FIGURE 6.

Effect of CDKAL1 silencing on insulin granule protein content. A, glucose-stimulated insulin secretion upon CDKAL1 knockdown. INS-1 cells were transfected with control (ctr) or CDKAL1-specific (kd) siRNA oligos. 48 h after transfection, cells were either kept at rest with 0 mm glucose (white bar, Rest) or stimulated with 25 mm glucose (black bar, Stim.) for 2 h. The amount of insulin released in the medium and remaining in the cells was determined by RIA. Total insulin (ins) (left panel) was calculated as insulin_(medium) + insulin_(cell). Secreted insulin (right panel) was calculated as insulin_(medium)/total insulin. The values in control-transfected cells at 0 mm glucose equal 1. n = 6, each independent experiment performed in triplicate. Error bars in A–C show mean ± S.D., and asterisks indicate statistical significance as determined by Student's t test. **, p ≤ 0.01; *, 0.01< p < 0.05. B, INS-1 cells were transfected and treated as in A. Proinsulin content in the medium and cells was determined by ELISA. The amount of total proinsulin in control-transfected cells at 0 mm glucose equals 1. n = 3, each independent experiment was performed in triplicate. C, cell extracts from INS-1 cells 48 h after CDKAL1-siRNA or control-siRNA oligos transfection were analyzed by immunoblot with anti-insulin Ab; two independent experiments (Exp) are shown (upper panel). In the lower panel, quantification of signals for proinsulin/insulin after CDKAL1 knockdown and normalization for the corresponding signal of γ-tubulin. Values in control-transfected cells equal 1. n = 5. D, immunoblot analysis of cell extracts as in C, for the indicated granule proteins. CDKAL1 was detected with Ab1. ICA512-TMF is the transmembrane fragment generated by convertase-mediated cleavage of the luminal domain of ICA512. γ-Tubulin was used as loading control. Three independent experiments are shown.