Figure 1.
Expression of CA-Lyn promotes the survival of nutrient-deprived U87 human GBM cells.
U87 human GBM cells expressing CA-Lyn, DN-Lyn or LV control were plated in DMEM with L-glutamine and 10% FBS. After 12 h, nutrient deprivation was induced by replacing the medium with L-glutamine- and FBS-free DMEM with 1% BSA. A, After 48 h of nutrient deprivation, whole cell lysates were western blotted with the indicated antibodies. B-D, After 7 days of nutrient deprivation, cell viability was determined by trypan blue exclusion (B–D) and apoptosis by FACs analysis of Annexin-V-labeled cells (E), blotting for cleaved caspase-7 (F), or FACS analysis for Annexin-V and propidium-iodide-labeled cells (G). B–E, and G, Conditions were assayed in replicas of 3 or 4, and the data analyzed and plotted as the mean±SEM. D & E, Statistical analysis using one-way ANOVA. G, Statistical analysis using t-test.
Figure 2.
Expression of CA-Lyn increases EdU incorporation in nutrient-deprived U87 GBM cells.
A–C, U87 cells expressing CA-Lyn, DN-Lyn or the LV control were plated and then starved of L-glutamine and FBS as described in Figure 1A. A, After 7 days of starvation cells were acetone fixed, stained with propidium iodide and analyzed for DNA content by FACS analysis. B, EdU was added after 41/2 days of nutrient deprivation and 18 hours later propidium-iodide was added and EdU incorporation analyzed by FACS. A & B, Conditions were assayed in replicas of 3 and the data analyzed as the mean±SD and plotted as a bar graph. C. 20 h after initiation of nutrient deprivation PP2 (200 nM) or DMSO (vehicle control) was added to the media. On day 5, viable adherent cells were counted by trypan blue exclusion. Conditions were performed in replicas of 3 or 4, and the data analyzed and presented as the mean±SEM.
Figure 3.
Expression of CA-Lyn increases the numbers of autophagosomes per cell and the levels of LC3B-II protein in nutrient-deprived U87 GBM cells.
U87 GBM cells expressing CA-Lyn, DN-Lyn or the LV control and GFP downstream of the IRES were transduced with RFP-LC3 lentiviral vector then plated and subjected to nutrient deprivation as in Figure 1A. A-B, After 5 days, cells were fixed and stained with DAPI nuclear dye. Representative photomicrographs (Mag X40; Scale bar 10 µm) are shown in (A) with arrows indicating autophagosomes (puncta). The numbers of puncta per cell, counted in at least 25 cells for each construct, are shown in (B) in which data are presented as the mean ± SEM with analysis using one-way ANOVA. C-D, At the indicated time-points after initiation of nutrient deprivation, whole cell lysates were western blotted with the indicated antibodies.
Figure 4.
Expression of CA-Lyn increases levels of pAMPK in nutrient-deprived GBM cells.
A-D, U87 GBM cells expressing CA-Lyn, DN-Lyn or the LV control were plated and subjected to nutrient deprivation as described in Figure 1A. A-B, 15 h prior to detergent lysis (day 5), cells were treated with vehicle, chloroquine (CQ; 10 µM) (A), or 3-methylamine (3-MA; 5 µM), and then whole cell lysates western blotted with the indicated antibodies. C & D, Cells were detergent lysed after five days of nutrient deprivation and blotted with the indicated antibodies. The densitometric readings for the LC3B-II band were normalized to GAPDH, the pS6 band normalized to total S6, and the pAMPK band normalized to total AMPK.
Figure 5.
Expression of CA-Lyn in GBM cells results in significantly larger tumors whereas expression of DN-Lyn results in smaller tumors.
Cells were harvested, resuspended in PBS, and 1×105 cells in 5 µl injected with stereotactic assistance into the nude mouse brain. At 35 days, mice were euthanized, brains harvested, fixed and analyzed. (A, B) Representative H&E sections of tumors generated on injection of U87 cells expressing control LV, CA-Lyn or DN-Lyn (n = 6/group) are shown in (A). Arrows denote the tumors (Mag X1.25; Scale bar 5 mm). Tumor volumes for each group are presented as the mean±SEM and analyzed using one-way ANOVA in (B). C-D, Representative TUNEL-stained tumor sections are shown in (C) (Scale bar 50 µm). Arrows denote TUNEL-positive cells. TUNEL-positive cells were counted in at least 2 slides for each tumor and the percentage of TUNEL-positive cells is presented as the mean ± SEM with analysis using a Mann Whitney test (D). E-F, Ki67 staining of tumor sections is shown (Mag X40) in (E). Percentage of Ki67 positive cells is presented as the mean±SEM with analysis using one-way ANOVA (F).
Figure 6.
Expression of CA-Lyn in GBM cells results in larger tumors containing autophagic vacuoles.
Xenograft tumors expressing LV, CA-Lyn or DN-Lyn were generated as described in the legend for Figure 5 and after euthanasia on day 35 tumors were fixed in EM fixative, followed by transmission EM as described in the Materials and Methods. A-E, Three different representative cells from CA-Lyn tumors; panel B-higher magnification of the box in Panel A, and panel D-higher magnification of the box in panel C. F & G, Representative tumor cell from LV tumor; panel G-higher magnification of the box in panel F. H & I, Representative tumor cell from DN-Lyn tumor; panel I-higher magnification of the box in panel H. Scale bars in each panel denote 1 µm. Arrows denote examples of autophagic vacuoles. J, Quantitation of autophagic vacuoles/tumor cell shows a significant increase in the CA-Lyn tumors as compared to the LV tumors (p value <0.0001; t-test). K, Scattergram denoting the number of autophagic vacuoles/tumor cell.