Celastrol inhibit VCaP cell growth <i>in vitro</i>.

<p>(<b>A</b>) Cells (1×10⁵) were plated in 35 mm dishes in complete medium and treated with different concentration of Celastrol for 24 h. Proliferation of the 8 groups of VCaP cells were measured using a Coulter counter. Cells were trypsinized and counted in triplicate. Cell numbers for each group are divided by the total cell number in the control group without Celastrol treatment. The experiment was repeated three times. Mean +/− standard deviation is shown. Asterisks indicate statistically significant differences between Celastrol treated group and the control. (<b>B</b>) 1×10⁵ of VCaP, LNCaP, PC3 and DU145 were exposured to 0.5 µM Celastrol for 24 h, cell numbers were counted for each group and divided by the total cell numbers in control groups to obtain the survival ratio.The experiment was repeated three times. Asterisks indicate statistically significant differences between different cell lines and the VCaP cells.</p

Celastrol targets AR-ERG-p536 in T/E fusion expressing PCa cells at protein level.

<p>(<b>A</b>) Western blot shows that Celastrol 2 h treament can significantly target the p536 expression in VCaP cells. Celastrol concentrations are shown on the top of each lane. Short time treatment of Celastrol does not affect AR, AR3, ERG expression, and total p65, IKBα as well as other well known Celastrol targets such as total AKT, phosphorylated AKT-473 and Hsp90. (<b>B</b>) When Celastrol treatments last for 24 h, p536 signaling was almost abolished in all groups. In groups in which Celastrol concentration is equal or higher than 0.5 µM, significant decreased AR and ERG as well as AR3 expression were seen. When concentration reaches 2 µM, 24 h treatment can affect multiple signaling such as phospho-AKT. β-actin was used as the control.</p

Celastrol can significantly inhibit CCL2 expression both at transcriptional and protein level.

<p>(<b>A</b>) VCaP cells were treated with Celastrol for 2 h, the inhibition of CCL2 at RNA level was shown in a dose-depedent manner by real-time PCR; (<b>B</b>) when Celastrol treatments were done for 24 h, even 0.05 µM can dramatically decrease CCL2 expression; (<b>C</b>) when exposed to Celastrol for 24 h, secreted CCL2 protein in culture medium by VCaP cells are significantly decreased. ELISA data are shown. Each concentration was tested in triplicate and the experiment was repeated three times. Asterisks indicate statistically significant differences between Celastrol treated group and the control by t-test.</p

Celastrol inhibit VCaP cell growth <i>in vivo</i>.

<p>(<b>A</b>) Nude mice were injected subcutaneously with VCaP cells expressing luciferase. After one week mice were treated with 0.5 mg/kg Celastrol or vehicle only. Luciferase flux of tumors prior to euthanasia and tumor and body weights at termination of treatment are shown. Asterisks indicate statistically significant differences between treated group and control group. (<b>B</b>) Inhibition of AR, ERG and p536 by quantitative Western blot normalized to β-actin. Representative Western blot of VCaP tumor extracts with antibodies against AR, ERG or p536 is shown. In each western blot, lanes 1–4 are the control tumors and lanes 5–8 are Celastrol treated tumors. β-actin is a loading control. (<b>C</b>) A tissue microarray (TMA) was prepared for IHC analysis for these mice tumors collected. Representative samples are shown. Images in the left two panels are for a control mouse tumor and images in the right two panels are for a Celastrol treated mouse tumor. H&E, AR, ERG and p536 staining are shown both at magnification of 40× and 100×. Dramatically decreased AR, ERG and p536 expression caused by Celastrol treatment are seen.</p

Celastrol targets T/E fusion, AR and AR3 expression at transcriptional level by quantitative Real-time PCR.

<p>When VCaP cells were treated with different concentration of Celastrol for 24 h, inhibition of targeted genes were evaluated by quantitative real-time PCR. β-actin was used as the control gene. T/E fusion expression(<b>A</b>), AR(<b>B</b>) and AR3 (<b>C</b>) expression are shown in each figure. The inhibition of these genes at RNA levels by Celastrol are significant in a dose-depedent manner. Asterisks indicate statistically significant differences between Celastrol treated group and the control by t-test.</p

Proteins that induced strong T-cell responses in mice immunized with S19.

<p>Proteins that induced strong T-cell responses in mice immunized with S19.</p

Protection against <i>B. abortus</i> 544 infection induced by recombinant <i>B. abortus</i> proteins immunization.

a<p>Units of protection were obtained by subtracting the mean log₁₀CFU/spleen of the vaccinated group from the mean log₁₀CFU/spleen of the control (PBS) immunized group.</p>*<p>p value<0.05.</p

IFN-γ secretion by splenocytes stimulated with the recombinant <i>B. abortus</i> proteins.

<p>Spleen cells from mice immunized with S19 or PBS were stimulated in vitro with recombinant proteins, ConA (positive control), or complete medium (negative control). Supernatants were collected and IFN-γ was determined by using an ELISA Quantikine Mouse kit (R&D Systems). All assays were performed in triplicate and the concentrations for IFN-γ in the culture supernatants were calculated by using the quantification formula. Significant differences between S19-immunized mice and PBS-immunized mice are indicated as follows: *, P<0.001.</p

Humoral immune response induced by CobB, AsnC, and Cu-Zn SOD.

<p>BALB/c mice (n = 5) were inoculated intraperitoneally with purified recombinant proteins plus adjuvant. At 0, 15, 30, and 45 days post the immunization, serum samples were collected and IgG antibody titers were determined by ELISA. The values are mean titers ± SD of antibodies from the five mice.</p

SDS-PAGE of the recombinant proteins.

<p>The expression of recombinant fusion proteins were analyzed by SDS-PAGE. M, molecular size protein markers; lane 1–12, the purified fusion proteins: BAB1_0063, BAB1_0116, BAB1_0381, BAB1_0512, BAB1_0553, BAB1_0560, BAB1_0597, BAB1_0722, BAB1_0812, BAB1_0917, BAB1_1108, BAB1_1124.</p