Figure 5
Figure 5. GRK6 kinase silencing induces selective apoptosis of myeloma cells. (A) KMS11 myeloma cells were infected with lentivirus driving the expression of 3xFLAG-tagged GRK6 (+), or with control lentivirus (−), and were assessed by anti-FLAG immunoblot (left panel) and immunoprecipitation (right panel), verifying abundant tagged GRK6 protein production. (B) KMS11 cells stably infected with 3xFLAG-GRK6 expressing LV, or with control LV, were treated with Lipofectamine 2000 (L2K) transfection reagent and control or GRK6 3′UTR siRNA. GRK6 3′UTR siRNA induced native GRK6 mRNA suppression in control cells, but did not suppress 3xFLAG-GRK6 (which lacks GRK6 3′ UTR sequence), as shown by QRT-PCR at 48 hours (right panel). The effect of GRK6 3′ siRNA on KMS11 viability at 96 hours was assessed in parallel by MTT assay (left panel). Ectopic 3xFLAG-GRK6 rescues KMS11 from GRK6 3′UTR siRNA-induced lethality, verifying that the siRNA's lethality is due to on-target GRK6 silencing. Both KMS11 lentiviral lines were tested with NS and UBB siRNA, revealing equivalent transfection efficiency and minimal L2K toxicity. (C) The lethality of GRK6 silencing in KMS11 myeloma cells was further verified by 4 GRK6 siRNAs (GRK6-2, -9, -3, -C) that target GRK6 exons: after transfection of KMS11, GRK6 mRNA knockdown was assessed by QRT-PCR at 48 hours and viability was assessed by annexin V + propidium iodide binding and by MTT assay (at 96 hours). Cellular inhibition was due largely to apoptosis. (D) Lentiviruses nos. 66 to 68 expressing distinct short hairpin RNA (shRNA) targeting GRK6 were screened for induction of GRK6 knockdown in KMS11 by immunoblot at 48 hours. LV no. 66 and no. 68 both induced GRK6 knockdown in contrast to no. 67 and control LV expressing nonsilencing (NS) shRNA. LV no. 66 induced similar suppression of GRK6 in MCF7 and SF767 cells. (E) OPM1 myeloma cells infected with GFP-expressing LV no. 66 were mixed 1:1 with OPM1 cells infected with control LV. Cells in which GRK6 was targeted by shRNA no. 66 were progressively eliminated from culture, as determined by serial flow cytometry for the GFP marker, despite the presence of healthy control OPM1, which became enriched. (F) Comparison of the effect of GRK6-shRNA no. 66 on the viability of myeloma (KMS11 and OPM1) and nonmyeloma (MCF7 and SF767) cell lines. Cells were infected in parallel with equal titer LV producing NS-shRNA, GRK6-shRNA no. 66, or GFP cDNA; percentage cellular infection was determined by GFP expression. Specific apoptosis induction by GRK6 silencing at 96 hours was determined by annexin V and 7-amino-actinomycin D binding, comparing control LV (producing NS-shRNA) with LV GRK6-shRNA no. 66. (G) The effect of GRK6 inhibition on cellular viability is summarized for a spectrum of human myeloma and nonmyeloma cells. Results were obtained as shown in panel F and are plotted as the GRK6-shRNA attributable apoptosis normalized to the percentage infection obtained with each cell line. Error bars are calculated from 5% absolute error in assessments of infection and apoptosis.

GRK6 kinase silencing induces selective apoptosis of myeloma cells. (A) KMS11 myeloma cells were infected with lentivirus driving the expression of 3xFLAG-tagged GRK6 (+), or with control lentivirus (−), and were assessed by anti-FLAG immunoblot (left panel) and immunoprecipitation (right panel), verifying abundant tagged GRK6 protein production. (B) KMS11 cells stably infected with 3xFLAG-GRK6 expressing LV, or with control LV, were treated with Lipofectamine 2000 (L2K) transfection reagent and control or GRK6 3′UTR siRNA. GRK6 3′UTR siRNA induced native GRK6 mRNA suppression in control cells, but did not suppress 3xFLAG-GRK6 (which lacks GRK6 3′ UTR sequence), as shown by QRT-PCR at 48 hours (right panel). The effect of GRK6 3′ siRNA on KMS11 viability at 96 hours was assessed in parallel by MTT assay (left panel). Ectopic 3xFLAG-GRK6 rescues KMS11 from GRK6 3′UTR siRNA-induced lethality, verifying that the siRNA's lethality is due to on-target GRK6 silencing. Both KMS11 lentiviral lines were tested with NS and UBB siRNA, revealing equivalent transfection efficiency and minimal L2K toxicity. (C) The lethality of GRK6 silencing in KMS11 myeloma cells was further verified by 4 GRK6 siRNAs (GRK6-2, -9, -3, -C) that target GRK6 exons: after transfection of KMS11, GRK6 mRNA knockdown was assessed by QRT-PCR at 48 hours and viability was assessed by annexin V + propidium iodide binding and by MTT assay (at 96 hours). Cellular inhibition was due largely to apoptosis. (D) Lentiviruses nos. 66 to 68 expressing distinct short hairpin RNA (shRNA) targeting GRK6 were screened for induction of GRK6 knockdown in KMS11 by immunoblot at 48 hours. LV no. 66 and no. 68 both induced GRK6 knockdown in contrast to no. 67 and control LV expressing nonsilencing (NS) shRNA. LV no. 66 induced similar suppression of GRK6 in MCF7 and SF767 cells. (E) OPM1 myeloma cells infected with GFP-expressing LV no. 66 were mixed 1:1 with OPM1 cells infected with control LV. Cells in which GRK6 was targeted by shRNA no. 66 were progressively eliminated from culture, as determined by serial flow cytometry for the GFP marker, despite the presence of healthy control OPM1, which became enriched. (F) Comparison of the effect of GRK6-shRNA no. 66 on the viability of myeloma (KMS11 and OPM1) and nonmyeloma (MCF7 and SF767) cell lines. Cells were infected in parallel with equal titer LV producing NS-shRNA, GRK6-shRNA no. 66, or GFP cDNA; percentage cellular infection was determined by GFP expression. Specific apoptosis induction by GRK6 silencing at 96 hours was determined by annexin V and 7-amino-actinomycin D binding, comparing control LV (producing NS-shRNA) with LV GRK6-shRNA no. 66. (G) The effect of GRK6 inhibition on cellular viability is summarized for a spectrum of human myeloma and nonmyeloma cells. Results were obtained as shown in panel F and are plotted as the GRK6-shRNA attributable apoptosis normalized to the percentage infection obtained with each cell line. Error bars are calculated from 5% absolute error in assessments of infection and apoptosis.

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