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Silencing of STRN4 suppresses the malignant characteristics of cancer cells.

Wong M, Hyodo T, Asano E, Funasaka K, Miyahara R, Hirooka Y, Goto H, Hamaguchi M, Senga T - Cancer Sci. (2014)

Bottom Line: We previously reported that STRN4 directly associated with protein kinases, such as MINK1, TNIK and MAP4K4, which are associated with tumor suppression or tumor progression.However, it remains unclear whether STRN4 is associated with tumor progression.Our results demonstrate a possible role of STRN4 in tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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STRN4 depletion induces anoikis. (a) Cells were transfected with siRNA and subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were performed, and the data are shown as the mean ± SD (*P < 0.05, compared with Ctrl siRNA-transfected cells). (b) Flag-KP4 and STRN4-KP4 cells were transfected with siRNA and then subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were performed, and the data are shown as the mean ± SD (*P < 0.05; NS, not significant). (c) siRNA-transfected cells were cultured in suspension for 24 h and then subjected to TUNEL assay. Representative images of TUNEL assay results are shown. The graph shows the percentage of apoptotic cells from three independent experiments. The data are shown as the mean ± SD (*P < 0.05, compared with Ctrl siRNA-transfected cells). (d) Flag-KP4 and STNR4-KP4 cells were subjected to anoikis assay. The graph shows the percentage of apoptotic cells from three independent experiments. The data are shown as the mean ± SD (*P < 0.05, NS, not significant).
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fig03: STRN4 depletion induces anoikis. (a) Cells were transfected with siRNA and subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were performed, and the data are shown as the mean ± SD (*P < 0.05, compared with Ctrl siRNA-transfected cells). (b) Flag-KP4 and STRN4-KP4 cells were transfected with siRNA and then subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were performed, and the data are shown as the mean ± SD (*P < 0.05; NS, not significant). (c) siRNA-transfected cells were cultured in suspension for 24 h and then subjected to TUNEL assay. Representative images of TUNEL assay results are shown. The graph shows the percentage of apoptotic cells from three independent experiments. The data are shown as the mean ± SD (*P < 0.05, compared with Ctrl siRNA-transfected cells). (d) Flag-KP4 and STNR4-KP4 cells were subjected to anoikis assay. The graph shows the percentage of apoptotic cells from three independent experiments. The data are shown as the mean ± SD (*P < 0.05, NS, not significant).

Mentions: We next assessed the anchorage-independent growth of the STRN4-depleted cells. Among the cancer cell lines we tested, KP4, PK9, HCT116, TE1 and SKOV3 formed colonies when cultured in the soft agar; therefore, we used these cell lines for the colony formation assay. siRNA-transfected cells were cultured in soft agar, and colony formation was evaluated 2 weeks later. The STRN4 siRNA-transfected cells formed significantly fewer colonies than the control siRNA-transfected cells (Fig. 3a). To confirm that the suppression of colony formation was mediated by STRN4 depletion, we performed a rescue experiment. STRN4-expressing KP4 cells were transfected with siSTRN4-3 and subjected to colony formation. The exogenous expression of STRN4 clearly inhibited the suppression of colony formation by siSTRN4-3 transfection (Fig. 3b). Anoikis is a form of cell apoptosis induced by the detachment of cells from the extracellular matrix. We hypothesized that the suppression of colony formation by STRN4 knockdown was associated with the promotion of anoikis. Cells transfected with siRNA were cultured in suspension for 48 h, and apoptotic cells were then detected via TUNEL assay. As shown in Figure 3(c), STRN4 depletion clearly increased the ratio of apoptotic cells after 48 h of suspension culture. A rescue experiment confirmed that the induction of anoikis was mediated by STRN4 depletion (Fig. 3d).


Silencing of STRN4 suppresses the malignant characteristics of cancer cells.

Wong M, Hyodo T, Asano E, Funasaka K, Miyahara R, Hirooka Y, Goto H, Hamaguchi M, Senga T - Cancer Sci. (2014)

STRN4 depletion induces anoikis. (a) Cells were transfected with siRNA and subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were performed, and the data are shown as the mean ± SD (*P < 0.05, compared with Ctrl siRNA-transfected cells). (b) Flag-KP4 and STRN4-KP4 cells were transfected with siRNA and then subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were performed, and the data are shown as the mean ± SD (*P < 0.05; NS, not significant). (c) siRNA-transfected cells were cultured in suspension for 24 h and then subjected to TUNEL assay. Representative images of TUNEL assay results are shown. The graph shows the percentage of apoptotic cells from three independent experiments. The data are shown as the mean ± SD (*P < 0.05, compared with Ctrl siRNA-transfected cells). (d) Flag-KP4 and STNR4-KP4 cells were subjected to anoikis assay. The graph shows the percentage of apoptotic cells from three independent experiments. The data are shown as the mean ± SD (*P < 0.05, NS, not significant).
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fig03: STRN4 depletion induces anoikis. (a) Cells were transfected with siRNA and subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were performed, and the data are shown as the mean ± SD (*P < 0.05, compared with Ctrl siRNA-transfected cells). (b) Flag-KP4 and STRN4-KP4 cells were transfected with siRNA and then subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were performed, and the data are shown as the mean ± SD (*P < 0.05; NS, not significant). (c) siRNA-transfected cells were cultured in suspension for 24 h and then subjected to TUNEL assay. Representative images of TUNEL assay results are shown. The graph shows the percentage of apoptotic cells from three independent experiments. The data are shown as the mean ± SD (*P < 0.05, compared with Ctrl siRNA-transfected cells). (d) Flag-KP4 and STNR4-KP4 cells were subjected to anoikis assay. The graph shows the percentage of apoptotic cells from three independent experiments. The data are shown as the mean ± SD (*P < 0.05, NS, not significant).
Mentions: We next assessed the anchorage-independent growth of the STRN4-depleted cells. Among the cancer cell lines we tested, KP4, PK9, HCT116, TE1 and SKOV3 formed colonies when cultured in the soft agar; therefore, we used these cell lines for the colony formation assay. siRNA-transfected cells were cultured in soft agar, and colony formation was evaluated 2 weeks later. The STRN4 siRNA-transfected cells formed significantly fewer colonies than the control siRNA-transfected cells (Fig. 3a). To confirm that the suppression of colony formation was mediated by STRN4 depletion, we performed a rescue experiment. STRN4-expressing KP4 cells were transfected with siSTRN4-3 and subjected to colony formation. The exogenous expression of STRN4 clearly inhibited the suppression of colony formation by siSTRN4-3 transfection (Fig. 3b). Anoikis is a form of cell apoptosis induced by the detachment of cells from the extracellular matrix. We hypothesized that the suppression of colony formation by STRN4 knockdown was associated with the promotion of anoikis. Cells transfected with siRNA were cultured in suspension for 48 h, and apoptotic cells were then detected via TUNEL assay. As shown in Figure 3(c), STRN4 depletion clearly increased the ratio of apoptotic cells after 48 h of suspension culture. A rescue experiment confirmed that the induction of anoikis was mediated by STRN4 depletion (Fig. 3d).

Bottom Line: We previously reported that STRN4 directly associated with protein kinases, such as MINK1, TNIK and MAP4K4, which are associated with tumor suppression or tumor progression.However, it remains unclear whether STRN4 is associated with tumor progression.Our results demonstrate a possible role of STRN4 in tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Show MeSH
Related in: MedlinePlus