Figure 1
From: GRK2 promotes growth of medulloblastoma cells and protects them from chemotherapy-induced apoptosis
GRK2 knockdown slows growth and proliferation of UW228 and Daoy medulloblastoma cell lines. (A–C) Tva-expressing UW228 (A) and Daoy (B) cells were infected with human GRK2 knockdown miRE cassettes using the viruses pRDAV-mCherry-miRE.GRK2.2441 (miRE.2441), pRDAV-mCherry-miRE.GRK2.3441 (miRE.3441) or control pRDAV-mCherry-miRE.Ren.713 (miRE.Ren; miRE.Ctrl). They were then flow-sorted for mCherry-expressing cells, expanded for 8 days, re-seeded (2.2 × 104 cells/35 mm dish UW228 and 2.5 × 104 cells/35 mm dish Daoy) and grown for counting over 6 additional days in 10% FBS DMEM (UW228) or 1% FBS DMEM (Daoy). Live cells (cells excluding trypan blue) from three independent biological replicates (different passage cells, separate infections, different batch viruses) were counted manually every other day in each sample. Data points are means/SD of the three biological replicates. P-values were calculated over time using ordinary 2-way ANOVA with alpha 0.05, between cells infected with miRE.Ctrl versus miRE.GRK2.3441, and between cells infected with miRE.Ctrl versus miRE.GRK2.2441 (p < 0.001 for each pair over time). (C) GRK2 knockdown for A-B was validated by western blot using anti-GRK2 mouse monoclonal antibody (Santa Cruz cat #sc-13143) that detects a single GRK2 band. (D,E) UW228 (D) and Daoy (E) cells were transfected with 25 nM control or GRK2 siRNA mixed with 2.5 nM FAM-labelled siRNA and stained with apoBrdU-APC and 7AAD. FAM-positive cells were analyzed by flow cytometry 30 h after transfection. The left panels are representative flow cytometry curves of one of four independent experiments (D) and one of three (E) biological replicates, and right panels are western blots showing the level of GRK2 knockdown for cells shown in the flow cytometry curves. The anti-GRK2 antibody used in panel D is Santa Cruz cat#sc-13143 used in C. Panel E western blot used rabbit polyclonal, Santa Cruz cat #sc562, (1:6,000) that detected two bands here, of which the top one is GRK2 (arrow). Middle panels D-E show mean ± SEM of percent cells in S-phase in these experiments (n = 4 independent experiments for D, n = 3 biological replicates for (E), with p-values calculated using two-tailed unpaired t-tests.
