Fig 1: The α7-nicotinic acetylcholine receptor (α7-nAChR) promotes cell viability, metastasis, and cancer stem cell (CSCs)-like phenotypes in hepatocellular carcinoma (HCC). (A) α7-nAChR-knockdown (KD) inhibited the cell viability of Hep-J5 cells, as analyzed by an SRB assay after 24 and 48 h of incubation. * p < 0.05, ** p < 0.01, p < 0.001 versus the wild-type (WT) groups (n = 3). (B) Graphical representation of the inhibitory effect of α7-nAChR-KD on the mRNA expressions of α7-nAChR, Bcl-xL, MCL-1, MMP-9, and Survivin in Hep-J5 cells. (C) α7-nAChR-KD cells exhibited slower area closure than did Hep-J5 WT cells in the in vitro Scratch-wounding cell migration assay (n = 3) after 48 h. (D–F) similar results were obtained in Mahlavu cells. (G) Representative photo-image showing the effect of α7-nAChR-KD on the invasive potential of Hep-J5 and Mahlavu cells. (H) Differential expressions of TGR5, RhoA, ROCK1, MMP2, and MMP9 proteins in Hep-J5 and Mahlavu WT and α7-nAChR-KD cells. (I) α7-nAChR-KD Mahlavu and Hep-J5 cells exhibited a significantly reduced ability to form tumorspheres compared to WT cells. The assays were performed in triplicate, and data shown are representative of three independent assays. β-Actin served as a loading control.
Fig 2: ROCK1 promotes the migration and invasion of NSCLC A549 and NCI-H1299 cells. (A) ROCK1 knockdown efficiency of the shRNA was measured by western blotting. (B and C) The role of ROCK1 in A549 and NCI-H1299 cell migration was evaluated by the wound healing assay. The wound closure distance was measured in 3 randomly selected fields. Scale bars: 200 µm. (D and E) The Transwell assay was employed to evaluate the effects of shROCK1 on cell invasion; the number of invading cells was counted from three independent experiments. Scale bars: 200 µm. (F) A549 and (G) NCI-H1299 cells were seeded in fibronectin-coated plates and cultured for 10-180 min; the adherent cells were trypsinized and counted at each time point. (H and I) Cells were cultured in 12-well plates (1×103 cells per well) for 24 h, then trypsinized with a diluted trypsin solution (trypsin:PBS, 1:19) for different time intervals (20-180 sec). The remaining adherent cells were digested and counted. Each experiment was repeated three times. All data are presented as the mean ± standard deviation (*P<0.05, **P<0.01, ***P<0.001). ROCK1, Rho-associated protein kinase 1; NSCLC, non-small-cell lung cancer.
Fig 3: Mechanism of ROCK1 promoting tumor cell migration, invasion and progression in NSCLC. ROCK1 reduces the activation/phosphorylation of PTEN and then phosphorylates PI3K/AKT, resulting in FAK phosphorylation and leading to accelerated cell migration/invasion and promotion of NSCLC progression. ROCK1, Rho-associated protein kinase 1; NSCLC, non-small-cell lung cancer; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; FAK, focal adhesion kinase.
Fig 4: Mitotic neuroepithelial cells in the mouse PNP have constricted apical surfaces with enriched myosin. A.3D reconstructions showing the shape of illustrative EGFP mosaically-labelled neuroepithelial cells positive or negative for pHH3. EGFP lineage-tracing was induced with Nkx1.2CreERT2. Cyan rings indicate the apical surface, the arrowhead indicates a sub-apical protrusion. Scale = 10 μm. B.Wholemount immunofluorescence showing neuroepithelial apical localisation of ROCK1 and pHH3. Arrow indicates apical surface of a mitotic cell in a 3D reconstruction. Scale = 100 μm (full PNP) and 25 μm (inset).C-D.Surface-subtracted wholemount immunofluorescence showing neuroepithelial apical localisation of MHC-IIB. Scale = 20 μm ∗ inCindicate cells shown inD(fire LUT in which faint staining is in blue and bright staining in yellow) to illustrate variability in cap/cortical MHC-IIb irrespective of apical area.E.Correlations between apical area and MHC-IIb intensity. Each embryo's MHC-IIb values are normalised to its average staining intensity (set to 100%) to correct for inter-individual differences. Yellow lines in insets show the cap (top graphs) versus cortex (bottom graphs) regions analysed. pHH3+ = 90 cell and pHH3- = 401 cells from 8 embryos with 17-19 somites.F.Schematic illustrating the proposed association between apical area and apical MHC-IIb. pHH3+ cells progressively accumulate MHC-IIb (grey) as they constrict their apical surface. Following division, pHH3- cells can either constrict or dilate.
Fig 5: Decreased CDC42 induces entosis by promoting the ROCK1/2 signaling pathway and E-cadherin expression in Pca cells (×400 magnification). (A) No entosis morphology or increased ROCK1/2 or E-cadherin expression was observed in Pca cells treated with Akt inhibitor (MK2206; 10 µM) or ERK1/2 inhibitor (SCH772984; 3 µM) for 4 weeks, respectively. (B) IC50 values of the CDC42 inhibitor ML141 in the Pca cell lines. (C) Entosis-like morphology of Pca cells under ML141 pressure (2 µM for 4 weeks). (D) siRNA knockdown of CDC42 expression at the mRNA and protein level. *P<0.05 vs. control (Student's t-test) (E) CDC42 siRNA and ML141 (2 µM) promote ROCK1/2 expression and E-cadherin expression (following treatment for 1 week). *P<0.05 and **P<0.01 vs. control (one-way analysis of variance followed by Fisher's least-significant difference test). CDC42, cell division cycle 42; ROCK, Rho kinase; Pca, prostate cancer; Akt, protein kinase B; ERK, extracellular-signal-regulated kinase; IC50, half-maximal inhibitory concentration; siRNA, short interfering RNA; E-cadherin, epithelial cadherin; C, control; T, treatment; M, ML141 treatment; S/Si, siRNA treatment; Sc, scrambled.
Supplier Page from Abcam for Anti-ROCK2 + ROCK1 antibody [EP786Y]