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Id3 induces an Elk-1-caspase-8-dependent apoptotic pathway in squamous carcinoma cells.

Chen YS, Aubee J, DiVito KA, Zhou H, Zhang W, Chou FP, Simbulan-Rosenthal CM, Rosenthal DS - Cancer Med (2015)

Bottom Line: Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor.We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar.Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, District of Columbia, 20057.

No MeSH data available.


Related in: MedlinePlus

(A) Immunoblot result showing inducibility of Id3 in A431 stable cell lines (pooled clones and four representative clones) by tetracycline (1 μg/mL) for 24 h. GAPDH was used as a loading control. Clone 5 (Cl5) was chosen to proceed with experiments described. (B) A431/Id3 cells were cultured in low serum (0.1% FBS) and viable cell numbers were recorded for 13 days for both uninduced (no Tet) and induced (Tet) groups. (C) A growth curve for A431/Vc cells was determined under the same conditions as A431/Id3 cells. (D–G) A431/Id3 cells were cultured in 0.1% serum and cell cycle analysis performed to determine percentages of cells in phases of the cell cycle. (H) GFP-expressing A431/Id3 and Ds-Red-expressing A431/Vc were cocultured in 0.1% serum. Growth curves were determined and statistical analyses performed to determine any significant differences in growth. (I) Immunoblot showing Id3 and caspase-3 cleavage products for A431/Id3 cells. FBS, fetal bovine serum. P values ≤ 0.05 are considered statistically significant and represented by asterisks.
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fig01: (A) Immunoblot result showing inducibility of Id3 in A431 stable cell lines (pooled clones and four representative clones) by tetracycline (1 μg/mL) for 24 h. GAPDH was used as a loading control. Clone 5 (Cl5) was chosen to proceed with experiments described. (B) A431/Id3 cells were cultured in low serum (0.1% FBS) and viable cell numbers were recorded for 13 days for both uninduced (no Tet) and induced (Tet) groups. (C) A growth curve for A431/Vc cells was determined under the same conditions as A431/Id3 cells. (D–G) A431/Id3 cells were cultured in 0.1% serum and cell cycle analysis performed to determine percentages of cells in phases of the cell cycle. (H) GFP-expressing A431/Id3 and Ds-Red-expressing A431/Vc were cocultured in 0.1% serum. Growth curves were determined and statistical analyses performed to determine any significant differences in growth. (I) Immunoblot showing Id3 and caspase-3 cleavage products for A431/Id3 cells. FBS, fetal bovine serum. P values ≤ 0.05 are considered statistically significant and represented by asterisks.

Mentions: To investigate the effects of Id3 in malignant SCC cells, a Tet-inducible system was used to induce Id3 expression. First, stable cell lines were verified for Id3 protein induction by Tet (Fig. 1A) and a robust induction of Id3 mRNA (seven- to eightfold) was confirmed by semiquantitative reverse transcriptase-mediated PCR (RT-PCR) as well as by qRT-PCR (Fig. S1). A Tet-induction time course was performed in A431/Id3 clone 5 and A431 vector control (A431/Vc) cell lines. The induction of Id3 in the A431/Id3 cl5 cell line can be seen as early as 2 h after addition of Tet, and a decrease observed after 16 h. There was no detectable Id3 protein in A431/Vc cells throughout the time period tested (Fig. S2). Normalized Id3 protein expression levels relative to GAPDH loading control are shown in Figure S2.


Id3 induces an Elk-1-caspase-8-dependent apoptotic pathway in squamous carcinoma cells.

Chen YS, Aubee J, DiVito KA, Zhou H, Zhang W, Chou FP, Simbulan-Rosenthal CM, Rosenthal DS - Cancer Med (2015)

(A) Immunoblot result showing inducibility of Id3 in A431 stable cell lines (pooled clones and four representative clones) by tetracycline (1 μg/mL) for 24 h. GAPDH was used as a loading control. Clone 5 (Cl5) was chosen to proceed with experiments described. (B) A431/Id3 cells were cultured in low serum (0.1% FBS) and viable cell numbers were recorded for 13 days for both uninduced (no Tet) and induced (Tet) groups. (C) A growth curve for A431/Vc cells was determined under the same conditions as A431/Id3 cells. (D–G) A431/Id3 cells were cultured in 0.1% serum and cell cycle analysis performed to determine percentages of cells in phases of the cell cycle. (H) GFP-expressing A431/Id3 and Ds-Red-expressing A431/Vc were cocultured in 0.1% serum. Growth curves were determined and statistical analyses performed to determine any significant differences in growth. (I) Immunoblot showing Id3 and caspase-3 cleavage products for A431/Id3 cells. FBS, fetal bovine serum. P values ≤ 0.05 are considered statistically significant and represented by asterisks.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472214&req=5

fig01: (A) Immunoblot result showing inducibility of Id3 in A431 stable cell lines (pooled clones and four representative clones) by tetracycline (1 μg/mL) for 24 h. GAPDH was used as a loading control. Clone 5 (Cl5) was chosen to proceed with experiments described. (B) A431/Id3 cells were cultured in low serum (0.1% FBS) and viable cell numbers were recorded for 13 days for both uninduced (no Tet) and induced (Tet) groups. (C) A growth curve for A431/Vc cells was determined under the same conditions as A431/Id3 cells. (D–G) A431/Id3 cells were cultured in 0.1% serum and cell cycle analysis performed to determine percentages of cells in phases of the cell cycle. (H) GFP-expressing A431/Id3 and Ds-Red-expressing A431/Vc were cocultured in 0.1% serum. Growth curves were determined and statistical analyses performed to determine any significant differences in growth. (I) Immunoblot showing Id3 and caspase-3 cleavage products for A431/Id3 cells. FBS, fetal bovine serum. P values ≤ 0.05 are considered statistically significant and represented by asterisks.
Mentions: To investigate the effects of Id3 in malignant SCC cells, a Tet-inducible system was used to induce Id3 expression. First, stable cell lines were verified for Id3 protein induction by Tet (Fig. 1A) and a robust induction of Id3 mRNA (seven- to eightfold) was confirmed by semiquantitative reverse transcriptase-mediated PCR (RT-PCR) as well as by qRT-PCR (Fig. S1). A Tet-induction time course was performed in A431/Id3 clone 5 and A431 vector control (A431/Vc) cell lines. The induction of Id3 in the A431/Id3 cl5 cell line can be seen as early as 2 h after addition of Tet, and a decrease observed after 16 h. There was no detectable Id3 protein in A431/Vc cells throughout the time period tested (Fig. S2). Normalized Id3 protein expression levels relative to GAPDH loading control are shown in Figure S2.

Bottom Line: Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor.We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar.Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, District of Columbia, 20057.

No MeSH data available.


Related in: MedlinePlus