Fig. 2. Knockdown of BTG2^/TIS21 expression recovers Twist1 activity.

a Regulation of Twist1 expression by BTG2^/TIS21 gene was re-examined via measuring its target gene expression by real-time PCR after forced expression of BTG2^/TIS21 gene in MDA-MB-231 cells. Note the reciprocal regulation of E-cadherin and N-cadherin expressions without significant change of Twist1 mRNA expression by BTG2^/TIS21 gene. GAPDH served as a control for amplification. b The same experiment was performed in MDA-MB-468 cells and similar regulation was observed in the BTG2^/TIS21 expresser. c To confirm the activity of BTG2^/TIS21 gene in the regulation of Twist1 target genes, MDA-MB-231 cells were transfected with siControl or siTIS21 (100 nM) for 24 h, and then transduced with either Ad-LacZ or Ad-TIS21 (100 moi). Total cellular RNAs were isolated 24 h later and subjected to real-time PCR analysis, and the expressions of E-cadherin, N-cadherin, and BTG2^/TIS21 were measured along with GAPDH as an internal control. p < 0.05 was considered as statistically significant. Note the significant changes of E-cadherin and N-cadherin expressions by the level of BTG2^TIS21 gene expression. d The same experiment was performed in MDA-MB-468 cells and the similar regulation of Twist1 target gene expression by BTG2^TIS21 was also found in the cells. Immunoblot analyses reveal the knockdown efficiency of siTIS21 in the MDA-MB-231 (e) and MDA-MB-468 (f) cells. Anti-HA and anti-α-tubulin antibodies were applied for the experiment (n = 2). All data are expressed as mean ± SD after two independent experiments