Fig. 2: Different NHSL3 isoforms are involved in single and collective cell migration.

a Scheme of NHSL3 isoforms expressed in MCF10A cells. Exons 5 and 6 are omitted in a short isoform. Three transcriptional start sites account for different exons 1, labelled E1, E1’ and E1”. Locations of the gRNA used for KO and siRNAs targeting specifically short and long isoforms are indicated. b MCF10A NHSL3 KO cells are stably transfected with plasmids expressing Flag-GFP or Flag-GFP tagged NHSL3 isoforms and analysed by Western blots using NHSL3 and GAPDH antibodies. Red stars indicate the location of NHSL3 isoforms. c Cells are tracked for 6.5 h and migration persistence is extracted from trajectories of single cells. n  =  60 cells, mean ± SEM. i3S is the only isoform that rescues the increased persistence of NHSL3 KO cells. d Quantification of different NHSL3 mRNAs in MCF10A cells using qRT-PCR and representation of their respective abundance. Proportions of NHSL3 isoforms are deduced from the PCR amplicons labelled a to e in the scheme, mean ± SEM. e Quantification of long and short isoforms at the protein level. The different isoforms are labelled at their C-terminus by knocking in GFP-Flag in exon 7 (KI). Lysates from parental MCF10A and KI cells are subjected to GFP immunoprecipitation. Lysates, immunoprecipitates and dilution thereof are subjected to NHSL3 Western blot. The short isoform is about 100-fold less abundant than long isoforms. f MCF10A cells stably expressing i3S are transfected with siRNAs targeting short or long isoforms and analysed by Western blots. g qRT-PCR of short and long isoforms in MCF10A cells transfected with siRNAs, n = 3, mean ± SEM. h Migration persistence of single MCF10A cells transfected with siRNAs. Tracking 6.5 h, n  =  60 cells, mean ± SEM. i Collective migration of NHSL3 KO cells stably expressing the different NHSL3 isoforms, assessed as in Fig. 1f. i2 is the only isoform that significantly rescues the decreased speed of follower cells in NHSL3 KO cells. Statistical significance is calculated with custom-made R programmes for single cell migration or with Kruskal-Wallis test for collective migration. Significant p-values are highlighted in yellow in the tables. Three biological repeats of each experiment gave similar results. Source data are provided as a Source Data file.