Decreased levels of PRMT1 alters BRCA1 promoter binding <i>in vivo.</i>

<p>(<b>a</b>) HeLa cells were transfected with different concentrations of PRMT1 siRNA (10, 25, 50 nM) following manufacturer's instructions. Results are representative of two independent experiments. (<b>b</b>) HeLa cells transfected with 50 nM Luc or PRMT1 siRNA were collected for ChIP analysis. Anti-BRCA1 (10 µg), anti-IgG (10 µg), and anti-histone H3-phosphorylated at S10 (H3-pS10, 5 µg) antibodies were used for ChIP analysis. PCR products were run on a 2% agarose gel and visualized with ethidium bromide staining. Results are representative of two independent experiments.</p

BRCA1 methylation status alters protein-protein interactions at the 504-802 region.

<p>(<b>a</b>) Schematic of BRCA1 504-802 primary sequence depicting important protein-protein interactions and domains that could be affected by the methylation of this region. (<b>b</b>) MDA-MB-231 cells were treated with AdOx (30 µM) in order to observe BRCA1 methylation inhibition upon treatment. Two milligram of MDA-MB-231 whole cell protein extract was immunoprecipitated with anti-BRCA1 or anti-IgG antibodies, separated on a 4-20% gel by SDS-PAGE, and western blotted using antibodies against Sp1 and BRCA1 proteins. Input represents 1/10 of immunoprecipitated material. Results are representative of two independent experiments. (<b>c</b>) MDA-MB-231 cells were treated with AdOx (30 µM) and whole cell extract separated on a 4-20% gel by SDS-PAGE, and probed with anti-Sp1 antibody. Densitometry was averaged from three independent immunoblots.</p

HTLV-1 Tax interacts with Drosha <i>in vitro</i>.

<p><b>A</b>) HeLa whole cell lysates (1 mg) were incubated with purified GST-fusion proteins (∼5 µg) bound to Glutathione-Sepharose beads: GST-Alone, GST-Tax wild type, GST-Tax N-terminus, 1–244, GST-Tax C-terminus, 244–336, and GST-Tax truncated, 288–353. Beads were washed gently, proteins eluted, and western blotted for the presence of Drosha. CEM whole cell extract serves as a positive Drosha control. <b>B</b>) 293T cell lysates were prepared from cells alone or cells transfected with pACH.Tax (5 µg) or pACH.M22 (5 µg) in the presence of the proteasome inhibitor PSI (10 µM). Approximately 1 mg of lysate was incubated with α-IgG, α-Drosha, or α-Tax overnight at 4°C. The next day, 50 µl of a 30% Protein A + G slurry was used to IP Drosha or Tax and subsequently western blotted for the presence of Drosha and Ubiquitin. Multiple isoforms of Drosha as detected by the antibody are indicated as Drosha I (170 kDa), Drosha II (156 kDa), and Drosha III (115 kDa). <b>C</b>) Graphical depiction of the GST-Tax constructs and the regions which they span for the Tax protein. Relative Drosha binding is based on densitometry counts. <b>D</b>) C81 cells were treated with the proteasome inhibitors PSI and ALLN at increasing concentrations (0.1, 1.0, and 10 µM) for 48 hours. Cell lysates were western blotted for the presence of Drosha. β-Actin serves as a loading control. Drosha recovery as a percentage of CEM Drosha levels is indicated from densitometry counts normalized to β-Actin using ImageJ.</p

Suppression of endogenous miRNAs dysregulated by HTLV-1.

<p><b>A</b>) 293Ts were transfected with pACH.Tax (5 µg) and 24 hours later were transfected with pGFP-Drosha (10 µg). Cells were collected 48 hours post transfection and were western blotted (50 µg) for IKK-β, BRM and β-Actin. <b>B</b>) 293T cells were transfected with Tax (5 µg) or antagomiRs (100 nM) targeting miR-199a-3p, miR-132, or Let7i. Cells were collected 72 hours post transfection, and western blotted (50 µg) for IKK-β, p65, p50, GSK-3β, BRM, and β-Actin. Densitometry counts of western blots for each indicated protein normalized to β-Actin, plotted as a percent change (measured as arbitrary counts) between antagomiR treated cells and 293T cells alone. Densitometry counts were calculated using ImageJ.</p

Tax prevents primary miRNA cleavage by Drosha.

<p>293T cells were transfected with Tax expression vectors (1 µg): Tax WT, TD1 (Δ1–37), TD55 (Δ55–92), TD99 (Δ99–142), TD150 (Δ150–198), TD254 (Δ254–287), or TD319 (Δ319–353). <b>A</b>) Forty-eight hours post transfection, total RNA was isolated by TRIzol extraction and expression of miR326 was determined by quantiMiR PCR kit using primers specific to miR326. Values shown are normalized to U6 and shown relative to control. <b>B</b>) Primary transcripts encoding miR326 were detected by RT-qPCR against a region upstream of the miRNA hairpin. Quantities were normalized to GAPDH and efficiency of Drosha processing was determined by charting the expression of mature miRNA over the expression of the primary transcript. Data is shown with the efficiency in the control cells set to 100%. <b>C</b>) A graphical depiction of the Tax mutant constructs TD1 and TD55 with deleted regions and the corresponding domains and motifs of full length Tax. The function of each domain is indicated in brackets and arrows, as well as the associated interacting cellular proteins below each region. The proposed Drosha binding region is indicated by the grey shaded box.</p

Loss of Drosha increases viral replication.

<p><b>A</b>) 293T cells were transfected with HTLV-1 pACH (5 µg) and 24 hours later were transfected with siRNAs against Luciferase (150 nM), Drosha (50, 150, and 300 nM), DGCR8 (50, 150, 300 nM), and Ago2 (50, 150, 300 nM) or were treated with 0.1, 1.0, or 2.5 µM Acriflavine (ACF). At 72 hours post-transfection, supernatants were collected and assessed for viral replication by RT (reverse transcriptase) assay. Data was collected in triplicate from at least 2 independent experiments. <b>B</b>) 293T cells from panel A were assayed by western blot for the efficiency of siRNA knockdown of the target proteins. β-Actin serves as a positive loading control.</p

The RNAi machinery is dysregulated with HTLV-1 infection.

<p><b>A</b>) Uninfected (H9, Jurkat, CEM), HTLV-1 infected, Tax-positive (C81, MT2, MT4), and HTLV-1 infected, Tax-negative (ED-, MT1) T-cell lines were screened for endogenous protein levels of Drosha, DGCR8, Dicer, and Ago2. Seventy-five micrograms of total lysates were used for western blots. Multiple bands for DGCR8 are indicative of isoforms at 86 and 65 kDa. β-Actin serves as a loading control. <b>B</b>) Total lysates of HTLV-1 infected, Tax-negative cell lines ED- and MT1 were titrated (50, 75 and 100 µg) and screened for endogenous protein levels of Drosha, DGCR8, Dicer, and Ago2. Multiple bands for DGCR8 are indicative of isoforms at 86 and 65 kDa. β-Actin serves as a loading control. <b>C</b>) Whole cell lysates (50 µg) from HTLV-1 infected primary CD4⁺ T-cells were run on a 10% Tris-glycine SDS-PAGE gel and western blotted for Drosha. Data is representative of three independent experiments. β-Actin serves as a loading control. <b>D</b>) Total RNA was isolated with TRIzol from CEM, MT2, ED-, and MT1 cell lines. RT-PCR was performed for cellular Drosha transcript levels. GAPDH serves as a housekeeping gene loading control. <b>E</b>) Uninfected 293T cells were transfected with pcDNA (0.1, 1.0, and 10 µg), pACH (0.1, 1.0, and 10 µg), pcTax (0.1, 1.0, and 10 µg), or pACH.M22 (0.1, 1.0, and 10 µg) and were screened for protein levels of Drosha, Dicer, Ago2, Ago1, and Tax. Seventy-five micrograms of total lysates was used for western blots. β-Actin serves as a loading control. <b>F</b>) Total RNA was isolated with TRIzol from the transfected cells in panel E. RT-PCR was performed for cellular Drosha transcript levels. GAPDH serves as a housekeeping gene loading control.</p

BIOder inhibits VEEV cell death <i>in vivo</i>.

<p><b>A</b>) Female C3H/HeN mice were treated subcutaneously with either DMSO or various concentration of BIOder (10 mg/kg, 20 mg/kg, 40 mg/kg) every day for 5 days. Mice were weighed daily and the average % of the mouse weight is plotted in panel A. <b>B</b>) <b>and </b><b>C</b>) Female C3H/HeN mice were infected intranasally with 5×LD50 (2×10⁷ pfu) of VEEV TC-83. Groups of 10 mice were treated SQ with vehicle, BIO (50 mg/kg) or BIOder (20 mg/kg) on days −1, 1, 3, and 5 and were monitored for survival for 14 days. Kaplan-Meier curves for survival between DMSO and BIO (panel B). Kaplan-Meier curves for survival between DMSO and BIOder (panel C). Significance was determined using Mantel-Cox Log-rank test. P-value of 0.057 between control and BIOder. This experiment was performed one time.</p

Modulation of GSK-3β Activity in Venezuelan Equine Encephalitis Virus Infection

<div><p>Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3β is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer's. Hence inhibition of GSK-3β in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3β inhibitors and GSK-3β siRNA experiments indicated that GSK-3β was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC₅₀ of ∼0.5 µM and a CC₅₀ of >100 µM. BIOder was a more potent inhibitor of GSK-3β than BIO, as demonstrated through <em>in vitro</em> kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3β is found in three distinct complexes in VEEV infected cells, whereas GSK-3β is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3β inhibitors for modulating VEEV infection.</p> </div

BIOder treatment alters expression of apoptotic genes to promote survival of U87MGs.

<p><b>A</b>) U87MGs were treated with 1 µM BIO, 1 µM BIOder, or DMSO and infected with VEEV TC-83. RNA was harvested from infected cells 24 hours post infection and analyzed by RT-PCR for expression of the indicated anti- and pro-apoptotic genes. <b>B</b>) Band intensities corresponding to triplicate samples were quantified and represented as fold change in gene expression over the DMSO control, with the DMSO control being set as a fold change of 1.0.</p