Journal: Oncotarget

Article Title: Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration.

doi: 10.18632/oncotarget.13261

Figure Lengend Snippet: Figure 3: Stimulus-dependent translocation of endogenous XB130 and Tks5 to the cell membrane indicates distinct signaling roles. A. Immunoblots of cytoplasm (C) and membrane (M) fractionated BEAS-2B cell lysates. Cells were treated with or without 50 ng/mL EGF, 500 nM PDBu or 0.1 μM NNK. XB130 and WAVE2 expression and translocation from the cytoplasm to the cell membrane are more dependent on EGF stimulation, whereas, Tks5 and N-WASP expression and translocation are more dependent on PDBu and NNK stimulation. B. Ratio of normalized membrane expression to normalized cytoplasm expression. Expression of Na+/K+ ATPase was used to normalize membrane fractions and expression of GAPDH was used to normalize cytoplasmic fractions. Data is summarized from three independent experiments and presented as mean ± SD. * represents p < 0.01 compared to the corresponding no treatment group.

Article Snippet: Small interfering RNA (siRNA) targeting human AFAP1L2, human Tks5 and control siRNA and rabbit anti-N-WASP (H100) (1:750), WAVE2 (H110) (1:500), PAK1 (C-19) (1:250) and mouse anti-Tks5 (M300) (1:500) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Translocation Assay, Membrane, Western Blot, Expressing

Journal: Oncotarget

Article Title: Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration.

doi: 10.18632/oncotarget.13261

Figure Lengend Snippet: Figure 4: XB130 colocalizes robustly with WAVE2 at lamellipodia but not at podosomes with N-WASP, after stimulation. A.-B. Co-immunofluorescence staining of XB130 (green), actin (blue) and either WAVE2 (A) or N-WASP (B) (red). BEAS2B cells were treated with or without 50 ng/mL EGF, 500 nM PDBu or 0.1 μM NNK. No treatment control shows normal stress fibers. Stimulation with EGF, PDBu and NNK produces actin-rich ruffled areas at the cell membrane, which are indicative of lamellipodia via WAVE2 staining (A). These areas are also enriched with XB130 (A and B). PDBu and NNK induce formation of podosomes (white arrows) which are enriched by N-WASP but not XB130 (B ). D. Mander’s overlap co-efficient (MOC) of the cell periphery displays the relative colocalization of XB130 with WAVE2, Tks5 or N-WASP, where 0 represents no colocalization and 1 represents perfect colocalization. XB130 colocalizes robustly with WAVE2 at the lamellipodia and to a lesser extent with Tks5 and N-WASP, indicating it translocates to and is involved in lamellipodia formation. Data is summarized from 10 different cells per group from 3 different experiments and presented as mean ± SD. * represents p < 0.01 for XB130/N-WASP and XB130/Tks5 MOCs compared to XB130/WAVE2 MOCs.

Article Snippet: Small interfering RNA (siRNA) targeting human AFAP1L2, human Tks5 and control siRNA and rabbit anti-N-WASP (H100) (1:750), WAVE2 (H110) (1:500), PAK1 (C-19) (1:250) and mouse anti-Tks5 (M300) (1:500) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunofluorescence, Staining, Control, Membrane

Journal: Oncotarget

Article Title: Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration.

doi: 10.18632/oncotarget.13261

Figure Lengend Snippet: Figure 8: Schematic diagram of the role of XB130 and Tks5 in Rac1 and Cdc42-associated cytoskeletal remodeling for lung epithelial cell migration. Cell migration requires cytoskeleton remodeling mediated by the Arp2/3 complex, which results in the formation of branched, F-actin rich structures (red ball and sticks), such as lamellipodia and podosomes. This diagram shows the A. Top- down view and B. Side-view of a cell displaying lamellipodia and podosome. We demonstrated a novel mechanism for lung epithelial cell migration, in which extracellular factors stimulate a sub-population of XB130 to dissociate from Tks5 and translocate to the cell periphery to promote Rac1-activated signaling of WAVE2-associated lamellipodia formation for cell extension and Tks5 mediation of Cdc42 activity via PAK1 interaction for the promotion of N-WASP-associated podosome assembly and function for ECM-dependent cell migration. Dashed black lines represent translocation of XB130 and Tks5 to the cell membrane.

Article Snippet: Small interfering RNA (siRNA) targeting human AFAP1L2, human Tks5 and control siRNA and rabbit anti-N-WASP (H100) (1:750), WAVE2 (H110) (1:500), PAK1 (C-19) (1:250) and mouse anti-Tks5 (M300) (1:500) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Migration, Activity Assay, Translocation Assay, Membrane