Fig. 3: RAD-TGTs detect CRISPR-KO of mechanosensing proteins in single and mixed cell populations.

a SuperPlots of the fold change of CY5 fluorescence intensity of individual WT, TLN1 KO, or CD44 KO U251 cells on 12 pN (top) or 54 pN (bottom) RAD-TGTs with WDV-Echi. Symbols represent the median Cy5 fold change for each replicate experiment. Identical symbols indicate the samples were collected on the same day. The horizontal red line is at the median fold change for all cells analyzed. For 12 pN experiments, n = 8 (WT), 3 (CD44KO), or 6 (talinKO) and for 54 pN experiments n = 5(WT) and 3 (CD44KO and talinKO). A two-tailed paired t-test was used for statistics, *p = 0.0360 (12 pN CD44KO), **p = 0.0028 (12 pN talinKO), and *p = 0.0322 (54 pN talinKO). Medians were paired based on the day each experiment was performed. b Histogram of a mixed population of U251 WT, Talin1, and CD44 KO (gray solid), overlaid with histograms of cell lines tested individually (outlines only). c Violin plots of individual populations, mixed population, and the sum of individual populations. d Cartoon of U251 WT and CD44 KO cells and accompanying histogram of the two cell types mixed together (gray) or individual. The composition of the population is determined by measuring the GFP signal on different applied gates of the CY5 histogram as CD44 KO also expresses GFP. The composition of the lowest 20%, highest 20%, and the entire population is displayed. e Scatter plots of U251 and CHO-K1 cells plated on surfaces containing a 12 pN Cy5 labeled RAD-TGT and 54 pN A488 labeled RAD-TGT for both each cell individually and in a mixed population. Source data are provided as a Source Data file. All cartoons and schematics were created with Biorender.com.