Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 1 Identification of SRC family kinase (SFK) members in porcine spermatozoa. Semen samples from several boars were lysed and run in a 10% SDS–PAGE, and western blotting was performed using anti c-Lyn (A), anti c-Yes (B), and anti p-Y416 SFK (C) antibodies as described. Positive controls of different porcine tissues: liver, lung, and brain (A, lanes 4, 5 and 6), or somatic cell types such as rat pancreatic acini (B, lane 4) and neuroblasts (B, lane 5 and 6) were also included.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: SDS Page, Western Blot

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 2 Enzymatic activation of SFK during porcine spermatozoa capacitation. Spermatozoa samples incubated under noncapacitating (TBM) or capacitating conditions (TCM) during 4 h were lysated and run in a 10% SDS–PAGE, and western blotting analysis was performed using anti p-Y416 SFK or anti-actin as a protein loading control. Panel A shows a representative experiment for SFK1 and SFK2 tyrosine phosphorylation at the indicated times. Values shown at the bottom graphs in Panel B are meanGS.E.M. of three independent experiments. Quantification of bands was performed by scanning densitometry. Asterisk shows statistical differences between samples incubated under capacitating conditions in TCM with those incubated in TBM at the same time point.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: Activation Assay, Incubation, SDS Page, Western Blot, Control, Phospho-proteomics

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 3 SFK inhibition enhances spontaneous acrosome reaction in porcine spermatozoa. Spermatozoa samples were incubated under noncapacitating (TBM) or capacitating conditions (TCM) in the presence or absence of the SFK inhibitor SU6656 (10 mM) for 4 h. Flow cytometry was then performed using FITC-PNA as a specific marker for spermatozoa acrosome status and propidium iodide (PI) as a marker for cell death. Results are expressed as the percentage of PNA-positive and PI-negative spermatozoaGS.E.M. (nZ6). Asterisk shows statistical differences of samples incubated with or without SU6656 at the same time point.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: Inhibition, Incubation, Flow Cytometry, Marker

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 4 SFK inhibition enhances ionophore-induced acrosome reaction in porcine spermatozoa. Spermatozoa were incubated under capacitating conditions (TCM) with the ionophore A23187 (1 mM) in the presence or absence of the SFK inhibitor SU6656 (10 mM) for 4 h. Flow cytometry was then performed using FITC-PNA as a specific marker for spermatozoa acrosome status and propidium iodide (PI) as a marker for cell death. Results are expressed as the percentage of PNA-positive and PI-negative spermatozoaGS.E.M. (nZ4). Asterisk shows statistical differences of samples incubated with or without SU6656 at the same time point.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: Inhibition, Incubation, Flow Cytometry, Marker

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 5 Effect of SFK inhibition in the F-actin content of porcine spermatozoa under capacitating conditions. F-actin content of boar spermatozoa was evaluated by flow cytometry using FITC–phalloidin as a specific marker for F-actin, as described in the Materials and Methods section. (A) Shows typical cytograms obtained after FITC– phalloidin staining of spermatozoa under different conditions: noncapacitating (TCM time 0) and capacitating (TCM 2 h), and spermatozoa induced to undergo acrosome reaction with 10 mM calcium ionophore (TCM CA23187). Cytograms show an increase in FITC–phalloidin staining representative of F-actin formation after incubation on TCM for 2 h at 38.5 8C and that F-actin content decreases close to basal level after the induction of acrosome reaction. (B) Shows quantification by flow cytometry of F-actin content of spermatozoa incubated under capacitating conditions (TCM) in the absence (solid bars) or presence (striped bars) of the SFK inhibitor SU6656 (10 mM) for 4 h. Data are expressed as the average percentage of spermatozoa showing high fluorescence intensity (high FITC–phalloidin staining) GS.E.M. (nZ5). Asterisk shows statistical differences between sperma- tozoa incubated in the presence or absence of SU6656 at the same time.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: Inhibition, Cytometry, Marker, Staining, Incubation

Journal: Oral oncology

Article Title: IL6 is associated with response to dasatinib and cetuximab: Phase II clinical trial with mechanistic correlatives in cetuximab-resistant head and neck cancer

doi: 10.1016/j.oraloncology.2017.03.011

Figure Lengend Snippet: Targeting both EGFR and SFK pathways shows enhanced HNSCC cell killing in vitro compared to single agent targeting. PECAPJ49 cells (A) or CAL33 cells (B) were treated with 0–50 nM dasatinib in the presence or absence of 100 nM cetuximab for 96 h in 24 well plates. The cells were washed, fixed and stained with crystal violet to determine cell proliferation. Solubilized crystal violet for each well was transferred to a 96-well plate and absorbance was read at 570 nM. Results are the mean ± SD of the average of two independent experiments. ANOVA, *p < 0.05; **p < 0.01.

Article Snippet: Tissues were evaluated by immunohistochemistry (IHC) for pSFK and pSTAT3 expression using anti-Phospho-SFK PY-416 (PK1109 1:50; Calbiochem) antibody and anti-Phospho-STAT3 PY-705 antibody (D3A7 1:500; Cell Signaling Technology).

Techniques: In Vitro, Staining