Journal: Reproduction (Cambridge, England)

Article Title: EGF receptor kinase activity is required for gap junction closure and for part of the decrease in ovarian follicle cGMP in response to luteinizing hormone.

doi: 10.1530/REP-10-0288

Figure Lengend Snippet: Observation of the time course of nuclear envelope breakdown in intact follicle-enclosed oocytes in response to an EGFR agonist (epiregulin), or to LH in the presence or absence of an EGFR kinase inhibitor (AG1478). A. A mouse follicle on a Millicell membrane; the prophase-arrested nucleus and 2 nucleoli are visible. B. Nuclear envelope breakdown at various times after treatment of follicles with LH (350 nM), epiregulin (100 nM), or AG1478 (500 nM) for 1 hour followed by AG1478 + LH. Numbers in parentheses indicate the number of follicles.

Article Snippet: Epiregulin and amphiregulin (mouse) were from R&D Systems (Minneapolis, MN).

Techniques: Membrane

Journal: Reproduction (Cambridge, England)

Article Title: EGF receptor kinase activity is required for gap junction closure and for part of the decrease in ovarian follicle cGMP in response to luteinizing hormone.

doi: 10.1530/REP-10-0288

Figure Lengend Snippet: cGMP in follicles under experimental conditions affecting EGFR kinase or MAP kinase activity. A. Measurement of follicle cGMP under the following conditions: no treatment (basal), 1 hour LH (350 nM), 1 hour epiregulin (100 nM), 1 hour AG1478 (500 nM) followed by 1 hour AG1478 + LH, 1 hour U0126 (10 μM) followed by 1 hour U0126 + LH. Bars indicate mean ± s.e.m., and numbers in parentheses indicate the number of samples. The cGMP concentration after 100 nM epiregulin treatment is significantly different from that for basal and LH conditions (p < .0001). Likewise, the cGMP concentration after AG1478 + LH treatment is significantly different from that for basal (p < .0001) and LH (p = .01) conditions. The cGMP concentration after U0126 + LH treatment is not significantly different from that after LH alone (p = .7), but is significantly different from the basal concentration (p = .0004). B. Demonstration that in follicles stimulated with LH, treatment with 500 nM AG1478 as described above reduces EGFR phosphorylation on tyrosine 1068 to a level that is indistinguishable from the basal level without LH. 20 μg of follicle lysates, and 2 μg of EGF-treated AG431 cells, were used for immunoblotting with an antibody specific for pY1068 EGFR and then with an antibody specific for total EGFR. The graph shows the ratio of the intensities of the ~170 kDa band measured with the 2 antibodies, normalized to the no treatment condition (mean ± S.D. for 2 independent experiments). Since the follicles were cultured for 24 hours in the absence of EGFR ligands or serum prior to preparing the samples, the basal signal seen with the pY1068 antibody is probably not due to EGFR kinase activity; it could result from phosphorylation of the EGFR by other kinases or to cross-reactivity of the antibody with non-phosphorylated EGFR.

Article Snippet: Epiregulin and amphiregulin (mouse) were from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Concentration Assay, Western Blot, Cell Culture

Journal: Reproduction (Cambridge, England)

Article Title: EGF receptor kinase activity is required for gap junction closure and for part of the decrease in ovarian follicle cGMP in response to luteinizing hormone.

doi: 10.1530/REP-10-0288

Figure Lengend Snippet: Gap junction communication in follicles under experimental conditions affecting EGFR kinase activity. Gap junction communication was assessed by injecting the oocyte with Alexa Fluor 350 and imaging the follicle 20 minutes later. A. Images of follicles that were treated as follows prior to injection of the fluorescent tracer: no treatment (basal), 1 hour LH (350 nM), 1 hour epiregulin (100 nM), 1 hour epiregulin (1 μM) + amphiregulin (1 μM), 1 hour AG1478 (500 nM) followed by 1 hour AG1478 + LH. B. Ratios of fluorescence in the mural granulosa cells over that in the inner cumulus cells (MG/IC), at 20 minutes after injection, for each of the conditions indicated in A. Bars indicate mean ± s.e.m., and numbers in parentheses indicate the number of follicles. The MG/IC ratio after treatment with 100 nM epiregulin is significantly different from that after LH (p = .05), and from the basal ratio (p = .005). Likewise, the MG/IC ratio after treatment with 1 μM epiregulin + 1 μM amphiregulin is significantly different from that after LH (p = .0003); the difference from the basal ratio is marginally significant (p = .06). The MG/IC ratio for AG1478 + LH is significantly different from that for LH (p < .0001), but not from the basal ratio (p = 1.0).

Article Snippet: Epiregulin and amphiregulin (mouse) were from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Imaging, Injection, Fluorescence

Journal: Disease Models & Mechanisms

Article Title: Tellu – an object-detector algorithm for automatic classification of intestinal organoids

doi: 10.1242/dmm.049756

Figure Lengend Snippet: Performance of Tellu regarding the quantification of intestinal organoid size in response to different growth factors. (A) Representative images of manual area calculation using ImageJ (top image) or the equivalent bounding box in Tellu (bottom image). Plotted are the organoid sizes on days 1 and 6 of culture (data as shown in Fig. 3 ) obtained through manual (Manual) or automated (Tellu) measurements ( n =595). (B) Representative images of organoids grown for 5 days in control medium (NR), in the presence of EGF or EREG (100 ng ml −1 ), or in the presence of EGF and EREG together (100 ng ml −1 each). Scale bar: 250 μm. (C,D) Comparison of manual versus Tellu quantification of organoid size (Manual: n =50 for NR, n =55 for EGF, n =63 for EREG, n =67 for EGF+EREG; Tellu: n =50 for NR, n =70 for EGF, n =73 for EREG, n =81 for EGF+EREG).

Article Snippet: In experiments comparing the effects of the EGF receptor ligands EGF (R&D Systems 236-EG) and EREG (R&D Systems 968-EP), both ligands were used at 100 ng ml −1 .

Techniques: Comparison