Fig 1: Expression of miR‐205 in MCF10A‐ErbB2, SKBR3, and MDA‐MB‐453 cells treated with ErbB2 signaling pathway inhibitors. Real‐time quantitative RT‐PCR analysis of miR‐205 expression in MCF10A‐ErbB2, SKBR3, and MDA‐MB‐453 cells treated with the indicated inhibitors. (A, D) Cells were treated with PI3K inhibitor LY294002 (50 μm), p38 MAPK inhibitor SB203580 (10 μm), MEK inhibitor U0126 (10 μm), or PD98059 (20 μm) for 48 h. (B, D) Cells were treated with Raf‐1 kinase inhibitor ZM‐336372 (1–5 μm) or ERK inhibitor SCH772984 (1 μm) for 48 h. Data are normalized to DMSO control and represented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 by Student's t‐test compared with DMSO. (C) Analysis in MCF10A, SKBR3, and MDA‐MB‐453 cells Data are mean ± SEM of three independent experiments. *P < 0.01 by Student's t‐test compared with MCF10A.
Fig 2: Inhibition of the Raf1 pathway negatively impacts viral DNA and protein accumulation. (A-C) MRC5 fibroblasts were treated with 2.17 uM of Regorafenib or Sorafenib, or DMSO control at the time of infection with AD169, at an MOI of 3.0 for indicated amount of time. (A) Viral DNA was quantified by qPCR of the viral gene, IE1 (avg±SD, n=12). (B) Samples in (A) were compared by area under the curve (AUC) to show the significance between treatments over the course of infection (avg±SD, n=12). (C) Viral protein expression levels at 24-, 72-, and 96 hours post-infection (hpi) were assessed by western blot. GAPDH was used as a loading control.
Fig 3: Phosphorylation of S621 enhances Raf1 binding to 14-3-3 during HCMV infection. (A) Overexpression of pLenti-Flag-Raf1-WT or pLenti-Flag-Raf1-S621A were compared to pLenti-empty vector (EV) control MRC5 fibroblasts and assessed by Western blot using antibodies specific to flag, Raf1 and S621- or S338-phosphorylated Raf1, and total or phospho-MEK and -ERK. GAPDH was used as a loading control. (B) EV, Flag-Raf1-WT and -S621A cells were mock (M) or HCMV (H) infected with AD169-WT at an MOI of 3.0 for 72 hours prior to Flag-pulldown and co-precipitation of endogenous 14-3-3. (C) Ratio of 14-3-3 to Raf1 in (B) was quantified. (D-E) Cells were infected at an MOI of 3.0, for 96 hours (avg±SEM, n=3) (D), or an MOI of 0.01 for 10 days (avg±SEM, n=6) (E), and viral titers were assessed by TCID50.
Fig 4: Expression of miR‐205 in MCF10A‐RafCAAX cells treated with ERK inhibitor. (A) Western blot analysis of MCF10A‐RafCAAX, MCF10A‐ErbB2, and MCF10A‐neo cells with anti‐Raf‐1 antibodies. β‐Actin was used as a control for loading. (B) Real‐time quantitative RT‐PCR analysis of miR‐205 expression in MCF10A‐RafCAAX and MCF10A‐neo cells. Data are mean ± SEM of three independent experiments. (C) Real‐time quantitative RT‐PCR analysis of miR‐205 expression in MCF10A‐neo and MCF10A‐RafCAAX cells treated with MEK inhibitor U0126 (10 μm) or PD98059 (20 μm), Raf1 kinase inhibitor ZM‐336372 (1 μm), or ERK inhibitor SCH772984 (1 μm) for 48 h. Data are mean ± SEM of three independent experiments. *P < 0.01 by Student's t‐test compared with MCF10A‐neo.
Fig 5: Pharmacological inhibition of Raf1 attenuates HCMVTB40/E cell-to-cell spread in fibroblasts and epithelial cells. (A-B) MRC5 fibroblasts or (C) ARPE19 epithelial cells were treated with 2.17 uM of Regorafenib or Sorafenib, or DMSO control at the time of infection with (A) AD169-GFP or (B-C) TB40/E-mCherry, at an MOI of 0.01. Viral spread was calculated by cells positive for (A) GFP or (B) mCherry on day 12 in MRC5hTs or (C) mCherry on days 15 and 30 (avg±SEM, n=48).
Supplier Page from Abcam for Anti-Raf1 (phospho S621) antibody