Journal: Cell host & microbe

Article Title: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells.

doi: 10.1016/j.chom.2009.03.003

Figure Lengend Snippet: Figure 1. KSHV and EBV MiRNA-Mediated Translational Repression of MICB (A) FACS analysis of ULBP3 and of MICB expres- sion by RKO cells transduced with lentivirus encoding either the KSHV miRNA cluster or the EBV pri-miR-BART2. The gray empty histogram represents the staining of ULBP3 or of MICB in the viral miRNA-transduced cells. The black empty histogram represents the staining of cells transduced with a control miRNA (miR-K12-12). The filled gray histogram represents the staining of the secondary mAb only. (B) Western blot analysis of MICB protein levels expressed by the cells stained in (A). Quantifica- tion of relative intensity was performed by ImageJ software. (C) Quantitative real-time PCR analysis of the MICB mRNA levels in the transduced RKO cells. Relative mRNA abundance is shown as a percentage of the level of HPRT mRNA (encoding hypoxanthine guanine phosphoribosyltransfer- ase), and error bars (SD) are derived from tripli- cates. MiR-K12-12 served as negative control.

Article Snippet: Membranes were incubated for 2 hr with 1 mg/ml anti-MICB mAb (R&D Systems), washed with 0.4% Tween in PBS, and then incubated for 0.5 hr with an anti-mouse-HRP secondary antibody (Jackson Laboratory).

Techniques: Transduction, Staining, Control, Western Blot, Software, Real-time Polymerase Chain Reaction, Derivative Assay, Negative Control

Journal: Cell host & microbe

Article Title: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells.

doi: 10.1016/j.chom.2009.03.003

Figure Lengend Snippet: Figure 2. MiR-K12-7 and MiR-BART2-5p Are the Viral MiRNAs that Mediate MICB Downregulation (A) FACS analysis of MICA expression by 293T cells transduced with lentivirus encoding the above indicated KSHV miRNA. The gray empty histogram represents the staining of MICA expression by the transduced cell. The black empty histogram represents the staining of miR-K12-1, which was used as a control. The filled gray histogram represents the staining of the secondary mAb only. (B) FACS analysis of MICB expression by RKO cells transduced with lentivirus encoding the indicated KSHV miRNA. The gray empty histogram represents the stain- ing of MICB expression by the transduced cell. The black empty histogram represents the staining of miR-K12-1 or miR-K12-2 miRNAs that were used as controls. The filled gray histogram represents the staining of the secondary mAb only. The red histogram shows the MICB downregulation mediated by miR-K12-7.

Article Snippet: Membranes were incubated for 2 hr with 1 mg/ml anti-MICB mAb (R&D Systems), washed with 0.4% Tween in PBS, and then incubated for 0.5 hr with an anti-mouse-HRP secondary antibody (Jackson Laboratory).

Techniques: Expressing, Transduction, Staining, Control

Journal: Cell host & microbe

Article Title: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells.

doi: 10.1016/j.chom.2009.03.003

Figure Lengend Snippet: Figure 4. The MICB Downregulation by MiR-K12-7 and MiR-BART2-5p Results in Reduced NKG2D-Mediated Killing (A and B) Cells expressing miR-K12-7 or miR- BART2-5p exhibit reduced NK killing. Bulk NK cells were preincubated either with anti-NKG2D mAb (white) or with isotype control mAb (gray). Labeled RKO cells expressing miR-K12-7 (A), miR-BART2-5p (B), or control miRNA (miR-K12- 12) (A and B) were then added and incubated for 5 hr. Shown are mean values ± SD. Statistically significant differences are indicated (*p % 0.03, by one-tailed t test). Error bars (SD) are derived from triplicates.

Article Snippet: Membranes were incubated for 2 hr with 1 mg/ml anti-MICB mAb (R&D Systems), washed with 0.4% Tween in PBS, and then incubated for 0.5 hr with an anti-mouse-HRP secondary antibody (Jackson Laboratory).

Techniques: Expressing, Control, Labeling, Incubation, One-tailed Test, Derivative Assay

Journal: Cell host & microbe

Article Title: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells.

doi: 10.1016/j.chom.2009.03.003

Figure Lengend Snippet: Figure 5. MiR-K12-7 and MiR-BART2-5p Are Expressed and Are Functional during Authentic Viral Infection (A) Quantitative real-time PCR analysis of viral miRNA expression using custom-designed Taqman RT primer and real-time PCR probe. The KSHV-infected BCBL1 cell line was assayed for the expression of miR-K12-7. The EBV-trans- formed 721.221 cell line was assayed for the expression of miR-BART2-5p. The cell lines served as negative controls for each other. RKO cells transduced with the relevant lentivirus were used as positive controls. Error bars (SD) are derived from triplicates. (B) FACS analysis of the expression of MICA and MICB by KSHV-infected and EBV-infected cell lines. Left side shows staining of BCBL1 cells transduced with an anti-miR-K12-7-sponge (gray empty histogram) or with anti-miR-BART2-5p sponge as control (black empty histogram). The filled gray histogram is of secondary mAb staining only. Right side shows staining of 721.221 cells transduced with an anti-miR-BART2-5p sponge (gray empty histogram) or with anti-miR-K12-7 sponge as control (black empty histogram). The filled dark gray histogram is the staining of MICB expression by parental 721.221. The filled gray histogram is of secondary mAb staining only. (C) The specific effect of the antiviral miRNA sponges was tested in a dual luciferase reporter assay. 293T cells were transduced with miR- K12-7 (left) or with miR-BART2-5p (right). In addi- tion, each 293T cell was transduced with either a relevant sponge (white) or an irrelevant sponge (gray). Firefly luciferase activity was normalized to Renilla luciferase activity and then normalized to the average activity of the control reporter. Shown are mean values ± SD. Statistically signifi- cant differences are indicated (*p < 0.006, by one-tailed t test). Error bars (SD) are derived from triplicates.

Article Snippet: Membranes were incubated for 2 hr with 1 mg/ml anti-MICB mAb (R&D Systems), washed with 0.4% Tween in PBS, and then incubated for 0.5 hr with an anti-mouse-HRP secondary antibody (Jackson Laboratory).

Techniques: Functional Assay, Infection, Real-time Polymerase Chain Reaction, Expressing, Transduction, Derivative Assay, Staining, Control, Luciferase, Reporter Assay, Activity Assay, One-tailed Test

Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: VPA upregulates the expression of MICA and MICB in pancreatic cancer cells. Pancreatic cancer cells were incubated with or without 1 mM VPA for 24 h. (A) Quantitative real-time RT-PCR analysis of MICA and MICB mRNA expression. (B) Flow cytometry analysis and quantification of MICA and MICB protein expression on the surface of pancreatic cancer cells. MFI, mean fluorescence intensity. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. ** P < 0.01.

Article Snippet: PANC-1, MIA PaCa-2, and BxPC-3 cells were cultured to 80-90% confluence, trypsinized, washed twice with phosphate buffer solution (PBS), re-suspended in PBS at 1 × 10 6 cells/100 μl, incubated with PE-anti-human MICA and MICB antibody or an isotype control antibody for 30 min, and then analyzed on a Becton Dickson LSR II flow cytometer (BD, Franklin Lakes, NJ, USA).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Flow Cytometry, Fluorescence

Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: PI3K/Akt signaling is required for VPA-induced upregulation of MICA and MICB in pancreatic cancer cells. (A, B) Quantitative real-time RT-PCR analysis. The VPA-induced upregulation of MICA and MICB mRNA expression were inhibited by the HER2/HER3 inhibitor lapatinib and the PI3K inhibitor LY294002, but not by the ATM/ATR inhibitor caffeine. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. * P < 0.05; ** P < 0.01; ns P > 0.05. (C) Flow cytometry analysis. The VPA-induced upregulation of MICA and MICB protein expression on the cell surface were inhibited by the PI3K inhibitor LY294002. MFI, mean fluorescence intensity; * P < 0.05. (D) Western blotting analysis. Transfection of the PI3KCA siRNA inhibited PI3KCA protein expression at 48 h post-transfection. NC, negative control; siR1-3, PI3KCA_siR sequence 1-3; ** P < 0.01. (E) Quantitative real-time RT-PCR analysis. VPA-induced upregulation of MICA and MICB mRNA expression were attenuated in PI3KCA-knockdown cells; Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. ** P < 0.01. (F) Flow cytometric analysis. VPA-induced upregulation of MICA and MICB protein expression on the cell surface were attenuated in PI3KCA-knockdown cells. MFI, mean fluorescence intensity; * P < 0.05; ** P < 0.01. (G) LDH release assay. The VPA-induced susceptibility of cancer cells to NK cell-mediated cell lysis was reduced in PI3KCA-knockdown cells. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. ** P < 0.01 and * P < 0.05 for NC vs. siR2; ▲▲ P < 0.01 and ▲ P < 0.05 for NC vs. siR3. siR2, PI3KCA siR sequence 2; siR3, PI3KCA siR sequence 3.

Article Snippet: PANC-1, MIA PaCa-2, and BxPC-3 cells were cultured to 80-90% confluence, trypsinized, washed twice with phosphate buffer solution (PBS), re-suspended in PBS at 1 × 10 6 cells/100 μl, incubated with PE-anti-human MICA and MICB antibody or an isotype control antibody for 30 min, and then analyzed on a Becton Dickson LSR II flow cytometer (BD, Franklin Lakes, NJ, USA).

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Western Blot, Transfection, Negative Control, Sequencing, Knockdown, Lactate Dehydrogenase Assay, Lysis

Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: VPA sensitizes pancreatic cancer cells to NK cell-mediated lysis in vivo by upregulating the expression of MICA and MICB. (A, B) Excised xenograft tumors and growth curves for the xenografts; VPA enhanced the growth inhibitory effect of NK cells in vivo ; this effect was attenuated by the PI3K inhibitor LY294002. Data are mean ± SD of five mice per group; * P < 0.05 for NK-92 vs. NK-92 + VPA; Δ P < 0.05 for NK-92 + VPA + LY294002 vs. NK-92 + VPA. (C) Immunohistochemical staining and quantification of MICA and MICB expression in the xenograft tumors; VPA significantly upregulated the expression of MICA and MICB in the pancreatic cancer xenograft tumors in vivo , this effect was attenuated by the PI3K inhibitor LY294002.

Article Snippet: PANC-1, MIA PaCa-2, and BxPC-3 cells were cultured to 80-90% confluence, trypsinized, washed twice with phosphate buffer solution (PBS), re-suspended in PBS at 1 × 10 6 cells/100 μl, incubated with PE-anti-human MICA and MICB antibody or an isotype control antibody for 30 min, and then analyzed on a Becton Dickson LSR II flow cytometer (BD, Franklin Lakes, NJ, USA).

Techniques: Lysis, In Vivo, Expressing, Immunohistochemical staining, Staining