Figure 1
Smad3 translocation to the nucleus, analyzed using imaging flow cytometry.
(a) Relative levels of Smad3 nuclearization in human keratocytes. *p < 0.001 compared to CON+; **p < 0.001 compared to CON+ and CON− (Chi2 tests). (b) Workflow diagram of the IDEAS software. First, images of the keratocytes in focus were chosen and then the relevant cell population was selected, excluding the nonspecific labeling. The probe similarity algorithm for co-localization of the nucleus and Smad3 was then defined. The software calculated the correlation coefficient (Similarity Dilate) of the two probes, plotted as a frequency histogram. R3, keratocytes with Similarity Dilate >1, which implies nuclearization has occurred. (c) Representative images of ketatocytes labeled for Smad3 and the nucleus, as revealed by the imaging flow cytometry (green, AlexaFluor® 488, Smad3; purple, 7AAD, nucleus). Upper panel, cytosolic Smad3; lower panel, nuclearized Smad3. CON+, positive control of 10 ng/ml TGF-β2; CON−, negative control for naïve human keratocytes. All of the following conditions also included 10 ng/ml TGF-β2: 10 ng/ml IGF-1; 5 ng/ml halofuginone (HAL); 10 ng/ml IGF-1 plus 5 ng/ml halofuginone (combo HAL); 10 nM SAHA; 10 ng/ml IGF-1 plus 10 nM SAHA (combo SAHA).
