate viral effector proteins. Vectors encoding GFP and IE1 were included as controls. The cells were harvested at 24 hours after transfection for indirect immunofluorescence staining. Scale bar: 2 µm. (B) Quantification of cells positive for candidate and/or SLF2 protein. The expression patterns of more than 70 cells were counted from three independent biological replicates and displayed as percentage (%) of cells expressing SLF2, the candidate protein or both. (C-D) SLF2 degradation in HFFs with doxycycline-inducible expression of HA-tagged UL35. Control and UL35-HA inducible cells were stimulated with 1µg/ml of doxycycline (dox) for 24 hours. (C) Western blot analysis to detect endogenous SLF2 and SMC6 after doxycycline-induction of UL35-HA expression; β-actin served as a loading control. (D) Immunofluorescence analysis of the subcellular localization of SLF2 after induction of UL35-HA expression by doxycycline. UL35 expression was detected using a monoclonal antibody against the HA-tag (HA); SLF2 was detected with a SLF2 specific polyconal antibody (SLF2). Scale bar: 2 µm. (E) Quantification of the number of cells with SLF2 foci in the absence or presence of UL35-HA. Two independent biological replicates were performed and >70 cells were analysed. The means of the % were calculated +/- SD.

Article Snippet: Primary human foreskin fibroblasts (HFFs), isolated from human foreskin tissue as described previously, and human retinal pigment epithelial cells ARPE-19 (ATCC-CRL-2302) were cultivated in Dulbeccós modified eagle medium (DMEM) (Gibco) supplemented with 5% fetal bovine serum (FBS) (Capricorn) and penicillin-streptomycin (Sigma-Aldrich) [ ].

Techniques: Transfection, Expressing, Immunofluorescence, Staining, Control, Western Blot

Journal: bioRxiv

Article Title: Human cytomegalovirus antagonizes SMC5/6 driven genome silencing via UL35 instituted proteasomal degradation of SLF2

doi: 10.64898/2026.05.26.727865

Figure Lengend Snippet: (A-B) Coexpression of UL35a and SLF2 are not mutually exclusive. ARPE-19 cells were cotransfected with an expression plasmid encoding SLF2 together with plasmids for FLAG-UL35, FLAG-UL35a, UL83 or Vpr. (A) Indirect immunofluorescence analysis, performed at 24 hours post transfection, to detect SLF2 and the respective candidate proteins using a SLF2 specific polyconal antibody together with antibodies directed against UL83, Vpr, or the FLAG-tag. Scale bar: 2 µm. (B) Quantification of cells positive for candidate and/or SLF2 protein. The expression patterns of more than 60 cells were counted from three independent biological replicates and displayed as percentage (%) of cells expressing SLF2, the candidate protein or both. (C-E) Dox-inducible expression of UL35a is not sufficient for SLF2 degradation. Control cells or HFFs with doxycycline-inducible expression of either UL35a-FLAG or UL35-FLAG were incubated with 500 ng/ml of doxycycline for 24 hours followed by either the preparation of cell lysates for western blotting or the fixation of cells for subsequent immunofluorescence analysis. (C and D) Western blot analysis to detect endogenous SLF2 as well as UL35 and UL35a using a FLAG-specific antibody; β-actin was used as a loading control. Panel D includes the analysis of cell lysates that were harvested after incubation with MG132 (10 µM) or DMSO to investigate the rescue of SLF2 via proteasomal inhibition. Three independent biological replicates were performed. (E) Indirect immunofluorescence analysis of the subcellular localization of PML and SLF2 upon expression of either UL35 or UL35a. Three independent biological replicates were performed. Scale bar: 2 µm.

Article Snippet: Primary human foreskin fibroblasts (HFFs), isolated from human foreskin tissue as described previously, and human retinal pigment epithelial cells ARPE-19 (ATCC-CRL-2302) were cultivated in Dulbeccós modified eagle medium (DMEM) (Gibco) supplemented with 5% fetal bovine serum (FBS) (Capricorn) and penicillin-streptomycin (Sigma-Aldrich) [ ].

Techniques: Expressing, Plasmid Preparation, Immunofluorescence, Transfection, FLAG-tag, Control, Incubation, Western Blot, Inhibition