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1.
FIG. 4.

FIG. 4. From: Mechanoregulation of Proliferation .

Skp2 mRNA expression is regulated by mechanical tension in human vascular SMC and human fibroblasts. RT-PCR for Skp2 and actin mRNA in human vascular SMC and in human foreskin fibroblasts isolated from released (-tension) and fixed (+tension) collagen matrices (two different concentrations of the RT reaction products were loaded to confirm that the actin PCR was in the linear range).

Xiaogang Jiang, et al. Mol Cell Biol. 2009 September;29(18):5104-5114.
2.
FIG. 2.

FIG. 2. From: Mechanoregulation of Proliferation .

Mechanoregulation of smooth muscle proliferative signals in intact tissue. (a) Immunoblots of p21CIP1, p27KIP1, PCNA, and β-tubulin in the smooth muscle layer of mouse bladders that were either unobstructed or completely obstructed for the time indicated (each lane in this and subsequent bladder obstruction experiments represents an assessment of the protein or RNA from an individual mouse). (b) RT-PCR for p27KIP1 mRNA in the smooth muscle layer of mouse bladders that were either unobstructed or completely obstructed for the time indicated (two different concentrations of the RT reaction products were loaded to confirm that the p27KIP1 PCR was in the linear range). (c) Immunoblots of Skp2 and β-tubulin in the smooth muscle layer of mouse bladders that were either unobstructed or completely obstructed for the time indicated. (d) RT-PCR for Skp2 and actin mRNA in the smooth muscle layer of mouse bladders that were either unobstructed or completely obstructed for the time indicated (two different concentrations of the RT reaction products were loaded to confirm that the actin PCR was in the linear range).

Xiaogang Jiang, et al. Mol Cell Biol. 2009 September;29(18):5104-5114.
3.
FIG. 3.

FIG. 3. From: Mechanoregulation of Proliferation .

Skp2 is required for bladder smooth muscle proliferation both in tissue culture and in intact tissue. (a) Immunoblots for Skp2, p27KIP1, and tubulin in human bladder SMC that were adherent to a tissue culture dish and transfected with scrambled or two different Skp2-specific siRNAs. (b) Cell numbers at indicated time points of human SMC that were adherent to a tissue culture dish and transfected as in panel a. (c) The bladder walls of skp2+/+ and skp2−/− mice that were either unobstructed or partially obstructed for 14 days and then fixed and stained with hematoxylin and eosin.

Xiaogang Jiang, et al. Mol Cell Biol. 2009 September;29(18):5104-5114.
4.
FIG. 6.

FIG. 6. From: Mechanoregulation of Proliferation .

NFATc1 directly targets the Skp2 promoter. (a) Comparison of the NFAT-binding site and surrounding sequence in the human and mouse Skp2 promoters with the NFAT-binding site and surrounding sequence in the human IL-2 promoter, the prototypical NFAT-binding site (38). (b) Immunoblot of NFATc3 and NFATc1 in nuclear lysates and whole-cell lysates of adherent and suspended human bladder SMC. Lamin A/C and tubulin were used as loading controls. (c) Immunoblot of NFATc1 in whole-cell lysates of suspended human bladder SMC treated with calf intestinal alkaline phosphatase as indicated. Nuclear lysates of adherent cells were used as a reference. (d) Immunoblot for NFATc1 in nuclear lysates and whole-cell lysates from the smooth muscle layer of mouse bladders that were partially obstructed as indicated. Lamin A/C was used as a loading control. (e) ChIP assay of the endogenous Skp2 promoter in the bladder smooth muscle layer of mouse bladders that were either unobstructed or partially obstructed as indicated. Primers targeted to the 5′-untranslated region of the β-globin gene were used as a control for specificity.

Xiaogang Jiang, et al. Mol Cell Biol. 2009 September;29(18):5104-5114.
5.
FIG. 1.

FIG. 1. From: Mechanoregulation of Proliferation .

Mechanoregulation of smooth muscle proliferative signals in a tissue culture model. (a) Schematic of a tissue culture system to study the effects of mechanical tension on proliferation (see text for details). (b) Fluorescence staining with Alexa Fluor 488-phalloidin for actin in human bladder SMC in released (-tension) and fixed (+tension) collagen matrices. (c) BrdU and DAPI (4′,6′-diamidino-2-phenylindole) staining of human bladder SMC isolated from released (-tension) and fixed (+tension) collagen matrices. (d) Immunoblots of p21CIP1, p27KIP1, PCNA, and β-tubulin in human bladder SMC isolated from released (-tension) and fixed (+tension) collagen matrices. (e) RT-PCR for p27KIP1 and b-myb mRNA in human bladder SMC isolated from released (-tension) and fixed (+tension) collagen matrices (two different concentrations of the RT reaction products were loaded to confirm that the p27KIP1 PCR was in the linear range). The level of b-myb mRNA was used as a proliferative marker. (f) Immunoblots of Skp2 and β-tubulin in human bladder SMC isolated from released (-tension) and fixed (+tension) collagen matrices. (g) RT-PCR for Skp2 and actin mRNA in human bladder SMC isolated from collagen matrices that were either released from (-tension) or left fixed to (+tension) the tissue culture dish in either serum-containing (+FBS) or serum-free (-FBS) media (two different concentrations of the RT reaction products were loaded to confirm that the actin PCR was in the linear range). (h) Immunoblots of Skp2 and β-tubulin in human bladder SMC isolated from collagen matrices that were left fixed to (+tension) the tissue culture dish in either serum-containing (+FBS) or serum-free (-FBS) medium.

Xiaogang Jiang, et al. Mol Cell Biol. 2009 September;29(18):5104-5114.
6.
FIG. 7.

FIG. 7. From: Mechanoregulation of Proliferation .

NFATc1 regulates Skp2 expression. (a) Immunoblot for NFATc1 in human bladder SMC that were adherent to a tissue culture dish and treated with indicated concentrations of cyclosporine for 12 h. (b) RT-PCR for Skp2 and GAPDH mRNA in human bladder SMC that were adherent to a tissue culture dish and treated with cyclosporine as in panel a (two different concentrations of the RT reaction products were loaded to confirm that the GAPDH PCR was in the linear range). (c) Immunoblots for Skp2, p27KIP1, and tubulin in human bladder SMC that were adherent to a tissue culture dish and treated with cyclosporine as in panel a. (d) Immunoblots for Erk and phospho-Erk in human bladder SMC that were adherent to a tissue culture dish and treated with cyclosporine as in panel a. Cells grown in the absence of serum (FBS) were used as a positive control for dephosphorylated Erk. (e) Immunoblots for NFATc1 and tubulin and RT-PCR for Skp2 and GAPDH mRNA in human bladder SMC that were adherent to a tissue culture dish and transfected with scrambled or two different NFATc1-specific siRNAs (two different concentrations of the RT reaction products were loaded to confirm that the GAPDH PCR was in the linear range). (f) Immunoblots for NFATc1, Skp2, p27KIP1, and tubulin in human bladder SMC that were adherent to a tissue culture dish and then transfected as in panel e. (g) Cell numbers at indicated time points of human SMC that were adherent to a tissue culture dish and transfected as in panel e. (h) DAPI-stained nuclei of human bladder SMC 120 h after the cells were transfected as in panel e. Similar results were obtained with both NFATc1 siRNAs.

Xiaogang Jiang, et al. Mol Cell Biol. 2009 September;29(18):5104-5114.
7.
FIG. 5.

FIG. 5. From: Mechanoregulation of Proliferation .

A consensus NFAT-binding sequence mediates the mechanoregulation of Skp2 transcription, and NFAT activity is increased by increased mechanical tension in intact tissue. (a) Human bladder SMC were first allowed to develop tension in collagen matrices. The cells were then treated with actinomycin D and the matrices were either released from (-tension) or left fixed to (+tension) the tissue culture dish (“0 h”). Cells were harvested to perform RT-PCR for Skp2 and actin mRNA at the indicated time points (two different concentrations of the RT reaction products were loaded to confirm that the actin PCR was in the linear range). (b) Luciferase assays of human bladder SMC transfected with (i) pGL3-promoter (control), (ii) a construct in which 423 bp of the 5′ flanking region of the Skp2 gene drives a luciferase reporter (Skp2-luc), or (iii) a construct in which a consensus NFAT-binding site (AGGAAAA) (38) has been deleted from Skp2-luc (Skp2ΔNFAT-luc) isolated from collagen matrices that were either released from (-tension) or left fixed to (+tension) the tissue culture dish. (c) Luciferase assay as in panel B, except using human bladder SMC transfected with pGL3-promoter (control) or pGL3-promoter driven by five tandem repeats of the Skp2 NFAT-binding site (NFAT-luc). (d) Luciferase assay using the smooth muscle layer of bladders from mice transgenic for an NFAT-luc reporter that were either unobstructed (sham) or completely obstructed for 24 h (the mean values obtained from three mice for each condition).

Xiaogang Jiang, et al. Mol Cell Biol. 2009 September;29(18):5104-5114.

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