Figure 3
Expression of shRNA against SEMA4A is associated with reduced growth in myeloma cells but not K562 erythroleukemia cells. The erythroleukemia cell line, K562 (A), and the human myeloma cell lines, NCI-H929 (B) and MM1.S (C), were lentivirally transduced with six shRNA predicted to target SEMA4A plus a control shRNA directed against luciferase (shLUC). All shRNA vectors expressed GFP. Transduction efficiency was deliberately maintained at ∼50%. Top panels: SEMA4A expression was measured at the cell surface by flow cytometry and is expressed relative to expression in GFP-negative (GFP-) cells. K562 cells do not express SEMA4A, and thus expression is not shown. Bottom panels: GFP-positive (GFP+)/total cell ratio was measured from day 4 after transduction. GFP+/total cell ratio is then plotted relative to that ratio at day 4 and normalized to the ratio for the control hairpin, shLUC. Error bars indicate ±SD from a minimum of 2 replicates for each shRNA. (D) MM1.S, NCI-H929, and NCI-H929 Cas9 were lentivirally transduced as described in panel A with a control (shLUC/sgNone) or shRNA/sgRNA targeting SEMA4A. Left panel: at 144 hours' post–viral transduction, SEMA4A expression was measured at the cell surface by flow cytometry and is expressed relative to expression in untransduced cells (UT). Right: Cell viability was also assessed by flow cytometry to determine the percentages of dead (Annexin V and LIVE/DEAD Fixable Violet positive), early apoptotic (Annexin V positive and LIVE/DEAD Fixable Violet negative), and alive (Annexin V and LIVE/DEAD Fixable Violet negative) cells. A one-way analysis of variance comparing MM1.S alive cells F(3,8) = 42.45, P < .0001, NCI-H929 alive cells F(3,8) = 101.2, P < .0001, and NCI-H929 Cas9 alive cells F(3,8) = 56.84, P < .0001 was performed. Dunnett's multiple comparisons correction is shown. (E) NCI-H929 constitutively expressing Cas9 were lentivirally transduced with sgRNA targeting exon six of SEMA4A (sgSEMA4A) or a nontargeting control sgRNA (sgNone). All sgRNA vectors expressed blue fluorescent protein (BFP), and transduction efficiency was deliberately maintained at ∼50%. After lentiviral transduction, cells were cultured ± the stromal cell line, HS5, or were supplemented ± interleukin-6 (IL-6). The BFP-positive (BFP+)/total cell ratio was measured from day 4 after transduction and is plotted relative to proportion at day 4 and normalized to the ratio of each control sgRNA, sgNone. Error bars are ±SD of replicates; n = 3. **P < .01; ***P < .001; ****P < .0001. NS, not significant.

Expression of shRNA against SEMA4A is associated with reduced growth in myeloma cells but not K562 erythroleukemia cells. The erythroleukemia cell line, K562 (A), and the human myeloma cell lines, NCI-H929 (B) and MM1.S (C), were lentivirally transduced with six shRNA predicted to target SEMA4A plus a control shRNA directed against luciferase (shLUC). All shRNA vectors expressed GFP. Transduction efficiency was deliberately maintained at ∼50%. Top panels: SEMA4A expression was measured at the cell surface by flow cytometry and is expressed relative to expression in GFP-negative (GFP-) cells. K562 cells do not express SEMA4A, and thus expression is not shown. Bottom panels: GFP-positive (GFP+)/total cell ratio was measured from day 4 after transduction. GFP+/total cell ratio is then plotted relative to that ratio at day 4 and normalized to the ratio for the control hairpin, shLUC. Error bars indicate ±SD from a minimum of 2 replicates for each shRNA. (D) MM1.S, NCI-H929, and NCI-H929 Cas9 were lentivirally transduced as described in panel A with a control (shLUC/sgNone) or shRNA/sgRNA targeting SEMA4A. Left panel: at 144 hours' post–viral transduction, SEMA4A expression was measured at the cell surface by flow cytometry and is expressed relative to expression in untransduced cells (UT). Right: Cell viability was also assessed by flow cytometry to determine the percentages of dead (Annexin V and LIVE/DEAD Fixable Violet positive), early apoptotic (Annexin V positive and LIVE/DEAD Fixable Violet negative), and alive (Annexin V and LIVE/DEAD Fixable Violet negative) cells. A one-way analysis of variance comparing MM1.S alive cells F(3,8) = 42.45, P < .0001, NCI-H929 alive cells F(3,8) = 101.2, P < .0001, and NCI-H929 Cas9 alive cells F(3,8) = 56.84, P < .0001 was performed. Dunnett's multiple comparisons correction is shown. (E) NCI-H929 constitutively expressing Cas9 were lentivirally transduced with sgRNA targeting exon six of SEMA4A (sgSEMA4A) or a nontargeting control sgRNA (sgNone). All sgRNA vectors expressed blue fluorescent protein (BFP), and transduction efficiency was deliberately maintained at ∼50%. After lentiviral transduction, cells were cultured ± the stromal cell line, HS5, or were supplemented ± interleukin-6 (IL-6). The BFP-positive (BFP+)/total cell ratio was measured from day 4 after transduction and is plotted relative to proportion at day 4 and normalized to the ratio of each control sgRNA, sgNone. Error bars are ±SD of replicates; n = 3. **P < .01; ***P < .001; ****P < .0001. NS, not significant.

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