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. 2012 Aug 1;287(39):32535–32545. doi: 10.1074/jbc.M112.402727

FIGURE 3.

FIGURE 3.

Elp1 and Elp3 regulate cell motility of melanoma-derived B16-F10 cells. A, generation and characterization of Elp1-deficient B16-F10 cells. Anti-Elp1, -Elp3, and -α-tubulin (loading control) Western blot analysis were carried out using cell extracts from B16-F10 infected with lentiviral constructs delivering small hairpin RNAs targeting two distinct sequences of the Elp1 transcript, or a control sequence as a negative control (“shRNA Elp1#1,” “shRNA Elp1#2,” and “shRNA control (CTR),” respectively). B, migration of control (CTR) or Elp1-depleted (Elp1#1 or Elp1#2) melanoma-derived cells was measured by wound healing assay. Pictures were taken at the indicated times after the wound. A quantification of the data obtained is illustrated on the right. For each experimental condition, the width of the wound was set to 100% at time 0 and the width in other time points expressed relative to that. The figure shows the data from a representative experiment performed in triplicates (mean values + S.D.). C, generation and characterization of Elp3-depleted melanoma-derived cells. mRNA levels from B16-F10 cells infected with lentiviral constructs delivering small hairpin RNAs targeting two distinct sequences of the Elp3 transcript, or a control sequence (shRNA Elp3#1, shRNA Elp3#2, and shRNA CTR, respectively), were assessed by qRT-PCR. Elp3 mRNA levels in control B16-F10 cells were set to 100%, and mRNA levels in other experimental conditions are relative to that. The figure shows the data from a representative experiment performed in triplicates (mean values + S.D.). D, same as B, but using Elp3-depleted melanoma-derived generated in C. E, wound healing assays were conducted with shRNA control, shRNA Elp3 B16-F10 cells or with shRNA Elp3 B16-F10 cells transfected with full-length Myc-ELP3. A quantification of the data obtained is illustrated on the right. For each experimental condition, the width of the wound was set to 100% at time 0 and the width in other time points expressed relative to that. The figure shows the data from a representative experiment performed in triplicates (mean values + S.D.). On the left, anti-Myc Western blots were carried out with protein extracts from the indicated cells collected at the end of the wound healing assay. “Mock” denotes experimental conditions in which cells were transfected with a control plasmid.