The Soft Agar Colony Formation Assay

Borowicz, Stanley; Scoyk, Michelle Van; Avasarala, Sreedevi; Rathinam, Manoj Kumar Karuppusamy; Tauler, Jordi; Bikkavilli, Rama Kamesh; Winn, Robert A.

doi:10.3791/51998

Cited by 391 publications

(337 citation statements)

References 13 publications

(6 reference statements)

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“…The soft agar assay was performed as described previously (26). Matrigel invasion was performed using Corning's Cell Invasion Assay protocol as described previously (21).…”

Section: Hur Knockdown Cell Line Generationmentioning

confidence: 99%

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth

et al. 2016

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Growing evidence shows that cancer cells use mRNA-binding proteins and miRNAs to posttranscriptionally regulate signaling pathways to adapt to harsh tumor microenvironments. In ovarian cancer, cytoplasmic accumulation of mRNA-binding protein HuR (ELAVL1) is associated with poor prognosis. In this study, we observed high HuR expression in ovarian cancer cells compared with ovarian primary cells, providing a rationale for targeting HuR. RNAi-mediated silencing of HuR in ovarian cancer cells significantly decreased cell proliferation and anchorage-independent growth, and impaired migration and invasion. In addition, HuR-depleted human ovarian xenografts were smaller than control tumors. A biodistribution study showed effective tumortargeting by a novel Cy3-labeled folic acid (FA)-derivatized DNA dendrimer nanocarrier (3DNA). We combined siRNAs against HuR with FA-3DNA and found that systemic administration of the resultant FA-3DNA-siHuR conjugates to ovarian tumorbearing mice suppressed tumor growth and ascites development, significantly prolonging lifespan. NanoString gene expression analysis identified multiple HuR-regulated genes that function in many essential cellular and molecular pathways, an attractive feature of candidate therapeutic targets. Taken together, these results are the first to demonstrate the versatility of the 3DNA nanocarrier for in vivo-targeted delivery of a cancer therapeutic and support further preclinical investigation of this system adapted to siHuR-targeted therapy for ovarian cancer.

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“…The soft agar assay was performed as described previously (26). Matrigel invasion was performed using Corning's Cell Invasion Assay protocol as described previously (21).…”

Section: Hur Knockdown Cell Line Generationmentioning

confidence: 99%

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth

et al. 2016

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“…Tumor cells, on the other hand, would remain capable of growing in the absence of anchorage dependence (18). The soft agar colony assay is able to determine the anchorage-independent growth of tumor cells, as well as the malignancy, and the stronger invasion capacity of tumor cells is associated with a greater number of cell colonies (19). The present study revealed that RUNX2-specific siRNA suppresses the colony formation of SAOS-2 in soft agar in a concentration-dependent manner (Fig.…”

Section: Discussionmentioning

confidence: 99%

RUNX2 RNA interference inhibits the invasion of osteosarcoma

2015

Oncology Letters

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Abstract.It has previously been demonstrated that the expression of the RUNX2 gene is increased in osteosarcoma tissues or cell lines; however, there is little research available on the effect of RUNX2 on osteosarcoma invasion. In the present study, small interfering (si)RNA to RUNX2 was designed and synthesized, and then transfected into SAOS-2 cells. The effects of RUNX2 RNA interference on the invasion of osteosarcoma cells were detected by the soft agar colony forming test and Transwell ® chamber assay. The expression of the associated proteins, vascular endothelial growth factor (VEGF), matrix metalloprotein-2 (MMP-2) and MMP-9, was detected by western blot analysis. The results revealed that the number of cell colonies was reduced dose-dependently by the siRNA and that the number of cells permeating through the filter membrane was decreased following transfection with the siRNA. The inhibition of RUNX2 caused a notable decrease in VEGF, MMP-2 and MMP-9 expression (0.16±0.04, 0.16±0.02 and 0.12±0.02) compared with the empty vector (0.86±0.22, 0.74±0.16 and 0.81±0.16) and blank control (0.78±0.12, 0.82±0.18 and 0.78±0.14) groups, respectively (P<0.01). It can therefore be concluded that RUNX2 siRNA inhibits the invasion of osteosarcoma cells by inhibiting the expression of VEGF, MMP-2 and MMP-9. IntroductionOsteosarcoma is a common type of malignant tumor in adolescents and children and thus, its etiology and pathogenesis have previously been unknown. It accounts for ~2.4% of all malignancies in pediatric patients, and ~20% of all primary bone cancers (1). The estimated annual incidence rate of osteosarcoma in patients <20 years of age in the USA is 5.0 per million individuals, and the disease is slightly more common in males (5.4 per million individuals) than females (4.0 per million individuals) (1). The prognosis of osteosarcoma is poor. Surgery is the main treatment, however, surgery alone may not prevent metastasis. However, surgery in combination with chemotherapy may prolong patient survival time (2). Certain studies have shown that RNA interference (RNAi) knockdown of cyclo-oxygenase 2 (3), ERK1/2 (4) or relaxin (5) inhibits the growth, invasion and migration of human osteosarcoma cells. The RUNX2 gene plays a crucial role in osteoblast differentiation and bone formation, and is closely correlated with the generation and development of osteosarcoma (6). Studies revealed that the expression of the RUNX2 gene was increased in osteosarcoma tissues or cell lines (7-10), but there is limited research available on the effect of RUNX2 on osteosarcoma invasion. In the present study, RUNX2 was downregulated in the osteosarcoma SAOS-2 cell line by RNAi, and its effects on cell invasion and the potential regulatory mechanism were studied. Materials and methodsExperimental materials. The human osteosarcoma SAOS-2 cell line (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) was stored in liquid nitrogen in the laboratory (Central Laboratory, Tonji Medical College, Wuhan, China). Dulbecco's modified E...

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“…To evaluate the self-renewal ability of mESC, the colony formation assay was performed using modifications of the method reported by Borowicz et al 72 Six-well plates were coated with mixture of 0.5 % noble agar and DMEM with/without Gln. After 30 minutes incubation, the viable mESCs suspended at DMEM with/without Gln were mixed with same volume of 0.6 % noble agar, and seeded the 2000 cells/well.…”

Section: Colony Formation Assaymentioning

confidence: 99%

Glutamine contributes to maintenance of mouse embryonic stem cell self-renewal through PKC-dependent downregulation of HDAC1 and DNMT1/3a

2015

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(2015) Glutamine contributes to maintenance of mouse embryonic stem cell self-renewal through PKCdependent downregulation of HDAC1 and DNMT1/3a, Cell Cycle, 14:20, 3292-3305, DOI: 10.1080/15384101.2015 Although glutamine (Gln) is not an essential amino acid, it is considered a critical substrate in many key metabolic processes that control a variety of physiological functions and are involved in regulating early embryonic development. Thus, we investigated the effect of Gln on regulation of mouse embryonic stem cell (mESC) self-renewal and related signaling pathways. Gln deprivation decreased Oct4 expression as well as expression of cell cycle regulatory proteins. However, Gln treatment retained the expression of cell cycle regulatory proteins and the Oct4 in mESCs, which were blocked by compound 968 (a glutaminase inhibitor). In addition, Gln stimulated PI3K/Akt pathway, which subsequently elicited PKCe translocation to membrane without an influx of intracellular Ca 2+ . Inhibition of Akt and PKC blocked Glninduced Oct4 expression and proliferation. Gln also stimulated mTOR phosphorylation in a time-dependent manner, which abolished by PKC inhibition. Furthermore, Gln increased the cellular population of both Oct4 and bromodeoxyuridine positive cells, suggesting that Gln regulates self-renewal ability of mESCs. Gln induced a decrease in HDAC1, but not in HDAC2, which were blocked by PKC inhibitors. Gln treatment resulted in an increase in global histone acetylation and methylation. In addition, Gln significantly reduced methylation of the Oct4 promoter region through decrease in DNMT1 and DNMT3a expression, which were blocked by PKC and HDAC inhibitors. In conclusion, Gln stimulates mESC proliferation and maintains mESC undifferentiation status through transcription regulation via the Akt, PKCe, and mTOR signaling pathways.

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The Soft Agar Colony Formation Assay

Cited by 391 publications

References 13 publications

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth

RUNX2 RNA interference inhibits the invasion of osteosarcoma

Glutamine contributes to maintenance of mouse embryonic stem cell self-renewal through PKC-dependent downregulation of HDAC1 and DNMT1/3a

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