Figure 3.

Distinct localization of active and inactive β1 integrins within FAs. (A) Representative confocal, STED, and STORM images of Hs578T cells colabeled with antiactive β1 integrin mAb 12G10 (OG488 or Cy3; Alexa Fluor 647) and antiinactive β1 integrin mAb AIIB2 (rhodamine or Alexa Fluor 405 through Alexa Fluor 647). White boxes indicate zoomed areas on the right. Bars: (main images) 5 µm; (insets) 2 µm. (B–D) Bars show mean Pearson’s correlation coefficients (± SD) obtained by confocal (B), STED (C), and STORM (D) images colabeled with mAb 12G10 and mAb AIIB2. Confocal, n = 10 images; STED, n = 20; STORM, n = 6. mAb 12G10 labelings targeted by two differently colored secondary abs were used as control for maximal colocalization. Confocal, n = 9 images; STED, n = 13; STORM, n = 6. (E) The three left bars show Hs578T cells labeled by mAbs AIIB2 (n = 18 images), K20 (n = 11), or 12G10 (n = 13) targeted with two differently colored secondary abs each as controls for maximal colocalization. The remaining bars show mean Pearson’s correlation coefficients (± SD) of STED images of Hs578T cells colabeled by mAb 12G10 and mAb 9EG7 (n = 12 images); mAb Huts-4 and mAb 9EG7 (n = 11); mAb K20 and mAb 9EG7 (n = 14); mAb K20 and mAb AIIB2 (n = 17); mAb 12G10 and mAb AIIB2 (n = 20); mAb 12G10 and mAb 13 (n = 11); mAb Huts-4 and mAb AIIB2 (n = 11); and mAb Huts-4 and mAb 13 (n = 5). The individual colocalizations were statistically compared with the control with the lowest coefficient (12G10), providing the most stringent comparison. The mAb AIIB2 versus mAb12G10 combination was duplicated from C to facilitate direct comparison. (F) Bars show mean percentages ± SD of overlapping nanoclusters segmented in STED images of Hs578T cells colabeled with mAb 12G10 and mAb AIIB2 (n = 20 images). Hs578T cells labeled using mAb K20 and two differently colored secondary abs both targeting mAb K20 were used as control for maximal overlap. n = 9 images. t test: *, P ≤ 0.05; ***, P ≤ 0.001.