Figure 3

CircZDHHC11 overexpression does not rescue the effects of miR-150 overexpression and circZDHHC11 regulates growth independent of its miR-150 binding sites. (A) Effective overexpression of circZDHHC11 was obtained in ST486 upon infection with a lentiviral vector containing the circZDHH11 sequence. Mean expression of a representative experiment is shown with upper and lower limits. The levels in absence of circZDHHC11 overexpression are set to 1. (B) Relative cell growth of ST486 was assessed upon overexpression of circZDHHC11 (green) or infection with a control vector (black). The percentage of GFP⁺ cells was followed post-transfection for three weeks and the GFP percentage was normalized to day 4 after transfection. Mean ± SD of two independent experiments is shown. (C) ST486 cells transfected with the lentiviral vector for circZDHHC11 overexpression (triangles) or control vector (circles) (both with GFP reporter) were transfected with the lentiviral vector for miR-150 overexpression carrying a BFP reporter (dark and light blue) or control (black and grey). Relative cell growth was assessed by following the percentages of BFP⁺ cells in the GFP⁺ fraction over three weeks post-transfection, with the percentages normalized to day 4 after transfection. Significance was determined by mixed model analysis; ***p < 0.001. (D-F) Same as (A–C) in DG75 cells. (G) Expression levels of circZDHHC11 in WT ST486 and the monoclonal NTCR1CR2 control and miR-150BSdel mutant cell lines were analyzed by RT-qPCR. (H–I) The effect of circZDHHC11 knockdown on growth of the monoclonal NTCR1CR2 and miR-150BSdel cell lines cells (sh1: light red, sh2: dark red, NT1: black, NT2: grey). Relative cell growth was assessed by following the percentage of RFP⁺ cells over three weeks post-transfection from three independent experiments, with the RFP percentage normalized to day 4 after transfection. Significance was determined by mixed model analysis; ****p < 0.0001.