Journal: Journal of integrative neuroscience

Article Title: Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

doi: 10.31083/j.jin2303047

Figure Lengend Snippet: Fig. 4. Netrin-1 contributed to the sprouting of sensory nerve fiber in the metastatic bone lesions through the netrin-1/DCC signaling pathway. RT-qPCR analysis of the expression of (A) DCC and (B) UNC5B in the DRG on day 21. n = 8 per group. (C) The

Article Snippet: The following primary antibodies were used: DCC (1:1000, ab273570; Abcam), UNC5B (1:1000, #13851; Cell Signaling Technology, Danvers, MA, USA), FAK (1:1000, #3285; Cell Signaling Technology), PhosphoFAK (Tyr397) (1:1000, #3283, Cell Signaling Technology), Rac1/Cdc42 (1:1000, #4651; Cell Signaling Technology), Phospho-Rac1/Cdc42 (Ser71) (1:1000, AF2393; Affinity Biosciences, Columbus, OH, USA) and GAPDH (1:2000, BL006B; Biosharp Life Sciences, Hefei, Anhui, China).

Techniques: Quantitative RT-PCR, Expressing

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Shear stress stimulates integrin β1 trafficking and increases directional migration of cancer cells via promoting deacetylation of microtubules.

doi: 10.1016/j.bbamcr.2020.118676

Figure Lengend Snippet: Fig. 3. Clathrin-independent internalization is necessary for LSS-induced directional cell migration, FA disassembly and polarized FAs assembly. (A) Inhibiting clathrin-independent internalization blocks LSS-induced cell migration. Cell monolayers were pretreated with nystatin (100 μM, 1 h) or DMSO and kept under static condition as control or exposed to LSS (1.8 dyn/cm2) in the presence of nystatin for 2 h. These cells were then wounded with a pipet tip and migration was recorded by time-lapse video microscopy (total 15 h, 15-min frame interval). Cell tracks were determined using the Image J software (‘Manual Tracking’ plug-in). Tracks of single cells at the wounded edge were shown. The wound edge was indicated by a white line. (B) Quantification of instant velocity in (A). Cell migration velocity was analyzed for each group and plotted as a function of time (0–15 h). (C–E) Quantification of the mean velocity, Euclidean distance, and Directionality ratio in (A). (F) Percentage of cells with within a 60° angle of directionality migration in (A). (G) Time-lapsed photomicrographs of FAs in MDA-MB-231 cells. GFP-vinculin cells were pretreated with nystatin (100 μM, 1 h) or DMSO, and then kept under static condition as control or exposed to LSS (1.8 dyn/cm2) in the presence of nystatin for 2 h before imaging (30 s intervals over a 10 min time course). The outlined regions in the left panel were enlarged in the right panels at the indicated time points. Scale bar = 10 μm. (H) Kymographs taken along red lines in (G) show FAs disassembly. (I) Inhibiting clathrin-independent internalization blocks LSS-induced FA polarized assembly. MDA-MB-231 were pretreated with nystatin (100 μM, 1 h) or DMSO and kept static as control or exposed to LSS (1.8 dyn/cm2, 2 h) in the presence of nystatin. p-Paxillin (red) and DNA (blue) in wounded monolayers were examined by immunofluorescence. Scale bar = 20 μm. (J) LSS induces the colocalization of vinculin and active integrin β1, and stimulates the accumulation of Cav-1 next to the FAs on the cell edge. GFP-vinculin cells were kept under static condition as control or exposed to LSS (1.8 dyn/cm2, 15 min) and fixed with 4% paraformaldehyde. The cells were stained for active integrin β1 (red) and Cav-1 (magenta), and imaged by confocal microscopy. Bottom panels show an enlargement of the area indicated by the white rectangle in the top panels. Scale bars = 10 μm. Data represent mean ± SD of three independent experiments. *p < 0.05, ***p < 0.001, n.s., no significant difference.

Article Snippet: Monoclonal antibodies against caveolin-1 (Cav-1), acetyl-α-tubulin (Lys40), polyclonal antibodies against clathrin heavy chain, phosphoFAK (p-FAK; Tyr397) and phospho-Paxillin (p-Paxillin, Tyr118) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Migration, Control, Microscopy, Software, Imaging, Staining, Confocal Microscopy