Fig. 3

Fig. 3 HMGB2 is a confinement-induced marker of invasion.

a, HMG family expression in interface cells from zebrafish melanoma scRNA-seq. b, Normalized hmgb2a/hmgb2b expression. P values are noted (Wilcoxon rank-sum test; two-sided). c, HMGB2 expression per cell in human melanoma scRNA-seq data from Jerby-Arnon et al.⁷. The arrow indicates interface cluster. d, Mean HMGB2 expression per cluster. P value calculated using Wilcoxon rank-sum test with Bonferroni’s correction; two-sided. e, Zebrafish melanoma stained for HMGB2 and Hoechst. f, Inset of region indicated in d. Elongated HMGB2-high cells are labelled. g, Correlation between nuclear circularity and HMGB2 intensity. Red dashed line, line of best fit by linear regression. h, Immunofluorescence targeting HMGB2 in confined A375 cells. i–k, HMGB2 intensity (i), Hoechst intensity (j) and HMGB2 intensity normalized to Hoechst (k) per cell. Unconfined, n = 49 cells from three images; confined, n = 97 cells from nine images. Horizontal lines, mean; box, s.e.m.; vertical lines, s.d. P value is indicated (two-sample t-test; two-sided). l, TurboID workflow. m, Nesprin 2 protein abundance; n = 3 replicates per condition. Horizontal line, median; hinges, first and third quartiles; whiskers, range. NLS, nuclear localization signal. n, HMGB2 expression in confined A375 cells. siNT, non-targeting siRNA; siSYNE2, SYNE2-targeting siRNA. o, Quantification of HMGB2 intensity. siNT unconfined, n = 72 cells from eight images; siNT confined, n = 94 cells from eight images; siSYNE2 unconfined, n = 48 cells from eight images; siSYNE2 confined, n = 64 cells from eight images. P value is indicated (analysis of variance with Tukey post hoc test; two-sided). p, Images showing acetylated tubulin (yellow) and Hoechst (magenta) in confined A375 cells. q, Quantification of acetylated tubulin intensity in confined cells. siNT, n = 66 cells from eight images; siSYNE2, n = 104 cells from eight images. P value is indicated (two-sample t-test; two-sided). Horizontal line, median; edges, upper and lower quartiles; whiskers, non-outlier minima and maxima (o,q). Scale bars, 50 µm (e), 10 µm (f), 25 µm (h,n,p). Illustrations in l were created using BioRender (https://biorender.com).

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