A) Immunoblotting evaluations of KIT and PKCθ expression and activation, in KIT-positive GIST cell lines (GIST882, GIST430 and GIST48) at 96 hours after infection by lentiviral KIT or PKCθ shRNA constructs. KIT shRNA infection inhibited KIT expression and activation in each cell line. PKCθ shRNA inhibition inhibited PKCθ expression and activation in each cell line, and also inhibited KIT expression and activation in the imatinib-resistant GIST48 and GIST430 cell lines. Control lanes, for each cell line, include uninfected cells (untreated lane) and cells infected with empty lentiviral vector. B) Comparison immunoblotting evaluations of KIT and PKCθ expression, after infection of KIT-positive Ewing's sarcoma (EWS502) cells with lentiviral KIT or PKCθ shRNA constructs. These studies show that PKCθ shRNA treatment does not inhibit KIT expression, in the absence of PKCθ expression, indicating that PKCθ shRNA-mediated KIT inhibition in GIST (2A) does not result from nonspecific interactions between the PKCθ shRNA and KIT mRNA. The GIST882 cells provide a positive control for KIT and PKCθ expression. EWS502 no-treatment controls include uninfected cells (untreated lane) and EWS502 infected with empty lentiviral vector. C) Immunoblotting evaluations of KIT-positive GIST cell lines (GIST882, GIST430 and GIST48) at 96 hours after infection by lentiviral KIT or PKCθ shRNA constructs. The immunoblotting assays evaluated affects of KIT and PKCθ knockdown on signaling intermediates (AKT, MAPK p42/44, S6, STAT1), proliferation markers (Cyclin A and PCNA), and cell cycle checkpoint proteins (p27, p21 and p53). The empty vector lane is a parallel control. The PI3-K and Actin immunostains show equivalence of lane loading. Control lanes, for each cell line, include uninfected cells (untreated lane) and cells infected with empty lentiviral vector.

A) Cell viability was evaluated in KIT-positive GIST882 (black bars), GIST48 (gray bars), and GIST430 (white bars) cell lines, at day 8 and day 12 after infection with lentiviral KIT and PKCθ shRNAs. Viability was analyzed using Cell-titer Glo® ATP-based luminescence assay. The data were normalized to the empty lentivirus control infections, and represent the mean values (± s.d.) from quadruplicate cultures. B) Apoptosis was evaluated in GIST882 (black bars) and GIST48 (gray bars) cell lines, at day 4 after infection with lentiviral KIT and PKCθ shRNAs. Caspase 3/7 activity was measured using a Caspase-Glo® luminescence assay. The data were normalized to the empty lentivirus control infections, and represent the mean values (± s.d.) from quadruplicate cultures. C) Cell cycle analyses were performed in GIST882 and GIST48 cells at 8 days and 4 days after infection by lentiviral KIT or PKCθ shRNA constructs, respectively. Apoptotic response was greater, in both cell lines, after treatment with PKCθ shRNA than after treatment with KIT shRNA.

Immunoblotting evaluations of GIST cell lines show strong PKCθ activation and expression in KIT-positive lines (GIST882, GIST430 and GIST48) but not in KIT-negative lines (GIST62 and GIST522). The Jurkat T-cell acute lymphoblastic leukemia provides a positive control for PKCθ activation and expression.

A) Immunoblotting evaluations of GIST882 cells, at 10 and 20 days after infection by lentiviral KIT or PKCθ shRNA constructs. KIT shRNA resulted in decreased expression of KIT, phospho-AKT, phospho-S6, and the proliferation markers Cyclin A and PCNA. PKCθ shRNA resulted in decreased expression of PKCθ, KIT, phospho-AKT, and Cyclin A. The PI3-K and β-actin immunostains show equivalence of lane loading. B) GIST882 cell culture appearance, when evaluated at 10 and 20 days after infection by lentiviral KIT or PKCθ shRNA constructs, showing growth inhibition compared to control GIST882 cultures infected with lentiviral empty constructs.