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Fig 6 - Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

Fig. 6 NHSL1 negatively regulates cell migration via the Scar/WAVE complex. a Speed of randomly migrating B16-F1 cells overexpressing either wildtype NHSL1 (NHSL1 WT, black circles) or the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or EGFP alone (red crosses) as control plated on fibronectin after selection using a bicistronic puromycin expression plasmid ensuring all cells analysed overexpressed NHSL1. Mean track speed (dt = 3, TR = 4; see 'Methods' for calculation). Results are mean ± SEM (error bars), of four independent biological experiments. Each data point represents the mean speed of a cell from a total number of 106, 104, 108 cells for control, NHSL1 WT and NHSL1 SW Mut, respectively. Oneway ANOVA: P = 0.0007; F(2,395) = 7.385; and Dunnett's multiple comparisons tests: ***P = 0.0003; ns P = 0.0655; b, c speed and persistence of randomly migrating wild-type B16-F1 cells expressing Myc alone as control (black circles) or CRISPR 2 cells expressing either the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or NHSL1 (NHSL1 WT, red crosses) or Myc alone as control (pink squares) plated on laminin after selection using a bicistronic blasticidin expression plasmid ensuring all cells analysed expressed NHSL1. Mean track speed (b) and persistence (c) (dt = 5, TR = 2; see 'Methods' for calculation). Each data point represents the mean speed of a cell. Results are mean ± SEM (error bars). b One-way ANOVA: P = 0.0077; F(3,302) = 4.044; and Dunnett's multiple comparisons test: **P = 0.0018; *P = 0.0364; ns P = 0.1215. c One-way ANOVA: P = 0.0034; F(3,302) = 4.648; and Dunnett's multiple comparisons tests: CRISPR 2 vs. WT control: *P = 0.0397; NHSL1 CRISPR2 vs. Rescue Myc-NHSL1 SW Mut: ns P = 0.9877; NHSL1 CRISPR2 vs. Rescue Myc-NHSL1 WT: ns P = 0.9442. d Mean Square Displacement analysis (log-log plot) of data shown in b, c. Results are mean values ± SEM (error bars). b-d n = 102 (wild-type cells Myc only), 55 (CRISPR 2 cells Myc only), 77 (CRISPR 2 Rescue Myc-NHSL1 SW Mut), 72 (CRISPR 2 Rescue Myc-NHSL1 WT) from five independent biological experiments. Source data are provided as a Source Data file.
NHSL1 negatively regulates cell migration via the Scar/WAVE complex. a Speed of randomly migrating B16-F1 cells overexpressing either wildtype NHSL1 (NHSL1 WT, black circles) or the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or EGFP alone (red crosses) as control plated on fibronectin after selection using a bicistronic puromycin expression plasmid ensuring all cells analysed overexpressed NHSL1. Mean track speed (dt = 3, TR = 4; see 'Methods' for calculation). Results are mean ± SEM (error bars), of four independent biological experiments. Each data point represents the mean speed of a cell from a total number of 106, 104, 108 cells for control, NHSL1 WT and NHSL1 SW Mut, respectively. Oneway ANOVA: P = 0.0007; F(2,395) = 7.385; and Dunnett's multiple comparisons tests: ***P = 0.0003; ns P = 0.0655; b, c speed and persistence of randomly migrating wild-type B16-F1 cells expressing Myc alone as control (black circles) or CRISPR 2 cells expressing either the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or NHSL1 (NHSL1 WT, red crosses) or Myc alone as control (pink squares) plated on laminin after selection using a bicistronic blasticidin expression plasmid ensuring all cells analysed expressed NHSL1. Mean track speed (b) and persistence (c) (dt = 5, TR = 2; see 'Methods' for calculation). Each data point represents the mean speed of a cell. Results are mean ± SEM (error bars). b One-way ANOVA: P = 0.0077; F(3,302) = 4.044; and Dunnett's multiple comparisons test: **P = 0.0018; *P = 0.0364; ns P = 0.1215. c One-way ANOVA: P = 0.0034; F(3,302) = 4.648; and Dunnett's multiple comparisons tests: CRISPR 2 vs. WT control: *P = 0.0397; NHSL1 CRISPR2 vs. Rescue Myc-NHSL1 SW Mut: ns P = 0.9877; NHSL1 CRISPR2 vs. Rescue Myc-NHSL1 WT: ns P = 0.9442. d Mean Square Displacement analysis (log-log plot) of data shown in b, c. Results are mean values ± SEM (error bars). b-d n = 102 (wild-type cells Myc only), 55 (CRISPR 2 cells Myc only), 77 (CRISPR 2 Rescue Myc-NHSL1 SW Mut), 72 (CRISPR 2 Rescue Myc-NHSL1 WT) from five independent biological experiments. Source data are provided as a Source Data file.
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