Figure 1. DRD2 is required for glioblastoma growth.

(A) Schema of genome-wide shRNA screen. Cells were serially passaged after infection with virus containing shRNA constructs. The relative abundance of each shRNA prior to and after passages was subsequently determined (see Methods). (B) Cy5 signal for each gene from three independent U87MG screens (denoted as A, B or C) was plotted against each other (on x-, y-, and z- axes, respectively). High degrees of correlation were seen (R²=0.89-0.92). Similar degrees of correlations were seen for Cy3 signals and for other cell lines. (C) PANTHER pathway analysis of pro-proliferative genes for U87MG and A172. * denotes p < 0.05. (D) PANTHER curated DRD2 pathway genes. * denotes pro-proliferative genes identified in our screen. (E) DRD2 silencing by independent sh-/siRNAs compromised clonogenic U87MG growth in vitro. Efficiency of DRD2 silencing was determined by RT-PCR (bottom panel). Cells were seeded at ~50% confluency, transfected or infected with siRNA or shRNA constructs for 48 hrs and re-plated in serial dilution for clonogenic survival assessment. (F) Clonogenic growth inhibition of long- and short-term passaged, patient-derived glioblastoma lines by haloperidol. Cells were incubated with haloperidol at the indicated concentrations for the duration of the clonogenic assay. (G) Multiple DRD2 antagonists suppressed U87MG clonogenic growth. S: spiperone (5 μM, 10 μM), H: haloperidol (5 μM, 10 μM), R: risperidone (10 μM, 25 μM), L: L-741,626 (10 μM, 25 μM). Cells were incubated with DRD2 antagonists at the indicated doses for the duration of the clonogenic assay. (H) DRD2 knockdown abolished U87MG subcutaneous xenograft growth. Top: 3×10⁵ cells transduced with inducible shRNA2 against DRD2 were injected subcutaneously into the flanks of Nu/Nu mice. 7 days later, the mice were randomized to drinking water with or without doxycycline (1 mg/ml). Tumor size was monitored every 5-7 days. Bottom: Inducible shRNA silencing of DRD2. U87MG cells with a stably integrated, inducible shRNA construct were treated with doxycycline or vehicle for 72 hrs. RNA was then extracted and DRD2 RT-PCR was performed. (I) Expression of an shRNA resistant form of DRD2 (termed DRD2RR) suppressed the anti-proliferative effects of shDRD2 in vivo. Top: U87MG cells transduced with shRNA2 were transfected with DRD2 or DRD2RR, and injected subcutaneously. 7 days post-injection, the mice were fed doxycycline (1 mg/ml) containing water. Tumor size was monitored every 5-7 days. Bottom: DRD2RR expression is resistant to the DRD2 silencing effects of shRNA2. U87MG cells with stably integrated shRNA2 and transfected with DRD2 or DRD2RR (noted here as RR) were treated with doxycycline for 72 hrs. Whole cell lysates were prepared from these cells for DRD2 immuno-blotting.