Figure 4.

Proinflammatory cytokines induce secretion of ISGs. (A) Pre- and post-expanded INS-1E cells labeled for insulin. The inset shows a zoomed-in region highlighting the improved resolution of expanded samples. (B) ISG counts before versus after expansion for 25 different cells (each circle represents the single cell; n = 3 independent experiments). (C) Control and cytokine-treated sample of INS-1E cells stained for ISGs in maintenance condition. Cells were acquired by confocal microscope using 405 and 488 excitation light, with 63x/NA1.4 objective lens. Scale bar 10 µm. (D) Profiles of intensity taken along the 2 × 15 µm² white square. The plot profiles show a more homogeneous distribution of IGs in the cytoplasm in the control (green) with respect to cytokine-treated samples (red). (E) Granule count in CTRL and CTK expanded samples (ExM), showing a statistically significant reduction of the insulin content in CTK samples. Data were presented as box plots with whiskers at the 5th and 95th percentiles, the central line at the 50th percentile, and the ends of the box at the 25th and 75th percentiles (n = 21 cells; 3 independent experiment; the total number of optical sections examined was 337 for CTRL samples and 344 for CTK samples). (F) Total proteins were extracted, and the expression of insulin were assessed by Western blotting (WB). GAPDH was used as a control for protein loading. Protein signals were quantified and corrected for the corresponding GAPDH value and expressed as fold change compared to untreated cells (CTRL) (n = 5 independent experiments). (**p ≤ 0.01; Non-parametric Mann Whitney test; significantly different from the control condition at 24 h of incubation).