Journal: BMC Cell Biology

Article Title: Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

doi: 10.1186/1471-2121-12-11

Figure Lengend Snippet: LPA and ATX/LPC induce OPN expression in SGC7901 cells . (A) Time course of OPN stimulation utilizing ATX (50 ng/ml) + LPC (20 μM) (ATX/LPC2). OPN expression was examined in SGC7901 cells by Western blot and real time-PCR. SGC7901 cells were treated with ATX, LPC, LPA, ATX/LPC1, ATX/LPC2 or DMEM/0.1% BSA. Total protein and RNA was extracted for Western blot (C) and real time -PCR (B), respectively. Figure1 D showed the densitometric ratio of OPN protein/α-tubulin. Statistical analysis was performed using Student's t test. *p < 0.05. The ATX/LPC concentration curve for stimulated motility provides a justification for the concentrations of ATX/LPC chosen throughout the manuscript. LPA or ATX/LPC stimulates OPN production in additional cell lines (eg, SMMC7721 in our study: ATX-LPA axis induces expression of OPN in hepatic cancer cell SMMC7721) as well as in SGC7901 cells.

Article Snippet: ATX (recombinant human ENPP-2/Autotaxin) and OPN antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: BMC Cell Biology

Article Title: Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

doi: 10.1186/1471-2121-12-11

Figure Lengend Snippet: ATX/LPC-regulated OPN expression is mediated by LPA2 receptor and activation of Akt and MAPK/ERK . SGC7901 cells were exposed to ATX, LPC, LPA, ATX/LPC2 or DMEM/0.1% BSA for 24 hours or 30 min, and then total RNA and protein were extracted for real time-PCR (A-D) and Western blot (E, F), respectively. (E) Immunoblotting was performed to detect the phosphorylation status of ERK and Akt. Total ERK and Akt levels were assessed as control. (F) The effects of the LPA inhibitor, ERK inhibitor and Akt inhibitor on ATX/LPC2-induced OPN expression in SGC7901 cells. SGC7901 cells were treated with Ki16425, PD98059, LY294002 or DMSO (0.1%; a solvent control) for 45 min prior to ATX/LPC2 treatment. 24 hours following stimulations, OPN expression was measured by Western blot analysis. All experiments were performed in triplicate and a representative result is shown here. Figure 2G showed the densitometric ratio of OPN protein/α-tubulin. Statistical analysis was performed using Student's t test. *p < 0.05.

Article Snippet: ATX (recombinant human ENPP-2/Autotaxin) and OPN antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Control, Solvent

Journal: BMC Cell Biology

Article Title: Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

doi: 10.1186/1471-2121-12-11

Figure Lengend Snippet: Requirement of OPN in migration induced by ATX-LPA axis protects against Taxol-induced apoptosis in SGC7901 cells . (A) Verification of siRNA knockdown of the OPN protein by Western blot showed a significant reduction of OPN protein in clone3 (SGC7901-siRNA-OPN). (B) The effect of OPN on ATX-LPA-induced migration was assayed using a Transwell assay. Either SGC7901, SGC7901-siRNA-vehicle, or SGC7901-siRNA-OPN cells (1 × 10 5 ) were added in the upper chambers of the transwells, incubated in DMEM/0.1% BSA or medium containing ATX, LPC, LPA, ATX/LPC2 and allowed to migrate for 48 hours at 37°C. Migrated cells on the lower chamber were quantified. (D) Flow cytometric analysis of apoptosis: SGC7901, SGC7901-siRNA-vehicle, or SGC7901-siRNA-OPN cells were treated with Taxol (50 nM) in the absence or presence of each group: ATX, LPC, LPA, ATX/LPC2 or DMEM/0.1% BSA for 24 hours. Apoptosis was analyzed by flow cytometry. Data shown in Figure 4C and E represents the mean ± SD from three individual experiments. Data are mean ± SD of triplicate determinations. Statistical analysis was performed using Student's t test. *p < 0.05.

Article Snippet: ATX (recombinant human ENPP-2/Autotaxin) and OPN antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Knockdown, Western Blot, Transwell Assay, Incubation, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Bim/Bcl-2 balance is critical for maintaining naive and memory T cell homeostasis

doi: 10.1084/jem.20070618

Figure Lengend Snippet: Naive Bcl-2–deficient T cells exhibit increased Bim-mediated death. LN cells from individual naive Bim + / + Bcl-2 + / + (filled circles); Bim + / − Bcl-2 + / − (open triangles); Bim + / − Bcl-2 − / − (filled squares); or Bim − / − Bcl-2 − / − mice (x marks; n = 3 mice/group) were cultured at 37°C and at various times were stained with antibodies against TCR-β and either CD4 or CD8 and propidium iodide, and cell death was assessed by flow cytometry. Results show the percentage of CD4 + (A) or CD8 + (B) T cells that were PI + (dead) ± the SEM. *, statistically significant difference in the death of CD4 + and CD8 + T cells from Bim + / − Bcl-2 − / − mice compared with all other groups (for CD4s P ≤ 0.009; for CD8s P ≤ 0.007). At 48 and 72 h, the death of CD4 + and CD8 + T cells from Bim − / − Bcl-2 − / − mice was significantly decreased compared with Bim + / + Bcl-2 + / + mice (for CD4s P ≤ 0.02; for CD8s P ≤ 0.05). Similarly, LN cells were cultured at 37°C and stained with antibodies against TCR-β, CD44, CD4 or CD8, and AnnexinV-FITC. Cell death was assessed by flow cytometry. Results show the percentage of naive (CD44 lo ) CD4 + (C) and CD8 + (D) T cells that were AnnexinV + (apoptotic or dead) ± the SEM. Death of naive CD4 + and CD8 + T cells from Bim + / − Bcl-2 − / − mice was significantly increased compared with all other groups (P ≤ 0.01). These data are representative of two independent experiments with similar results. (E) LN cells from either C57BL/6 (open squares) or Bim − / − (filled squares) mice were cultured with the indicated concentrations of ABT-737 for 24 h. Results show the percentage of CD4 + T cells (dead) ± the SEM (similar results were obtained with CD8 + T cells). (F) Groups of C57BL/6 mice ( n = 4 mice/group) were injected i.p. with either vehicle (open bars) or ABT-737 (shaded bars) for 14 d and killed, and cell numbers were quantified by flow cytometric analysis. Results show the absolute numbers of CD4 + T cells, CD8 + T cells, and B cells present in the LN ± the SEM. Similar results were obtained in the spleen.

Article Snippet: To assess viability of memory versus naive T cells, cells were stained with antibodies against CD4, CD8, CD44, and AnnexinV-FITC (R&D Systems) and analyzed by flow cytometry.

Techniques: Cell Culture, Staining, Flow Cytometry, Injection

Journal: The Journal of Experimental Medicine

Article Title: Bim/Bcl-2 balance is critical for maintaining naive and memory T cell homeostasis

doi: 10.1084/jem.20070618

Figure Lengend Snippet: Accumulation of memory T cells in Bim +/− Bcl-2 −/− mice is caused by increased proliferation rather than survival. Groups of Bim + / + Bcl-2 + / + and Bim + / − Bcl-2 − / − mice ( n = 3 mice/group) were killed, and LN cells from individual mice were cultured with the indicated concentrations of IL-7 (A) or -15 (B) for 36 h at 37°C. After culture, cells were stained with antibodies against TCR-β, CD8, and CD44 to identify endogenous memory phenotype T cells and with Annexin V-FITC (R&D Systems) to identify cells that were dead, and then analyzed by flow cytometry. Results show the percentage of inhibition of cell death ([{percentage of cells dead in medium – percentage of cells dead in cytokine} / percentage of cells dead in medium] × 100) ± the SD. Cell death without cytokine was 50.9 ± 2.5% for Bim + / + Bcl-2 + / + and 74.8 ± 5.4% for Bim + / − Bcl-2 − / − mice. *, statistically significant difference between Bim + / + Bcl-2 + / + and Bim + / − Bcl-2 − / − mice for IL-7 (P ≤ 0.001) and -15 (P ≤ 0.03). (C and D) Groups of Bim + / + Bcl-2 + / + ( n = 6) and Bim + / − Bcl-2 − / − mice ( n = 5) were infected with LCMV. At day 126 after infection, mice were injected once daily with BrdU for 14 d. On day 141, mice were killed and BrdU accumulation within memory T cells was assessed by staining cells for CD8, CD62L, MHC tetramer, and intracellular BrdU. Results show the percentage of tetramer + CD62L + CD8 + T cells that are BrdU + ± the SEM. Percentages of D b gp33 + (C) and D b np396 + (D) T cells that were BrdU + were significantly increased in Bim + / − Bcl-2 − / − mice compared with either Bim + / + Bcl-2 + / + or Bim − / − Bcl-2 − / − mice (P ≤ 0.002 for both). (E and F) Adoptive transfer experiments. T cells were purified from LN and spleens of 2-mo-old Bim + / + Bcl-2 + / + , Bim + / − Bcl-2 − / − , and Bim − / − Bcl-2 − / − mice. 2 × 10 6 T cells were transferred into Ly5.1 congenic recipient mice ( n = 12 mice/group); on days 1 ( n = 6 mice/group) and 10 ( n = 6 mice/group), recipient mice were killed and donor T cells were quantified in their LNs and spleens by flow cytometry. Results show the percentage of donor naive and memory CD4 + (E) or CD8 + (F) T cells (combined LN and spleen) remaining on day 10, relative to the number present on day 1 after transfer ± the SEM. (G and H) Purified T cells from 20-mo-old C57BL/6 mice were transferred into Ly5.1 congenic mice ( n = 8). Recipient mice were treated with either vehicle ( n = 4) or with ABT-737 ( n = 4) for 10 d, and the number of donor naive and memory T cells was quantified by flow cytometry. Results show the total number of donor CD4 + (G) and CD8 + (H) naive versus memory T cells in mice treated with either vehicle (open bars) or ABT-737 (shaded bars) ± the SEM. Numbers above the shaded bars indicate the fold decrease in cell number by ABT-737. Similar results were obtained in mice without cell transfer.

Article Snippet: To assess viability of memory versus naive T cells, cells were stained with antibodies against CD4, CD8, CD44, and AnnexinV-FITC (R&D Systems) and analyzed by flow cytometry.

Techniques: Cell Culture, Staining, Flow Cytometry, Inhibition, Infection, Injection, Adoptive Transfer Assay, Purification