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. 2014 Jan 23;16(7):933–945. doi: 10.1093/neuonc/not303

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© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Fig. 1. — Silencing and pharmacological inhibition of PRKD2 inhibits GBM cell growth. (A) Effect of PRKD2 RNAi on glioma cell proliferation. Two different siRNA constructs (siP5; siP6) were used to silence PRKD2 expression. Untreated cells and cells transfected with nontargeting siRNA (siScr) were used as controls. Cells were harvested and counted on day 6 post silencing. Results represent mean ± SD of relative cell numbers normalized to untreated cells from one representative experiment done in triplicate. The p53 status of the cells is indicated. Silencing efficacy is shown in the right panel. (B) Effects of pharmacological PRKD inhibition on GBM cell proliferation. The indicated p53^wt and p53^mut GBM cells were incubated in the presence of vehicle dimethyl sulfoxide (DMSO) or 0.5 and 1 µM CRT0066101 (added in DMSO). Cells were harvested and counted 3 days post CRT0066101/DMSO addition. Results represent mean ± SD of relative cell numbers normalized to untreated cells. Data from one representative experiment performed in triplicate are shown. (C) Analysis of cell cycle distribution and DNA synthesis of U87MG (upper panels) and GM133 (lower panels) cells. Two days post transfection control (untreated and siScr transfected) and PRKD2-silenced (siP5) cells were stained with propidium iodide (PI) (left panel) or bromodeoxyuridine (BrdU) (right panel) and analyzed by flow cytometry. Results are expressed as percentage of total cells and represent the mean values from 3 (PI) and 2 (BrdU) independent experiments. (**P < .01, *P < .05, one-way ANOVA). (D) Detection of Annexin V positive U87MG and GM133 cells. Four days post transfection cells (untreated, siScr, and siP5 transfected) were stained with APC Annexin V (‘A’) and PI and analyzed by fluorescence activated cell sorting. The percentages of A^-/PI⁻ (viable), A⁺/PI⁻ (early apoptotic), A⁺/PI⁺ (late apoptotic) and A⁻/PI⁺ are shown. Mean values from 2 experiments are presented. (E) PRKD2 silencing in glioma xenografts. Forty-eight hours after transfection, 1 × 10⁶ control or treated U87MG cells (siScr and siP5) were subcutaneously injected into the flank of SCID mice (n = 5 per group). Xenograft size was measured 3 times a week, and tumor volume was calculated. Results are expressed as mean ± SEM. Bar indicates delayed growth onset of siPRKD2 xenografts compared with siSCR xenografts (one-way ANOVA, **P < .01). Two animals (shown in grey circles) receiving silenced cells did not develop tumors until day 76 post inoculation.

Fig. 1. — Silencing and pharmacological inhibition of PRKD2 inhibits GBM cell growth. (A) Effect of PRKD2 RNAi on glioma cell proliferation. Two different siRNA constructs (siP5; siP6) were used to silence PRKD2 expression. Untreated cells and cells transfected with nontargeting siRNA (siScr) were used as controls. Cells were harvested and counted on day 6 post silencing. Results represent mean ± SD of relative cell numbers normalized to untreated cells from one representative experiment done in triplicate. The p53 status of the cells is indicated. Silencing efficacy is shown in the right panel. (B) Effects of pharmacological PRKD inhibition on GBM cell proliferation. The indicated p53^wt and p53^mut GBM cells were incubated in the presence of vehicle dimethyl sulfoxide (DMSO) or 0.5 and 1 µM CRT0066101 (added in DMSO). Cells were harvested and counted 3 days post CRT0066101/DMSO addition. Results represent mean ± SD of relative cell numbers normalized to untreated cells. Data from one representative experiment performed in triplicate are shown. (C) Analysis of cell cycle distribution and DNA synthesis of U87MG (upper panels) and GM133 (lower panels) cells. Two days post transfection control (untreated and siScr transfected) and PRKD2-silenced (siP5) cells were stained with propidium iodide (PI) (left panel) or bromodeoxyuridine (BrdU) (right panel) and analyzed by flow cytometry. Results are expressed as percentage of total cells and represent the mean values from 3 (PI) and 2 (BrdU) independent experiments. (**P < .01, *P < .05, one-way ANOVA). (D) Detection of Annexin V positive U87MG and GM133 cells. Four days post transfection cells (untreated, siScr, and siP5 transfected) were stained with APC Annexin V (‘A’) and PI and analyzed by fluorescence activated cell sorting. The percentages of A^-/PI⁻ (viable), A⁺/PI⁻ (early apoptotic), A⁺/PI⁺ (late apoptotic) and A⁻/PI⁺ are shown. Mean values from 2 experiments are presented. (E) PRKD2 silencing in glioma xenografts. Forty-eight hours after transfection, 1 × 10⁶ control or treated U87MG cells (siScr and siP5) were subcutaneously injected into the flank of SCID mice (n = 5 per group). Xenograft size was measured 3 times a week, and tumor volume was calculated. Results are expressed as mean ± SEM. Bar indicates delayed growth onset of siPRKD2 xenografts compared with siSCR xenografts (one-way ANOVA, **P < .01). Two animals (shown in grey circles) receiving silenced cells did not develop tumors until day 76 post inoculation.