Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway
Fig 5
The src kinase c-yes orchestrates the cl-CD95L-mediated PI3K/Akt signaling pathway and cell migration.
(A) Left panels: The leukemic cell line H9 was transiently transfected with Lck-GFP. 24 h after transfection, living cells were treated or untreated for the indicated times in the presence of cl-CD95L (100 ng/ml). 0 min (0’) condition corresponds to cells analyzed prior to the addition of 100 ng/mL of cl-CD95L. CD95 was stained using a mouse anti-CD95 mAb and a goat Alexa555-coupled anti-mouse IgG mAb. Right panels: Activated PBLs were incubated for the indicated times with 100 ng/ml of cl-CD95L, and then cells were fixed. Lipid rafts were stained using Alexa488-cholera toxin B subunit and CD95 was followed as previously mentioned. Images were acquired with a confocal microscope with a 63× objective. 0 min (0’) condition corresponds to cells analyzed prior to the addition of 100 ng/mL of cl-CD95L. Cell morphology was followed using phase contrast microscopy (Bars = 7.5 mm). The percentage of T-cells displaying a CD95 cluster was assessed (300 cells counted for each condition). Among the activated T-lymphocytes showing CD95 cluster, the amount of CD95-CAP co-localized with lipid rafts was assessed. (B) H9 T-cells were pre-incubated for 30 min with DMSO (vehicle), 10 mM of PP2, 5 mM of LY294002 (LY), 2.5 mM of wortmannin (Wort), or 40 mM of zVAD-fmk (zV) and then treated for 15 min with 100 ng/ml of cl-CD95L. Untreated condition corresponds to cells incubated with supernatant of pcDNA3.1(+)-transfected HEK cells. Cells were lyzed and Akt phosphorylation (S473) and whole Akt were assessed by immunoblots. (C) The H9 T-cell line (left panels) and activated PBLs (right panels) were incubated for indicated times with 100 ng/ml of cl-CD95L or APO1-3. APO1-3 is an agonistic anti-CD95 mAb incubated for 15 min with indicated cells to activate CD95 and trigger DISC formation. Then, cells were lyzed and CD95 was immunoprecipitated. The 0 min condition corresponds to unstimulated cells directly lyzed to next immunoprecipitate CD95. APO1-3 also serves for the immunoprecipitation step of CD95 (see Materials and Methods). The immune complex was resolved in a 10% SDS-PAGE and indicated immunoblots were performed. Total lysates were depicted to confirm that the same amount of protein was present for each immunoprecipitation. The black stars represent heavy chains of Ig. (D) Boyden chamber assays were performed as described in Materials and Methods. PS120CD95 (All PS120 cells have been reconstituted with wild type NHE1 (see Materials and Methods) were pre-incubated with or without a non-cytotoxic and non-cytostatic concentration of PP2 (10 mM) or DMSO (vehicle) for 30 min and then treated (cl-CD95L) or untreated (Untreated condition corresponds to cells incubated with supernatant of pcDNA3.1(+)-transfected HEK cells) for 24 h with 100 ng/ml of cl-CD95L. Data are representative of three independently performed experiments. The vehicle conditions in the presence or absence of cl-CD95L also serve in S9B Fig to decipher the effect of zVAD-fmk. (E) Activated PBLs were pre-incubated with 10 mM of the src inhibitor PP2, 5 mM of the PI3K inhibitor LY294002 (LY) or DMSO (vehicle) and then incubated in the presence of cl-CD95L (100 ng/ml) for 24 h. Untreated cells correspond to activated PBLs incubated for 24h with supernatant of pcDNA3.1(+)-transfected HEK cells. Then, endothelial transmigration of PBLs was assessed as described in Materials and Methods. (F) The silencing effect of the c-yes-targeting shRNAmir-pGIPZ vectors was analyzed by immunoblot in lentiviral-transduced PS120CD95 cells. 72 h after transduction, cells were lysed and 100 mg of lysate was loaded per line. β-actin serves as loading control. (G) The T-cell line H9 was transduced with scramble (scr) or c-yes (shYes#3)-targeting shRNAmir-containing lentivirus. 72 h after transduction, living cells were harvested and green cells were sorted by flow cytometry using FACSAria. Cells were immediately stimulated with cl-CD95L (100 ng/ml) (15 min) or left untreated (0 min), and the amounts of Akt phosphorylation (S473) were assessed by immunoblot. (H) Cell migration of indicated shRNAmir-transduced PS120CD95 was assessed using the Boyden chamber assay. Left panels: 72 h after transduction, cells were treated (cl-CD95L) or untreated (supernatant of pcDNA3.1(+)-transfected HEK cells) with 100 ng/ml of cl-CD95L for 24 h. For each experiment, five pictures of the migrating cells were taken, and a representative picture was depicted (Bars = 50 mm). Right panel: To quantitatively measure cell motility, Giemsa-stained migrating cells from the lower side of the membrane were lyzed and absorbance was measured at a wavelength of 560 nm. Values represent means and SD of three independently performed experiments. (I) H9 cells were transduced as mentioned in (G), and the green cells were incubated in the presence of cl-CD95L (100 ng/ml) or supernatant of pcDNA3.1(+)-transfected HEK cells (untreated) for 24 h. Then, the endothelial transmigration of the sh-RNA-expressing T-cells was assessed as described in Materials and Methods.