Fig 5.

KRT17 is necessary for GLI1-mediated oncogenesis in Ewing sarcoma. (A) Western blot analysis of KRT17 in A673 cells infected with a control shRNA (Luc) or two different shRNA constructs targeting KRT17. Tubulin was used as the loading control. (B) Growth assays (3T5) for the A673 cells described above for panel A. Student's t test showed no significant difference in growth curves. (C) Quantification of colonies formed in methylcellulose by A673 cells expressing a control shRNA (Luc) or two different KRT17 shRNAs, reexpressing an empty vector or an RNAi-resistant KRT17 cDNA construct. Error bars indicate standard deviations of duplicate assays. P values were determined by using Student's t test, comparing all conditions to the control knockdown/empty vector condition (∗∗, P ≤ 0.01). (D to F) Survival curves for immunodeficient mice subjected to subcutaneous or intratibial injections with A673 cells or SK-N-MC cells expressing a control shRNA (ERG) or a KRT17 shRNA. Five mice and 12 mice were used per condition for the A673 cells and the SK-N-MC cells, respectively. For the subcutaneous model, both flanks of each mouse were injected subcutaneously. Under the control conditions, one mouse died due to the anesthesia and was censored from the analysis. Therefore, 8 and 10 tumors were measured for the control knockdown and KRT17 knockdown groups, respectively. For the intratibial model, the right tibia of each mouse was injected, and therefore, 5 tumors for the A673 group and 12 tumors for the SK-N-MC group were measured for both the control (ERG) knockdown and the KRT17 knockdown groups. The mice in each group in the subcutaneous model were sacrificed once their tumors reached a size limit of 2 cm³. The mice in each group in the intratibial model for both A673 and SK-N-MC cells were sacrificed once their tumors reached a size limit of 1.5 cm³. Percent survival was plotted for both models as Kaplan-Meier survival curves by using GraphPad Prism. The P values determined by log-rank test (Mantel-Cox test) using GraphPad Prism are indicated. (G) Western blot analysis of control (ERG) shRNA- or KRT17 shRNA-expressing tumors from the subcutaneous injection model described above for panel D. KRT17 levels in the tumors were compared to levels in the parental A673 cells expressing either the control shRNA or KRT17 shRNA, used to inject mice. Tubulin was used as the loading control. (H) Quantification of colonies formed in methylcellulose by A673 cells expressing a control shRNA (Luc) or a GLI1 shRNA and reexpressing the empty vector, 3×FLAG-tagged GLI1, or 3×FLAG-tagged KRT17 cDNA constructs. Error bars indicate standard deviations of duplicate assays. The P value was determined by using Student's t test, comparing the GLI1 knockdown/empty vector conditions to the control knockdown/empty vector conditions (∗∗∗, P ≤ 0.001).