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. 2020 Feb 18;11:219. doi: 10.3389/fimmu.2020.00219

Figure 6.

Figure 6

Silica exposure induces cytoskeleton remodeling and alteration of MΦ polarization through activation of the RhoA/ROCK pathway. (A) Representative pictures of fluorescence microscopy: F-actin and nuclei were stained by Alexa Fluor 568-phalloidin (red) and DAPI (blue), respectively. M0-MDM were untreated or treated with 25 μg/cm2 of SiO2 for 10 min or polarized into M1 cells for 24 h. MDM were also pre-treated or not 1 h with the ROCK inhibitor Y27632 at 20 μM. (B–D) M0-MDM (Ct) from the same healthy donors were pre-treated or not with 20 μM Y27632 and then untreated or not with 25 μg/cm2 of SiO2 for the indicated time. The GTP-binding fraction of RhoA was pulled-down as described in Materials and Methods. (C,D) Western-blot analyzes of Phospho-MYPT1 expression were performed on whole-cell lysates. The relative levels of the proteins were determined by densitometry (Experiment on MDM from 4 to 8 different healthy donors). (E) Effect of Y27632 on the membrane expression of CD206, CD163, and CD204. MDM from the same healthy donors were pre-treated or not 1 h with the ROCK inhibitor Y27632 at 20 μM before exposure to 25 μg/cm2 of SiO2 for 4 h; data determined by flow cytometry are expressed as ratio of MFI (Experiment on MDM from 5 to 7 different healthy donors). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.