FIGURE 4.

HTS in primary human cells identifies p18 and p21 as inhibitors of beta cell cycle entry. a, Z-score plots of screen results from three independent donors of islets. Control cells not expressing SV40 large T antigen (TAg) (blue circles), TAg⁺ cells infected with control shRNA (orange circles), and TAg⁺ cells infected with library shRNAs (gray circles) are shown. Dotted red lines mark the region of both Z-scores greater than 2. The x axis represents Z-scores of double-positive (EdU, insulin) cells based on the absolute number; the y axis represents Z-scores for the percentage of total beta cells. b, screen images showing EdU-positive cells (red) in beta cells silenced for p18. Scale bar, 10 μm. c, histogram showing percentage of EdU⁺ insulin⁺ cells following p18 silencing over a 10-day assay period compared with non-targeting control (CON). d, Western blot showing knockdown of p18 following p18 silencing. e, histogram showing percentage of EdU⁺ insulin⁺ cells in each screen replicate following p18 silencing over a 10-day assay period compared with non-targeting control (CON). f, screen images showing EdU-positive cells (red) in beta cells silenced for p21 using lenti-shRNA constructs used in the screen. Scale bar, 10 μm. g, histogram showing percentage of EdU⁺ insulin⁺ cells following p21 silencing over a 10-day assay period compared with non-targeting control (CON). h, Western blot showing knockdown of p21 following silencing using lenti-shRNA constructs used in the screen. i, histogram showing percentage of EdU⁺ insulin⁺ cells in each screen replicate following p21 silencing over a 10-day assay period compared with non-targeting control (CON). For data in c, e, and i, * indicates p < 0.05, two-tailed unpaired t test.