Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: Life Science Alliance

Article Title: Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase

doi: 10.26508/lsa.202101120

Figure Lengend Snippet: (A) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h and then lysed. Equal amounts of the whole cell lysates (WCLs) were incubated with anti-lamin A/C or pre-immune serum (IgG) as the control. The immunonocomplexes were analyzed by immunoblotting (IB) with anti-PY or anti-lamin A/C antibodies. An equal amount of WCLs was analyzed by immunoblotting with anti-Src, anti-Src pY416, or anti-actin. The tyrosine phosphorylation of lamin A was quantified and expressed as -fold relative to the level of MCF7 without dasatinib. (B) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h. The cells were fixed and stained for lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The nuclear circularity (4π × area/perimeter 2 ) was determined. The P -values were calculated from at least 150 cells pooled from three independent experiments. The percentage of the cells with nuclear lobulation was measured (n ≥ 400). The values (mean ± SD) are from three experiments. *** P < 0.001. (C) MDA-MB-231 cells were infected with lentivirues capable of expressing FLAG-lamin A or the mutants (Y45F and Y45D) and selected in the medium with neomycin (neo). An equal amount of WCLs was analyzed by immunoblotting (IB) with antibodies as indicated. (D) The cells as described in panel C were fixed and stained for FLAG-lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The percentage of the cells with nuclear lobulation was measured (n ≥ 900). Values (means ± SD) are from three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available for this figure.

Article Snippet: The rabbit polyclonal anti-Src pY416 (MAB2685) antibody was purchased from R&D Systems.

Techniques: Incubation, Control, Western Blot, Phospho-proteomics, Staining, Infection, Expressing

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: TP53-dependence on the effect of doxorubicin and Src inhibitor combination therapy.

doi: 10.1177/1010428318794217

Figure Lengend Snippet: Figure 1. The paradoxical effect of Src inhibition: (a) additive growth inhibition by SU6656 during DOX treatment in 14 cancer cell lines. Cells were treated with 0.5-mM DOX or the combination of 0.5-mM DOX and 25-mM SU6656. For the combination of DOX and SU6656, cells were pre-treated with SU6656 for 1 h prior to DOX treatment. After 2 days, cell viability was measured using the MTTassay. Additive growth inhibition was evaluated by subtracting the viable value with the combination treatment from the viable value with the DOX treatment. Data represent the mean 6 S.D. (b) pSrc (Y416) levels in TCGA samples (TCGA RPPA score) from the cBioPortal open source. The significance of the statistical comparisons was based on the two-tail t-test. (c) Increased expression of pSrc in the DOX treated HCT116–/– tumor model. Phosphorylation of Src was analyzed using immunohistochemistry as described in ‘‘Materials and methods’’ section. (d) The TP53 status depends on the differential activation of Src. HCT116+/+and HCT116–/– cells were treated with 0.5 mM of DOX for 16 h. Indirect immunofluorescence was performed using an anti-pSrc antibody. (e) HCT116+/+, HCT116–/–, U2OS, and MG63 cells were treated with 0.5-mM DOX. After treatment for the indicated time, cell lysates were prepared and Western blotting was performed to assess the levels of pSrc, Src, pp53, and p53. (f) Synergism and antagonism of SU6656 in cell lines. The box and whisker plot represents the distribution of excess over bliss score for the combination of DOX and SU6656. Cells were treated with each drug or with the combination of DOX and SU6656, for 2 days (0.5- mM DOX, 25-mM SU6656). Excess over bliss for TP53 wild-type cell lines (n = 5; HepG2, HCT116+/+, U2OS, MCF7, and A549 cells) and TP53-altered cell lines (n = 7; DU145, MG63, SaOS2, MDA-MB468, HCT116–/–, 143B, and Huh7 cells) were compared by the Mann–Whitney test. Significant differences are marked by asterisks (*p\0.05). (g) Synergy scores in colon cancer (HCT116+/ +, HCT116–/–) and osteosarcoma (U2OS, MG63) cell lines for the combination of DOX and AZD0530. Cells were treated with increasing doses of DOX (0, 0.25, 0.5, 1, 2, and 4 mM) or AZD0530 (0, 0.25, 0.5, 1, 2, and 4 mM), respectively or the combination of DOX and AZD0530. Cell viability was measured with an MTTassay after 2 days of treatment. The synergy scores were calculated by the Loewe dose-additivity model. (h) The synergistic effect of AZD0530 with DOX in p53 silenced U2OS cells. (i) The antagonistic effect of AZD0530 with DOX in MG63 cells ectopically expressing p53. The upper and lower panels represent the Loewe excess and isobologram, respectively.

Article Snippet: Sections were washed in PBS, and treated with normal goat for 20min at room temperature to block non-specific binding,20 and incubated with an anti-phospho (Y416)-Src antibody (Novus Biologicals, Littleton, CO, USA) for 16 h at 4 C. The sections were incubated for 30min at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit IgG.

Techniques: Inhibition, Expressing, Phospho-proteomics, Immunohistochemistry, Activation Assay, Immunofluorescence, Western Blot, Whisker Assay, MANN-WHITNEY