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Results: 6

1.
Fig. 2

Fig. 2. From: Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis.

PI 3-kinase inhibitors inhibit branching morphogenesis in SMG cultures denuded of mesenchyme. (A) E13 SMG were isolated, and mesenchyme was enzymatically and physically removed. (B) SMG lacking mesenchyme cultured in Matrigel for 48 h with media containing 20 ng/ml EGF and 200 ng/ml FGF7 undergo branching morphogenesis. (C, D) SMG epithelial rudiments grown in Matrigel with media containing EGF and FGF7 in the presence of PI 3-kinase inhibitors for 48 h show reduced branching: 20 μM LY (C), and 50 nM wmn (D), suggesting that PI 3-kinase inhibitors act directly on the epithelium. Scale bar, 100 μm.

Melinda Larsen, et al. Dev Biol. ;255(1):178-191.
2.
Fig. 5

Fig. 5. From: Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis.

PIP3 stimulates branching in SMG in the presence of LY inhibitor. Eight pairs of E13 glands were treated with media containing 10 μM LY + carrier C1 (A and C) or 10 μM LY + C1 + PIP3 (B and D). PIP3 stimulated cleft formation in the presence of 10 mM LY (see arrow in high magnification view in D) relative to a paired gland in (C). (E) Clefts per gland were counted and graphed relative to the number of clefts/gland at time zero and expressed in AU. Scale bar, 100 μm (A, B) and 50 μm (C, D).

Melinda Larsen, et al. Dev Biol. ;255(1):178-191.
3.
Fig. 3

Fig. 3. From: Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis.

PI 3-kinase is expressed in embryonic SMG. (A) Extracts from E12 and E13 SMG were probed with an antibody recognizing the p85 isoform of PI 3-kinase, and a band of apparent molecular weight 85 kDa was observed in both extracts. E13 SMG were fixed, immunostained, and examined by confocal microscopy. Shown is a single section of a single bud labeled with: (B) anti-p85 antibody, (C) the basement membrane marker perlecan, and (D) rhodamine-phalloidin that binds actin filaments and demonstrates effective penetration of labeled molecules. (E) Merge of three channels. PI 3-kinase was primarily localized in the epithelium (e) with some localization in the mesenchyme (m). Scale bar, 20 μm.

Melinda Larsen, et al. Dev Biol. ;255(1):178-191.
4.
Fig. 6

Fig. 6. From: Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis.

PI 3-kinase inhibitors have effects on SMG distinct from those of other signaling molecules. After 44 h, compared with control SMG (A), 10 μM LY-treated E13 SMG (B) show fewer and larger rounded buds, while 50 μM PD98059-treated SMG (C) show a reduced number of buds. The combination of 10 μM LY and 50 μM PD98059 (D) showed no significant difference from LY alone, quantitated in (E). SMG treated with an inhibitor of EGFR signaling, PD153035 at 50 pM (F), show a decrease in bud number relative to control (A). (G) With the combination of LY (5 μM) and PD15035 (50 pM), there is little difference from LY alone, which can be observed quantitatively (H). (I) SMG treated with 1.5 μM SU5402, an inhibitor of FGFR1 signaling, show elongated stalks and abnormal bud structure. (J) SMG treated with both LY (5 μM) and SU5402 (2 μM) show a composite morphology reminiscent of both inhibitors, and an additive inhibitory effect when quantitated (K). Scale bar, 100 μm.

Melinda Larsen, et al. Dev Biol. ;255(1):178-191.
5.
Fig. 4

Fig. 4. From: Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis.

Western analysis of PI 3-kinase and downstream target Akt in E13 SMG. (A) Epithelium (E)- and mesenchyme (M)-enriched cell extracts were generated and analyzed by Western analysis. The epithelial marker E-cadherin and the mesenchymal marker vimentin were used to confirm the effectiveness of the separation. In agreement with immunolocalization, PI 3-kinase was expressed primarily in the epithelium, while Akt was in both cellular compartments. (B) Quantitation of the Western analysis in (A), graphed as the relative pixel density of each band relative to the actin control for each lane. (C) Whole SMG were treated with LY (25 μM) or wmn (50 nM) for 1.5, 3, 7, or 25 h. Extracts were analyzed by Western analysis with anti-pAkt (Ser473), Akt, and actin antibodies on blots that were stripped and reprobed. (D) Quantitation of the Western blot in (C), graphed as the relative pixel density of each band relative to the actin controls for each lane and normalized to control at each time point, indicates that the level of phosphorylation of Akt at Ser473 correlates with increasing time of incubation with LY. Treatment with wmn shows a similar effect at 25 h and at 1.5–7 h (data not shown).

Melinda Larsen, et al. Dev Biol. ;255(1):178-191.
6.
Fig. 1

Fig. 1. From: Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis.

Inhibitors of PI 3-kinase inhibit salivary gland branching morphogenesis in organ culture. (A) E13 SMG were treated with 20 μM LY294002 or control media and observed with time-lapse microscopy for 20 h. Shown are the first and last images of control- and LY-treated SMG (see http://wwwdir.nidcr.nih.gov/dirweb/cdbrb/DMD/larsen/DB/movies.asp for Movie 1). There is a significant inhibition of SMG branching in the presence of 20 μM LY294002 relative to control glands at 20 h. (B, C) The PI 3-kinase inhibitors LY and wortmannin (wmn) were added to SMG organ cultures at increasing concentrations. Dose-response of LY (B) and wmn (C) in E13 SMG cultures show the number of buds per gland on the y-axis. Effects of LY on SMG branching can be reversed upon removal of inhibitor from the growth media. Shown are SMG at 24 h: control (D), 10 μM LY (E), and 50 nM wmn (F). After 68 h, the control glands (G) have continued branching, and SMG treated with LY (H) remain inhibited. (I) SMG treated with LY for 24 h and then switched to control media (LY-wash) have resumed branching. (J) Quantitation of inhibition with 10 μM LY at 24 h and recovery of SMG after removal of LY from the media, graphed as buds/gland, normalized to the number of buds per gland at 2 h, and expressed in arbitrary units (AU) vs time. Differences between all samples (except LY and LY-wash at 24 h) were significant by one-way ANOVA at P < 0.05, and the difference between LY and LY-wash at 68 h was significant at P < 0.0001 by unpaired, two-tailed t test, assuming equal variance. Scale bars, 100 μm.

Melinda Larsen, et al. Dev Biol. ;255(1):178-191.

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