Journal: bioRxiv

Article Title: Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression

doi: 10.1101/2023.10.31.564923

Figure Lengend Snippet: a . Calu-3 cells were cultured at 18% (grey) or 1% (blue) O 2 oxygen for 24h prior to infecting with RSV (MOI 0.2) and the cultures maintained under normoxic or hypoxic conditions for the stated times and cells lysed for PCR quantification of intracellular viral RNA (RSV-F), NDRG1 mRNA and infectivity at 24h or 48hpi (mean ± SEM, n = 4-8, Mann–Whitney test, Two-sided). All data are expressed relative to 18% O 2 values at their respective time point. HIF-1α and β-actin protein expression were assessed by immunoblot at 48hpi. b . Real-time monitoring of Calu-3 cells transduced with HRE-GFP were treated with PHIs (Daprodustat, Molidustat or Roxadustat) or infected with RSV at MOI 0.2 or 2 (mean ± SEM, n = 5), where UF = uninfected. c . Calu-3 HRE-GFP reporter cells were treated with Daprodustat at 50 or 100 μM or infected with RSV at a range of MOIs for 48hpi and stained with anti-RSV-F-APC followed by flow cytometric analysis. d . Calu-3 cells were pre-treated with PHIs for 24h before infecting with RSV (MOI 0.2) and intracellular viral RNA (RSV-F) and infectivity measured at 48hpi (mean ± SEM, n = 6-8, One-way ANOVA with multiple comparisons, Two-sided). e . HEp-2 or BEAS-2B were pre-treated with Daprodustat (100 μM) for 24h before infecting with RSV (MOI 0.2) and intracellular viral RNA (RSV-F) and infectivity measured at 48 hpi (mean ± SEM, n = 4, Mann–Whitney test, Two-sided). HIF-1α and β-actin protein expression were assessed by immunoblot at 48hpi. UF = uninfected.

Article Snippet: Daprodustat (GSK: 1278863), Molidustat (Bay 85-3934), Roxadustat (FG-4592) were obtained from MedChemExpress.

Techniques: Cell Culture, Infection, MANN-WHITNEY, Expressing, Western Blot, Transduction, Staining

Journal: bioRxiv

Article Title: Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression

doi: 10.1101/2023.10.31.564923

Figure Lengend Snippet: a. Calu-3 cells were pre-treated with PHIs (Daprodustat, Molidustat, Roxadustat) 24h prior to infection with a reporter RSV expressing GFP (MOI 0.2) and viral replication measured every 8h using an Incucyte® Live-Cell Imaging System. GFP signal was normalised per image with respect to cell confluence (mean ± SEM, n = 4). b . Effect of PHI treatment on RSV infected Calu-3 cells. Calu-3 cells were treated with Daprodustat or Molidustat (50 μM) before and after infection (pre+post) or 24hpi or 48hpi where the arrows indicate addition of drugs and GFP expression measured using the Incucyte® Live-Cell Imaging System. GFP signal and confluence were measured and the integrated intensity values are represented (mean ± SEM, n = 6). c. Combined Daprodustat and Palivizumab treatment inhibits RSV replication. Calu-3 cells were treated with a range of Palivizumab doses in the presence or absence of Daprodustat (9.0 μM) prior to infection with RSV-GFP (MOI 0.2) and viral replication measured at 48hpi using an Incucyte® Live-Cell Imaging System. GFP signal was normalized per image and cell confluence (mean ± SEM, n = 4). UF = uninfected.

Article Snippet: Daprodustat (GSK: 1278863), Molidustat (Bay 85-3934), Roxadustat (FG-4592) were obtained from MedChemExpress.

Techniques: Infection, Expressing, Live Cell Imaging

Journal: bioRxiv

Article Title: Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression

doi: 10.1101/2023.10.31.564923

Figure Lengend Snippet: a . HEK293T effector cells (expressing beta strands 1-7 of GFP), engineered for a doxycycline-inducible RSV F protein, were cocultured with HEK293T target cells (expressing beta strands 8-11 of GFP). b . The inhibitory activity of Daprodustat or Molidustat was measured as green intensity signal 48h after co-culture (mean ± SEM, n = 3). c. HEK293T cells were treated with PHI (50 μM) in the presence of doxycycline and F expression analysed by flow cytometry using an APC-conjugated anti-RSV-F antibody at 24hpi. d . Calu-3 cells were infected with RSV (MOI 1) and at 48hpi RSV-F expression imaged.

Article Snippet: Daprodustat (GSK: 1278863), Molidustat (Bay 85-3934), Roxadustat (FG-4592) were obtained from MedChemExpress.

Techniques: Expressing, Activity Assay, Co-Culture Assay, Flow Cytometry, Infection

Journal: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Effect of VRK1 depletion of epigenetic modifications of H3K27 in the presence or absence of serum in A549 lung adenocarcinoma cells. The field figure is shown in Additional file : Fig. S3. A Effect of VRK1 depletion on histone H3K27 methylation in A549 cells. B Effect of VRK1 depletion on histone H3K9 acetylation in A549 cells. The quantification of H3K27 methylation (left) and acetylation (right) from 50 cells is shown below. The immunoblots of the cell extracts from the experiment are shown at the bottom. The lysate was divided in two for loading in gels to determine each of the H3K27 modifications. *** p < 0.001. siCt, siControl; siV-02, siVRK1-02; siV-03, siVRK1-03

Article Snippet: The VRK1 inhibitor (VRK-IN-1) [ ] was obtained from MedChemExpress (Monmouth Junction, NJ, USA; Cat. No. HY-126542) and used as recommended by the manufacturer.

Techniques: Methylation, Western Blot

Journal: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Effect of VRK1 depletion or inhibition with VRK1-IN-1 on epigenetic modifications of H3K4 and H3K9 in A549 cells the presence or absence of serum. A Effect of VRK-IN-1 on histone H3K4m3 levels. B Effect of VRK-IN-1 on histone H3K9ac levels. C Effect of VRK-IN-1 on histone H3K9m3 levels. Ctr: control. *** p < 0.001

Article Snippet: The VRK1 inhibitor (VRK-IN-1) [ ] was obtained from MedChemExpress (Monmouth Junction, NJ, USA; Cat. No. HY-126542) and used as recommended by the manufacturer.

Techniques: Inhibition, Control

Journal: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Interaction of HDAC and KAT with VRK1. A Left: reciprocal interaction of endogenous VRK1 with increasing amount of transfected HDAC-Flag. Right: dose-dependent interaction between VRK1-HA and increasing amounts of HDAC1-Flag. B Left: interaction of endogenous VRK1 with transfected PCAF-Flag. Right: reciprocal interaction of transfected VRK1-HA and PCAF-Flag. Transfections were performed in HEK293T cells

Article Snippet: The VRK1 inhibitor (VRK-IN-1) [ ] was obtained from MedChemExpress (Monmouth Junction, NJ, USA; Cat. No. HY-126542) and used as recommended by the manufacturer.

Techniques: Transfection

Journal: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Interaction of SETDB1 (KMT1E) and KDM with VRK1. A Left: reciprocal interaction of SETDB1 with endogenous VRK1. Right: reciprocal interaction of SETDB1-Flag (KMT1E) with transfected VRK1-HA. B Left: reciprocal interaction of KDM4A (JMJD2A)-HA (top) or KDM3A (JMJD1A)-V5 (bottom) with endogenous VRK1. Right: reciprocal interaction of JMJD2A-HA (top) or KDM3A-V5 (bottom) with transfected VRK1-HA. Transfections were performed in HEK293T cells

Article Snippet: The VRK1 inhibitor (VRK-IN-1) [ ] was obtained from MedChemExpress (Monmouth Junction, NJ, USA; Cat. No. HY-126542) and used as recommended by the manufacturer.

Techniques: Transfection

Journal: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Inhibitors of histone PTMs

Article Snippet: The VRK1 inhibitor (VRK-IN-1) [ ] was obtained from MedChemExpress (Monmouth Junction, NJ, USA; Cat. No. HY-126542) and used as recommended by the manufacturer.

Techniques: Concentration Assay

Journal: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Plasmids

Article Snippet: The VRK1 inhibitor (VRK-IN-1) [ ] was obtained from MedChemExpress (Monmouth Junction, NJ, USA; Cat. No. HY-126542) and used as recommended by the manufacturer.

Techniques: Plasmid Preparation, Expressing

Journal: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Primary antibodies

Article Snippet: The VRK1 inhibitor (VRK-IN-1) [ ] was obtained from MedChemExpress (Monmouth Junction, NJ, USA; Cat. No. HY-126542) and used as recommended by the manufacturer.

Techniques: FLAG-tag