Fig. 4: BTG2^/TIS21-induced loss of TWIST1 expression is not due to protein degradation.

a MG132 treatment failed to protect the BTG2^/TIS21-induced Twist1 loss. MDA-MB-231 (3 × 10⁵/60 mm dish) cells were transduced with either Ad-LacZ (100 moi) or Ad-TIS21(100 moi) virus for 48 h and then treated with MG132 (10 µM) for various time points to block proteasome activity. Twist1 protein was accumulated by the treatment in the LacZ expresser, whereas it was failed in the BTG2^/TIS21 expresser as opposed to accumulation of BTG2^/TIS21 expression that served as a positive control. α-Tubulin served as a loading control. b BTG2^/TIS21-induced Twist1 loss cannot be protected by NH₄Cl treatment. MDA-MB-231 (3 × 10⁵ cells) cells transduced with either Ad-LacZ or Ad-TIS21 were treated with 10 mM of NH₄Cl to block lysosome activity before immunoblot analysis at the time points. Twist1 protein was accumulated in the LacZ expresser by the treatment; however, it was lost in the BTG2^/TIS21 expresser. NH₄Cl treatment also failed to alter the expression of p62. NH₄Cl increased LC3B, the autophagosome-associated lipid form of LC3, which accumulates if their lysosomal degradation is inhibited, which served as a positive control. α-Tubulin served as a loading control. c Ubiquitination analysis. To investigate whether BTG2^/TIS21-induced TWIST1 loss is regulated by ubiquitination or not, 293TN cells were transfected with either v5-Twist1 (1.0 μg) and/or BTG2-HA (1.0 μg) before IP analysis with anti-v5 antibody and immunoblot assay with anti-ubiquitin antibody. v5-Twist1 expression was slightly increased in the vector expresser by MG132 treatment (compare lane 4 vs. lane 3 in the v5-panel); however, it was rather decreased in the BTG2^/TIS21 expresser (compare lane 6 vs. lane 5 in the v5 panel). The interaction between v5-Twist1 and BTG2^/TIS21-HA was almost similar and there was no difference in the ubiquitination after MG132 treatment. d No alteration of autophagy signal in the BTG2^/TIS21 expresser vs. LacZ control. MDA-MB-231 cells transduced with either Ad-LacZ or Ad-TIS21 were subjected to immunoblot analysis, to examine p62 and LC3B conversion. α-Tubulin as loading control. Blots are a representative of two independent experiment