Fig. 2
Effect of cLIUS on select genes, SOX9 localization, and SOX9 mRNA stability. Gene expression of a c-MYC and c-JUN and b SOX9 and RUNX2 in MSCs exposed to cLIUS at indicated frequencies were estimated by qRT-PCR (n = 3). MSCs plated at 1 × 105 cells/well were exposed to cLIUS (14 kPa) at frequencies: 5 MHz (2.5 Vpp), 2 MHz (6 Vpp), or 8 MHz (9.5 Vpp) at constant pressure amplitude of 14 kPa one time for 5 min. Non-cLIUS-stimulated MSCs served as controls (n = 3). Data are shown as the mean ± standard deviation (Welch’s t-test). c MSCs in coverslips (n = 3) at an initial seeding density of 1 × 105 cells/well were stimulated with cLIUS application at 14 kPa (5 MHz, 2.5 Vpp) for 5 min and fixed in 4% PFA. Confocal micrographs (× 60 magnification) of immunofluorescent staining of SOX9 (green) shows the localization of SOX9 in the MSCs under cLIUS (Scale bar = 20 μm). Nucleus was stained by Dapi (blue) and d quantified by ImageJ (n = 10–20). Data are shown as the mean ± standard deviation. e MSCs (2 × 104 cells per well) were treated with 5 μg/ml actinomycin D, followed by stimulation with cLIUS at 14 kPa (5 MHz, 2.5 Vpp), for 15 min (n = 3). Total RNA was collected after 0 min, 30 min, 1 h, and 2 h of actinomycin D treatment, and the fold change in mRNA transcripts of SOX9 were quantified by qRT-PCR. In a parallel experiment without actinomycin D treatment, total RNA was collected at indicated time points following cLIUS stimulation, and the fold change in SOX9 mRNA transcripts was quantified by qRT-PCR (n = 3). Non-cLIUS-stimulated samples served as controls at respective time points (n = 3). Data represent the average ± standard deviation of fold change in SOX9 mRNA levels normalized to time point 0. f Graphical representation of the amount of SOX9 mRNA transcripts in actinomycin D-treated MSCs with or without cLIUS stimulation normalized to non-actinomycin D-treated samples at respective time points