Y27632

HEK-293 and THP1 cells were transfected with siRNAs (200 nM) or plasmid (4 mg/mL) controls using Lipofectamine RNAiMAX or Lipofectamine 2000 reagent following the manufacturer’s recommendations (Invitrogen). At 24–48 h after transfection, the cells were stimulated with IFN-a (1000 U/mL, PBL Interferon Source) for 6 h. The RhoA/ROCK inhibitor Y27632 (30,60,90 μM, Beyotime) was added 45 min before stimulation with IFN-a (1000 U/mL, PBL Interferon Source). IFN-α was used to activate IFNAR as a type I IFN stimulus.

HEK-293 T cells were seeded into 6-well plates and 5 × 10⁵ HEK-293 T cells per well transfected with siRhoA (200 nM) or RhoA over-expression plasmids (4 m/mL), or their controls; 48 h after incubation, the cells were treated with IFN-a (1000 U/mL) for an additional 6 h or the RhoA/ROCK inhibitor Y27632 (60 μM, Beyotime), which was added for an additional 45 min before adding IFN-a. The cells then were harvested and lysed at different time points and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes for immunoblotting followed by protein detection with the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). The visualized proteins were scanned and the signal intensities quantified using Image J. The specified primary antibodies were directed against RhoA (Santa Cruz Biotechnology, diluted 1:200), total and phosphorylated STAT1 and STAT2, (Proteintech, diluted 1:3000), and β-actin (Abcam, diluted 1:5000). The secondary antibodies were horseradish-peroxidase (HRP)-linked anti-mouse IgG antibody (Proteintech 1:10,000) and HRP-linked anti-rabbit IgG antibody (Proteintech, diluted 1:10,000).

The hMSCs derived from umbilical cord were purchased from Nuwacell Co., Ltd. (Hefei, China) as stem cells product. Following the company's protocols, hMSCs were cultured in ncMission Basal Medium (RP02010-01, Nuwacell) supplemented with ncMission 25 × Supplement (RP020210-02, Nuwacell). Culture dishes were kept at 37°C in a humidified incubator with 5% CO₂. Complete medium was changed twice per week. When hMSCs reached 80% confluency, cells were released with 0.25% trypsin (SH30042.01, HyClone) and plated at a density of 5 × 10³/cm². The hMSCs at passages 6–7 were used in our experiments. To verify the effect of GTPase activity on cell morphology and nuclear deformation in this UCS platform, hMSCs were incubated in culture media with 10 μM Y27632 (SC0326, Beyotime) to inhibit Rho-kinase activity. The cells were pretreated 30 min before UCS, and Y27632 was present throughout the experiment.