The RasGAP moiety carries the tumor sensitizing activity of TAT-RasGAP_317-326.

<p><b>A.</b> CCRF-CEM cells were seeded in 6-well plates and directly treated with 10 μM TAT-RasGAP_317-326, TAT, TAT-Scrambled or TAT-Mutated in the absence or in the presence of 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death. <b>B-C.</b> The TC252 (B) and NB1-FBS (C) cell lines were treated similarly but in combination with 4-HC only. P10, TAT-RasGAP_317-326; TAT-S, TAT-Scrambled; TAT-M, TAT-Mutated; 4-HC, 4-hydroperoxycyclophosphamide. Means with the same letter are not significantly different.</p

Necrosis-like death induced by 4-HC in CCRF-CEM cells.

<p><b>A.</b> Three hundred thousand CCRF-CEM cells were seeded in 6-well plates and directly treated with 50 μM 4-HC. Cell death was evaluated after several time points (0, 3, 6, 12 and 24 hours) using 7-AAD and Annexin V-FITC staining. <b>B.</b> CCRF-CEM cells were seeded in 6-well plates and directly treated with the indicated doses of 4-HC, etoposide or vincristine in the presence or in the absence of 10 μM TAT-RasGAP_317-326. After 24 hours of drug incubation, 7-AAD and Annexin V-FITC staining was performed to evaluate cell death. 4-HC, 4-hydroperoxycyclophosphamide.</p

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

25 avril 19411941/04/25 (A41,N14694)

Mechanism of action and clinical use of cyclophosphamide, doxorubicin, etoposide and vincristine.

<p>Only the tumor types that were analyzed in this study are mentioned here. The listing was based on the protocols of the Children’s Oncology Group and the International Society of the Pediatric Oncology (EURO-E.W.I.N.G. 99; AALL0232; HR-NBL-1.5/SIOPEN). NB, neuroblastoma; ES, Ewing sarcoma; ALL, acute lymphoblastic leukemia; AML: acute myeloid leukemia.</p><p>Mechanism of action and clinical use of cyclophosphamide, doxorubicin, etoposide and vincristine.</p

Antibodies tested in this study.

<p>Antibodies tested in this study.</p

The effect of TAT-RasGAP_317-326 as a chemosensitizer of leukemia cells and non-tumor lymphocytes.

<p><b>A.</b> Two acute myeloid leukemia cell lines (THP-1 and M-07e) and one T acute lymphoblastic leukemia cell line (CCRF-CEM) were seeded in 6-well plates and directly treated with 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations in the presence or in the absence of 10 μM TAT-RasGAP_317-326. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death (last four columns). Alternatively (first column), the cells were treated with increasing concentrations of TAT or TAT-RasGAP_317-326 alone. After 24 hours, the evaluation of cell death was carried out using 7-AAD staining. <b>B.</b> Isolated lymphocytes from three distinct healthy subjects were treated as described in Fig. 2A. The dosages of chemotherapeutic agents used to treat the healthy lymphocytes from the three subjects are similar to those used to treat the T-ALL CCRF-CEM cells. Note that the graphs are derived from single experiments where lymphocytes are immediately used after their isolation (if cultured <i>in vitro</i>, they would experience high levels of spontaneous apoptosis that would prevent accurate measurement of anti-cancer drug- and peptide-induced death). T-ALL, T-acute lymphoblastic leukemia; AML, acute myeloid leukemia; 4-HC, 4-hydroperoxycyclophosphamide. * p<0.05 t-test after Bonferroni correction.</p

Antibodies that detect SHB in western blot fail to immunoprecipitate it.

<p>HEK 293T CE12 cells having one of their alleles tagged with V5 (V5-tag) or HEK 293T cells knock-out for Shb (KO) were used to prepare whole cell extracts, which were then used as material for the immunoprecipitations (40 μg of total protein). Shb was immunoprecipitated from the cell extracts (2 mg of total protein) using the 74483 and 530 antibodies from Santa Cruz. An anti-V5 mouse antibody was used for control immunoprecipitations. The samples were then analysed by western blot using a rabbit anti-V5 antibody. The arrowhead indicates the band corresponding to Shb. The bottom panel shows a lower exposure of the indicated area to better distinguish the heavy chain of the antibody used in the immunoprecipitation (indicated by *), against which the goat anti-rabbit secondary antibodies react (weakly in the case of mouse primary antibodies).</p

TAT-RasGAP_317-326 sensitizes neuroblastoma cells to chemotherapy and displays a direct killing effect on NB1-derived cell lines.

<p><b>A.</b> The light grey thick arrow on top of the figure represents the treatment regimen administrated to the patient from whom the three NB1-derived cell lines were established. The NB1 cells are derived from primary high risk neuroblastoma bone marrow samples. NB1-NBM and NB1-FBS were established at initial diagnosis and cultured in NBM and DMEM media, respectively. NB1-NBM-Re was established at a subsequent relapse and cultured in DMEM. The three NB1-derived cell lines were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120487#pone.0120487.g003" target="_blank">Fig 3</a>. <b>B.</b> The LAN-1 cell line is derived from a high risk neuroblastoma. LAN-1 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120487#pone.0120487.g003" target="_blank">Fig 3</a>. M, month; BM, bone marrow; 4-HC, 4-hydroperoxycyclophosphamide. * p<0.05 t-test after Bonferroni correction.</p

V5-tagged Shb is specifically detected with an anti-V5 antibody.

<p>The V5 signal corresponding to Shb-V5 was detected by western blot. HEK 293T cells were transfected with empty plasmid or a Stag-Shb-V5 encoding plasmid. To test the specificity of the signals, the CE12 cell line bearing an endogenous V5-tagged Shb allele was knocked-down for Shb (siRNA^Shb) or treated with control siRNA (siRNA^SCR). Arrowheads point to Shb-V5. Equal amounts of proteins were loaded (50 μg) on each lane.</p

Assessment of nine anti-Shb antibodies.

<p>Anti-Shb antibodies were tested by western blotting for their ability to recognize their antigen in HEK 293T cells transfected with either empty plasmid or a Stag-Shb-V5 plasmid, or in CE12 cells, treated with control or Shb-specific siRNA. Whole cell lysates are those that were used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188311#pone.0188311.g001" target="_blank">Fig 1</a>. Arrowheads point to Shb-V5; *, unspecific signals.</p