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Role of Slug transcription factor in human mesenchymal stem cells.

Torreggiani E, Lisignoli G, Manferdini C, Lambertini E, Penolazzi L, Vecchiatini R, Gabusi E, Chieco P, Facchini A, Gambari R, Piva R - J. Cell. Mol. Med. (2012)

Bottom Line: In this study we found that Slug-silenced cells changed in morphology, decreased in their migration ability, increased Sox9 and Sox5 and decreased Sox6 and STAT1 expression.On the contrary, the effect of Slug depletion on Runx2 was influenced by cell type.As a whole, our findings have important potential implication on bone tissue engineering applications, reinforcing the concept that manipulation of specific TF expression levels may elucidate MSC biology and the molecular mechanisms, which promote osteogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biochimica e Biologia Molecolare, Sezione di Biologia Molecolare, Università degli Studi di Ferrara, Ferrara, Italy.

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Related in: MedlinePlus

Silencing of Slug gene expression by siSlug in hMSCs. hMSCs were transfected with siSlug or a non-relevant siRNA (scr). (A) Slug expression was determined at mRNA level, and revealed by quantitative RT-PCR analysis. RT-PCR results were calculated using the DDCt method, using GAPDH as the housekeeping gene, and WJ-MSCs siSlug transfected sample as the calibrator. Statistical analysis was performed control and a non-relevant siRNA versus siSlug-silenced cells (*, o and ^ for hTP-MSC, hIC-MSC and hWJ-MSC, respectively), as described in Results. (B) Slug expression was determined at protein level, and revealed by Western blot. Ten μg of whole cell lysates were assayed on a 12% SDS-PAGE, and the proteins were visualized using Supersignal Femto Substrate (Pierce). Size markers are reported (kD). IP3K was used as loading control.
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fig02: Silencing of Slug gene expression by siSlug in hMSCs. hMSCs were transfected with siSlug or a non-relevant siRNA (scr). (A) Slug expression was determined at mRNA level, and revealed by quantitative RT-PCR analysis. RT-PCR results were calculated using the DDCt method, using GAPDH as the housekeeping gene, and WJ-MSCs siSlug transfected sample as the calibrator. Statistical analysis was performed control and a non-relevant siRNA versus siSlug-silenced cells (*, o and ^ for hTP-MSC, hIC-MSC and hWJ-MSC, respectively), as described in Results. (B) Slug expression was determined at protein level, and revealed by Western blot. Ten μg of whole cell lysates were assayed on a 12% SDS-PAGE, and the proteins were visualized using Supersignal Femto Substrate (Pierce). Size markers are reported (kD). IP3K was used as loading control.

Mentions: Specifically, hMSCs were transfected with siRNA sequence targeting Slug gene transcript [18]. Non-targeting siRNA was used as a negative control. We first checked that siRNA/Slug was efficient in decreasing Slug expression in all hMSCs. Quantitative real-time RT-PCR showed that basal Slug expression levels are higher in hTP- and hIC-MSCs than in hWJ-MSCs (Fig. 2A). Nevertheless, in all cases siRNA/Slug reduced Slug mRNA expression by about 80% (P < 0.05). The efficiency of Slug silencing was also validated at protein level in all hMSCs by Western blot analysis (Fig. 2B).


Role of Slug transcription factor in human mesenchymal stem cells.

Torreggiani E, Lisignoli G, Manferdini C, Lambertini E, Penolazzi L, Vecchiatini R, Gabusi E, Chieco P, Facchini A, Gambari R, Piva R - J. Cell. Mol. Med. (2012)

Silencing of Slug gene expression by siSlug in hMSCs. hMSCs were transfected with siSlug or a non-relevant siRNA (scr). (A) Slug expression was determined at mRNA level, and revealed by quantitative RT-PCR analysis. RT-PCR results were calculated using the DDCt method, using GAPDH as the housekeeping gene, and WJ-MSCs siSlug transfected sample as the calibrator. Statistical analysis was performed control and a non-relevant siRNA versus siSlug-silenced cells (*, o and ^ for hTP-MSC, hIC-MSC and hWJ-MSC, respectively), as described in Results. (B) Slug expression was determined at protein level, and revealed by Western blot. Ten μg of whole cell lysates were assayed on a 12% SDS-PAGE, and the proteins were visualized using Supersignal Femto Substrate (Pierce). Size markers are reported (kD). IP3K was used as loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822845&req=5

fig02: Silencing of Slug gene expression by siSlug in hMSCs. hMSCs were transfected with siSlug or a non-relevant siRNA (scr). (A) Slug expression was determined at mRNA level, and revealed by quantitative RT-PCR analysis. RT-PCR results were calculated using the DDCt method, using GAPDH as the housekeeping gene, and WJ-MSCs siSlug transfected sample as the calibrator. Statistical analysis was performed control and a non-relevant siRNA versus siSlug-silenced cells (*, o and ^ for hTP-MSC, hIC-MSC and hWJ-MSC, respectively), as described in Results. (B) Slug expression was determined at protein level, and revealed by Western blot. Ten μg of whole cell lysates were assayed on a 12% SDS-PAGE, and the proteins were visualized using Supersignal Femto Substrate (Pierce). Size markers are reported (kD). IP3K was used as loading control.
Mentions: Specifically, hMSCs were transfected with siRNA sequence targeting Slug gene transcript [18]. Non-targeting siRNA was used as a negative control. We first checked that siRNA/Slug was efficient in decreasing Slug expression in all hMSCs. Quantitative real-time RT-PCR showed that basal Slug expression levels are higher in hTP- and hIC-MSCs than in hWJ-MSCs (Fig. 2A). Nevertheless, in all cases siRNA/Slug reduced Slug mRNA expression by about 80% (P < 0.05). The efficiency of Slug silencing was also validated at protein level in all hMSCs by Western blot analysis (Fig. 2B).

Bottom Line: In this study we found that Slug-silenced cells changed in morphology, decreased in their migration ability, increased Sox9 and Sox5 and decreased Sox6 and STAT1 expression.On the contrary, the effect of Slug depletion on Runx2 was influenced by cell type.As a whole, our findings have important potential implication on bone tissue engineering applications, reinforcing the concept that manipulation of specific TF expression levels may elucidate MSC biology and the molecular mechanisms, which promote osteogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biochimica e Biologia Molecolare, Sezione di Biologia Molecolare, Università degli Studi di Ferrara, Ferrara, Italy.

Show MeSH
Related in: MedlinePlus