human retinal pigment epithelial cells arpe 19  (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

Journal: bioRxiv

Article Title: Nuclear DNA Damage Response Triggers Reorganization of Mitochondrial Nucleic Acids

doi: 10.64898/2026.05.23.727397

Figure Lengend Snippet: (A) Representative immunofluorescence images of A549 cells stained for TOMM20 and TFAM at the indicated times after 20 Gy ionizing radiation (IR). Enlarged TOMM20-positive mitochondrial structures containing concentrated TFAM signal are defined as mito-blobs. Scale bars, 20 µm; insets, 5 µm. (B) Quantification of mito-blobs per cell and 53BP1 foci per nucleus over time after 20 Gy IR. (C) Quantification of mito-blobs per cell 3 days after the indicated doses of IR. (D) Representative immunofluorescence images of ARPE-19 cells stained for TOMM20 and TFAM under untreated conditions or 3 days after 20 Gy IR. Scale bars, 20 µm; insets, 5 µm. (E) Quantification of mito-blobs per cell in ARPE-19 cells under the conditions shown in (D). (F) Representative immunofluorescence images of A549 cells stained for TOMM20 and TFAM after treatment with IR or the indicated cytotoxic agents. Drug-treated cells were treated with 100 nM cisplatin, etoposide, irinotecan, paclitaxel, pemetrexed, or 5-FU for 48 h. Scale bars, 20 µm; insets, 5 µm. (G) Quantification of mito-blobs per cell after the treatments shown in (F). IR was quantified 72 h after 20 Gy; drug-treated cells were quantified after 48 h treatment. Data are shown as individual cells with biological replicate means and mean +/- SEM or as box-and-whisker plots where indicated. n = 3 biological replicates except in (C), where one representative experiment is shown. ns, not significant; *p<0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2 tailed Welch’s t test.

Article Snippet: Human retinal pigment epithelial ARPE-19 cells (ATCC, CRL-2302) were immortalized with hTERT.

Techniques: Immunofluorescence, Staining, Whisker Assay

Journal: bioRxiv

Article Title: Nuclear DNA Damage Response Triggers Reorganization of Mitochondrial Nucleic Acids

doi: 10.64898/2026.05.23.727397

Figure Lengend Snippet: (A) Representative immunofluorescence images of A549 cells stained for DAPI and 53BP1 at the indicated times after 20 Gy IR. Scale bar, 10 µm. (B) Representative images showing TOMM20 staining and mito-blob (FASTKD2) in A549 cells 3 days after the indicated doses of IR. Scale bars, 20 µm; insets, 5 µm. (C) Representative immunofluorescence images of ARPE-19 cells stained for DAPI and 53BP1 under untreated conditions or 3 days after 20 Gy IR. Scale bar, 10 µm. (D) Quantification of 53BP1 foci per nucleus in ARPE-19 cells under the conditions shown in (C). (E) Representative immunofluorescence images of A549 cells stained for DAPI and 53BP1 after IR or the indicated cytotoxic treatments. Drug-treated cells were treated with 100 nM cisplatin, etoposide, irinotecan, paclitaxel, pemetrexed, or 5-FU for 48 h. Scale bar, 10 µm. (F) Quantification of 53BP1 foci per nucleus after the treatments shown in (E). IR was quantified 72 h after 20 Gy; drug-treated cells were quantified after 48 h treatment. Data are shown as individual nuclei with biological replicate means and mean +/- SEM. n = 3 biological replicates. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 by 2 tailed Welch’s t test.

Article Snippet: Human retinal pigment epithelial ARPE-19 cells (ATCC, CRL-2302) were immortalized with hTERT.

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Nuclear DNA Damage Response Triggers Reorganization of Mitochondrial Nucleic Acids

doi: 10.64898/2026.05.23.727397

Figure Lengend Snippet: (A) Quantification of mitochondrial 12S rRNA locus abundance normalized to B2M in A549 cells expressing doxycycline-inducible catalytically inactive mito-ApaLI (ApaLI Dead) or active mito-ApaLI (ApaLI Active), with or without 72 h doxycycline treatment. (B) Representative live-cell images of A549 cells expressing ApaLI Dead or ApaLI Active after DMSO or 3 days doxycycline treatment. Cells were stained with MitoTracker Deep Red (MTDR) and SYBR Green I. Scale bars, 20 µm; insets, 5 µm. (C) Quantification of mito-blobs per cell in A549 cells expressing ApaLI Dead or ApaLI Active under the indicated doxycycline and IR conditions. Doxycycline was applied for 72 h where indicated; IR-treated cells were quantified 3 days after 20 Gy IR. (D) Representative immunofluorescence images of ARPE-19 cells carrying the inducible AsiSI system at the indicated times after AsiSI induction. Cells were stained for TOMM20 and TFAM. Scale bars, 20 µm; insets, 5 µm. (E) Quantification of mito-blobs per cell at the indicated times after AsiSI induction. (F) Representative immunofluorescence images of A549 cells treated with ATR inhibitor (ATRi) for the indicated times. Cells were stained for TOMM20 and TFAM. Scale bars, 20 µm; insets, 5 µm. (G) Quantification of mito-blobs per cell at the indicated times after ATRi treatment. Data are shown as individual cells or biological replicate values with biological replicate and means and mean +/- SEM. n = 3 biological replicates. ns, not significant; *P < 0.05; **P < 0.01 by 2 tailed Welch’s t test.

Article Snippet: Human retinal pigment epithelial ARPE-19 cells (ATCC, CRL-2302) were immortalized with hTERT.

Techniques: Expressing, Staining, SYBR Green Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Nuclear DNA Damage Response Triggers Reorganization of Mitochondrial Nucleic Acids

doi: 10.64898/2026.05.23.727397

Figure Lengend Snippet: (A) Representative immunofluorescence images of ARPE-19 cells carrying the inducible AsiSI system at the indicated times after AsiSI induction. Cells were stained for DAPI and 53BP1. Scale bar, 10 µm. (B) Quantification of 53BP1 foci per nucleus at the indicated times after AsiSI induction. (C) Quantification of mitochondrial 12S rRNA locus abundance normalized to B2M in A549 cells treated with ATRi for the indicated times or with ddC for 48 h as positive control for mtDNA depletion. (D) Representative immunofluorescence images of A549 cells treated with ATRi for the indicated times. Cells were stained for DAPI and 53BP1. Scale bar, 10 µm. (E) Quantification of 53BP1 foci per nucleus at the indicated times after ATRi treatment. Data are shown as individual nuclei or biological replicate values with biological replicate means and mean +/- SEM. n = 3 biological replicates. ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2 tailed Welch’s t test.

Article Snippet: Human retinal pigment epithelial ARPE-19 cells (ATCC, CRL-2302) were immortalized with hTERT.

Techniques: Immunofluorescence, Staining, Positive Control

Journal: Materials Today Bio

Article Title: ROS-responsive transmembrane peptide-antibody conjugate eyedrops for the non-invasive treatment of choroidoretinopathy

doi: 10.1016/j.mtbio.2026.102819

Figure Lengend Snippet: Preparation and characterization of the conjugates (A) Schematic diagram of responsive release of the conjugates. (B) Particle size and distribution of aVEGFA, MPEG-aVEGFA, and the conjugates. (C) Particle size changes of aVEGFA, MPEG-aVEGFA, and the conjugates after incubation in hydrogen peroxide. (D) Transmission electron microscopy images of aVEGFA and the conjugates, scale bar = 100 nm. (E–G) Schematic diagram and quantitative results of in vitro experiments assessing corneal penetration ability of FITC-aVEGFA. (H, I) Schematic diagram and quantitative results of in vitro experiments assessing corneal penetration ability of FITC-aPDL1. (J, K) Cytotoxicity evaluation of TAT-MPEG-aVEGFA on HCE-T cells and ARPE19 cells. (L, M) Scratch assay results estimating angiogenesis inhibition, and quantitative analysis. n.s. (not significant) p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control. Abbreviations: aVEGFA, anti–vascular endothelial growth factor A antibody; TAT-MPEG, trans-activator of transcription peptide–methoxy polyethylene glycol; ARPE-19, human retinal pigment epithelial cell line; HCE-T, human corneal epithelial cell line.

Article Snippet: Murine melanoma (B16) cells were obtained from COBIOER Biotechnology (Nanjing, China), human retinal pigment epithelial cell line (ARPE-19) cells and human corneal epithelial cell line (HCE-T) cells were obtained from ProCell Biotechnology (Wuhan, China), HUVECs were obtained from SUNNCELL Biotechnology (Wuhan, China).

Techniques: Incubation, Transmission Assay, Electron Microscopy, In Vitro, Wound Healing Assay, Inhibition, Control