Supplementary Figure 2: Further confirmation of the thymocyte negative-selection criteria by force-dependent bond lifetimes, molecular stiffness, and cell pulling.

Related to Fig. 4. a, Mean ± s.e.m. of lifetime versus force plots of total bonds with indicated peptides presented by H2-K^b (green square and blue diamond) or H2-K^bm3 (red square), TCR bonds with three peptides presented by H2-K^bα3A2 (brown circle), and CD8 bond with VSV:H-2K^b (black triangle) measured using 2C TCR transgenic mice. The total bonds with the negative selecting ligands, SIYR:H-2K^b and dEV8:H-2K^bm3, exhibited catch bond behavior at low forces whereas those with the positive selecting ligands, dEV8, EVSV and p2Ca bound to H-2K^b, showed slip bond behavior^9–11,52. Like mOVA, the super agonist SIYR also formed catch bond at low forces with the 2C TCR in the absence of CD8, but the other two pMHC bimolecular interactions and the MHC–CD8 interaction formed slip bonds. The numbers of bond lifetime measurements per curve for different ligands, the results of statistical tests examining the trends of the curves and their differences are summarized in Supplementary Tables 1d, 2c and 3a,b, respectively. b, Molecular stiffness histograms of 2C TCR bimolecular complexes with the indicated peptides bound to H-2K^bα3A2, total complexes with these peptides bound to H-2K^b, and total complexes with dEV8:H-2K^bm3 and p2Ca:H-2K^b. Data (bar) were fitted by a single (black curve) or double (black curve = cyan curve + red curve) Gaussian. The fitting parameters and statistics for their comparisons are summarized in Supplementary Tables 4c and 5c, respectively. c, Representative images of thymocytes placed on wVSV, wCatnb, and wOVA tagged with a 13.1 pN MTP viewed in the bright-field (left column), fluorescence (middle column), and merged (right column) channels. Scale bar = 5 µm. d, Comparison of initial force signals from wVSV, wCatnb, and wOVA tagged with a 13.1 pN MTP. e, Comparison of force signal decays of OT1 thymocytes pulled on mOVA and wOVA tagged with a 13.1 pN MTP. f, Representative images of thymocytes pulled on indicated peptides presented by H-2K^b tagged with a 4.7 pN MTP viewed in the bright-field (left column), fluorescence (middle column), and merged (right column) channels. Scale bar = 5 µm. g, Comparison of normalized fluorescence intensity (points, left ordinate) and fraction of positive cells (bars, right ordinate) for indicated peptides bound to H2-K^b. Normalized fluorescence intensity was calculated by dividing the mean fluorescence intensity from a Cy5 positive cell by the background. Data are presented as mean ± s.e.m of all positive cells (each cell is represented by a point with N indicating the number of positive cells). h, Comparison of force signal decays of OT1 thymocytes pulled on wQ4H7 and wQ4R7 tagged by a 4.7 pN MTP. i, Representative images of OT1 thymocytes pulled on wOVA tagged with a 13.1 pN MTP treated with the indicated agents: DMSO control, actin polymerization inhibitor latruculin A, and ROCK inhibitor Y-27632. OT1 thymocytes before (top row) and 10 min after (bottom row) the drug treatment were viewed in the bright-field (left column), fluorescence (middle column), and merged (right column) channels. Scale bar = 5 µm. j, Comparison of force signal decays of OT1 thymcoytes pulled on wOVA over 20 min for the same treatments as in (i). Data in (e,h,j) are presented as mean ± s.e.m. of fluorescence intensity normalized by the initial value at 0 min (N = number of cells pooled from ≥at least two independent experiments). *and **** denote p < 0.05 and 0.0001, respectively, by two-way ANOVA.