Fig. 2

NIPSNAP1 expression is transcriptionally repressed by c-Myc-Miz1 with de-repression in response to serum deprivation. A Relative NIPSNAP1 mRNA levels in HCT116 cells after transduction with sh-Ctrl, sh-SP1, sh-HIF1a, sh-c-Jun, sh-FOXO1 or sh-c-Myc lentiviruses determined by qPCR at 0, 24 and 48 h following FBS withdrawal. B Western blotting measurements of NIPSNAP1 and P27 levels in HCT116 cells treated with sh-Ctrl or sh-c-Myc lentiviruses at 0, 24 and 48 h following FBS withdrawal. Actin was used throughout as a loading control. C HCT116 cells were transduced with sh-Ctrl or sh-Miz1 lentiviruses at 0, 24 and 48 h following serum (FBS) withdrawal. Cell lysates were subjected to Western blotting against Miz1 or actin (top) or total RNA used for qPCR-based measurement of NIPSNAP1 mRNA (bottom). D HCT116 cells were transduced with sh-Ctrl or sh-c-Myc in combination with empty Flag-vector or the Flag-Miz1 overexpression vector. Cell lysates were subjected to Western blotting against Flag or c-Myc (top) or total RNA used for qPCR-based measurement of NIPSNAP1 mRNA (bottom). E Schematic illustrating the putative c-Myc binding sites (BSs) in the NIPSNAP1 promoter, termed c-Myc-BS1 (− 1929 to − 1921 bp) and c-Myc-BS2 (− 279 to − 272 bp), respectively. F ChIP assays targeting c-Myc (left) or Miz1 (right) and the corresponding control IgG samples performed in HCT116 cells. Primers targeting c-Myc-BS1 and c-Myc-BS2 were used along with negative and positive control primers against the GAPDH and LAST promoters, respectively, the latter a known target of c-Myc/Miz1. G Schematic illustrating the design of pGL3-based luciferase reporter constructs containing the wildtype (WT) or mutant c-Myc binding sites in the NIPSNAP1 promoter. H Luciferase reporter assays conducted in HCT116 cells measuring the activity of the WT NIPSNAP1 reporter construct after co-transfection of the empty 3*Flag-vector or 3*Flag-c-Myc overexpression vector. Relative luciferase activity (left) and Western blotting to confirm ectopic c-Myc expression. I Luciferase reporter assays were conducted as per H after transduction with sh-Ctrl or sh-c-Myc and co-transfection with either the wildtype (WT) or mutant (Mut) c-Myc reporter constructs. J Luciferase reporter assays were conducted as per H after transduction with sh-Ctrl or sh-Miz1 and co-transfection with the empty 3*Flag-vector or 3*Flag-c-Myc overexpression vector. Relative luciferase activity (left) and Western blotting to confirm Miz1 knockdown and ectopic c-Myc expression. K Luciferase reporter assays were conducted as per H after transduction with either the wildtype (WT) or mutant (Mut) c-Myc reporter constructs at 0, 24 and 48 h following serum (FBS) withdrawal. A, C, D and H–K are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test, Images in B and F represents three independent experiments (*P < 0.05; **P < 0.01, ***p < 0.001)