siRNA knockdown of the key ER stress genes ATF-4 and DDIT3 leads to reduced sensitivity towards JC011 in NCCIT cells.

ATF-4 and DDIT3 were silenced via siRNA knockdown. Sensitivity towards JC011 was attenuated in DDIT3 knockdown and ATF-4 knockdown (P<0.05) NCCIT cells thereby confirming involvement of the PERK/ATF4/DDIT3 ER stress pathway in JC011 mediated cytotoxicity (A–E). qRT-PCR confirmation of ATF-4 and DDIT3 transcript knockdown (F). Cell viability figures were normalized to untreated controls and reported as mean ± S.D. of three independent experiments (n = 3). Statistical analysis was performed with the Student′s T-test.

Untreated zebrafish (A, G) and zebrafish treated with DMSO (B, H), JC11 (C, D, E, I and J) and 9-cis retinoic acid (F, K). DMSO treated zebrafish appeared morphologically similar to untreated zebrafish. Most zebrafish treated with JC11 showed normal morphology (C, I). A small percentage of zebrafish treated with JC11 had tail malformation (D, E, arrow), liver malformation (E, J, red circle) and intestinal malformation (E, yellow line). In=intestine, L=liver, T=trunk/tail.