Journal: bioRxiv

Article Title: Steady-state epithelial apical flatness is characterized by MLCK morphodynamics and asynchronous Ca 2+ oscillations, but not underlying ECM geometry

doi: 10.1101/2025.11.21.688964

Figure Lengend Snippet: (A) Table showing drugs used to target myosin II, inhibitor function, and concentration. (B) Schematic showing the localization of myosin II interaction with actin in an MDCK monolayer. (C) A side view of a single optical section of a MDCK confluent monolayer treated with DMSO immunostained with ZO-1 (red), β-catenin (green), and filamentous actin dye, phalloidin (grey), and DNA DAPI (blue) counterstain. (D) As in (C) but treated with 30 µM blebbistatin, 50 µM Y27632 (E), and 100 µM ML-7 (F). (G) Individual cell measurements of macro-height for DMSO and drug-treated MDCK monolayers. Average macro-height for DMSO, blebbistatin, Y27632, and ML-7-treated cells was 12.20 µm ± 0.89 µm s.d., 12.11 µm ± 1.11 µm s.d., 11.61 µm ± 1.28 µm s.d., 12.20 µm ± 1.49 µm s.d., respectively (n=45 cells for each treatment). (H) As in (G) but of micro-height. Average micro-height for DMSO, blebbistatin, Y27632, and ML-7-treated cells was 1.30 µm ± 0.27 µm s.d., 2.44 µm ± 0.84 µm s.d., 2.72 µm ± 0.89 µm s.d., 1.84 µm ± 0.80 µm s.d., respectively. (I) Flatness threshold indices of macro-and micro-height of treated and control MDCK confluent monolayers. Note: All myosin II inhibitor treatments resulted in a loss of flatness. (Scale bars: 5µm)

Article Snippet: MDCK cells were treated with 10μM of cytochalasin D (C8273, Sigma), or 5μM of withaferin A (W4394, Sigma), or 16.5μM of nocodazole (M1404, Sigma), 30μM blebbistatin (B0560, Sigma) or 50μM Y27632 (sc-281642; Santa Cruz Biotechnology), 100μM ML-7 (ab120848; Abcam), dissolved in DMSO for 2 hr in an incubator at 37°C, 5% CO 2 before processing.

Techniques: Concentration Assay, Control

Journal: Frontiers in Immunology

Article Title: Crystalline Silica Impairs Efferocytosis Abilities of Human and Mouse Macrophages: Implication for Silica-Associated Systemic Sclerosis

doi: 10.3389/fimmu.2020.00219

Figure Lengend Snippet: 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/cm 2 of SiO 2 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/cm 2 of SiO 2 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/cm 2 of SiO 2 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.

Article Snippet: Fasudil was obtained from MedchemExpress whereas the Rho-associated protein kinase (ROCK) inhibitor (+)-C-trans-4-(1-aminoethyl)-N- (4-pyridyl) cyclohexane carboxamide (Y27632) was purchased from Santa cruz Biotechnology, INC (Heidelberg).

Techniques: Activation Assay, Fluorescence, Microscopy, Staining, Binding Assay, Western Blot, Expressing, Membrane, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Crystalline Silica Impairs Efferocytosis Abilities of Human and Mouse Macrophages: Implication for Silica-Associated Systemic Sclerosis

doi: 10.3389/fimmu.2020.00219

Figure Lengend Snippet: RhoA/ROCK pathway inhibition enhances efferocytosis capacities of silica-exposed MDM and this effect is not due to a down expression of SR-B1 after RhoA/ROCK inhibition. (A,B) EI of MDM pre-treated or not with the ROCK inhibitors (A) Y27632 or (B) fasudil at 20 μM and treated with 25 μg/cm 2 of SiO 2 for 4 h and then exposed to apoptotic Jurkat cells for 90 min (Experiment on MDM from at least 5 different healthy donors). (C,D) Expression of SR-B1 expressed as (C) ratio of MFI and (D) percentage of positive cells of MDM treated or not with the ROCK inhibitor Y27632 and Fasudil at 20 μM for 4 h analyzed by flow cytometry (Experiment on MDM from 2 to 5 different healthy donors). * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: Fasudil was obtained from MedchemExpress whereas the Rho-associated protein kinase (ROCK) inhibitor (+)-C-trans-4-(1-aminoethyl)-N- (4-pyridyl) cyclohexane carboxamide (Y27632) was purchased from Santa cruz Biotechnology, INC (Heidelberg).

Techniques: Inhibition, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Crystalline Silica Impairs Efferocytosis Abilities of Human and Mouse Macrophages: Implication for Silica-Associated Systemic Sclerosis

doi: 10.3389/fimmu.2020.00219

Figure Lengend Snippet: RhoA/ROCK pathway inhibition by Y27632 enhances efferocytosis capacities of MDM from patients with systemic sclerosis. (A) EI of M0 MDM from healthy donors untreated or treated with 25 μg/cm 2 of SiO 2 for 4 h and of M0 MDM from SSc patients (Experiment on MDM from 20 different healthy donors and 20 different SSc patients). (B) EI of M0 MDM from SSc patients pre-treated or not with 20 μM Y27632 before exposure to apoptotic Jurkat cells (apoJ) for 90 min (Experiment on MDM from 6 different healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Fasudil was obtained from MedchemExpress whereas the Rho-associated protein kinase (ROCK) inhibitor (+)-C-trans-4-(1-aminoethyl)-N- (4-pyridyl) cyclohexane carboxamide (Y27632) was purchased from Santa cruz Biotechnology, INC (Heidelberg).

Techniques: Inhibition