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. 2018 Aug 14;11(5):1232–1243. doi: 10.1016/j.tranon.2018.07.014

Figure 2.

Figure 2

The potential involvement of Calponin 3 in cell invasion and migration. (A) The mRNA level of CNN3 in 32 previously reported colorectal cancer cell lines. Amplified CNN3 fragments were absent in 3 of the 32 CRC cell lines (i.e., in SNU-175, Caco-2, and SW-403). Of the remaining 29 cell lines, 10 (SNU-61, SNU-81, SNU-283, SNU-407, SNU-769B, SNU-C1, Colo-201, HT-29, SW-480, and WiDr) had relatively low expressions, whereas 19 of the CRC cell lines (SNU-503, SNU-769A, SNU-1033, SNU-1040, SNU-1047, SNU-1197, SNU-C2A, SNU-C4, SNU-C5, Colo-205, Colo-320, DLD1, HCT-8, HCT-15, HCT-116, LoVo, LS174T, NCI-H716, and SW-1116) exhibited strong expressions. (B) The protein level of CNN3 in two pairs of metastasis colorectal cancer cell lines. Calponin 3 was increased in metastatic CRC cell lines (SW-620, KM12SM, and KM12L4) than primary CRC cell lines (SW-480 and KM12C). (C) The efficacy of short hairpin RNA to knockdown Calponin 3 was accessed with Western blotting. The E-cadherin level was confirmed in comparison to Calponin 3. E-cadherin and Calponin 3 were inversely related. The result of 3D spheroid cell invasion assay indicated that knockdown of Calponin 3 resulted in decreased invasiveness in both (D) SNU-2404 and (E) SNU-2414 set. Cell proliferation was measured in parallel with 3D spheroid cell invasion assay to distinguish invasion ability from the growth rate in both (F) SNU-2404 and (G) SNU-2414 set. Error bars and P values are calculated with independently repeated experiments (N = 3, *P<.05, ***P<.001) Besides, peritoneal metastasis cells with downregulated Calponin 3 formed spheroid in different shape from original peritoneal metastasis cells. Cells were aggregated more tightly, and the spheroid maintained intact round shape compared to original peritoneal metastasis cells in both (H) SNU-2404 and (I) SNU-2414 set. (J, L) Downregulating Calponin 3 in SNU-2414B hindered the wound closing rate compared to SNU-2414B naive and SNU-2414B with control vector. The experiment was triplicated and averaged. (K) Cell proliferation was measured in parallel with wound healing assay to distinguish migration ability from growth rate. The experiment was triplicated and averaged. Error bars and P values are calculated with independently repeated experiments (N = 3, *P<.05, **P<.01, ***P<.001)