Fig 1: Senescence of cultured human beta-like cells induces functional maturation genes and increases interferon responsiveness. (A) EndoC-βH3 cells with or without CreER activation (non-Sen and Sen, respectively) stained for the senescence marker SA-βGal (top, blue) or for Insulin (bottom, red) and the proliferation marker Ki67 (green). Scale bar = 100 μm. (B) Percentage of Ki67+ non-senescent and senescent EndoC-βH3 cells stained as in (A). Mean of n = 6 replicates ± SEM, t test. (C) Glucose-stimulated insulin secretion (GSIS) of non-senescent and senescent EndoC-βH3 cells. Insulin levels in medium were measured by ELISA in cells exposed to low (1.5 mM) or high (20 mM) glucose levels. Mean of n = 3 replicates ± SEM, t test. (D) Levels of p21 mRNA in senescent and non-senescent cells as measured by mRNA-seq. Mean of n = 6 replicates ± SEM Padj < 0.001, DESeq2. (E) Gene sets downregulated in senescent EndoC-βH3 cells. x axis indicates log(Padj) (Metascape). (F) Gene sets upregulated in senescent EndoC-βH3 cells, measured by mRNA-seq, calculated by Metascape (left) or by MSigDB (right). (G) Scores of indicated signatures in non-senescent and senescent EndoC-βH3 cells. Boxes indicate interquartile values with indicated median of n = 6 replicates, t test. (H) Relative mRNA levels of indicated genes in non-senescent and senescent EndoC-βH3 cells measured by mRNA-seq. Mean of n = 6 replicates ± SEM, Padj < 0.001 for all comparisons, DESeq2. (I, J) Heat maps representing scaled log2 expression levels of PDX1 and MAF target genes (I) and of interferon-stimulate genes (J) in non-senescent and senescent EndoC-βH3 cells (n = 4 replicates). (K) mRNA levels of indicated interferon-stimulated genes in non-senescent and senescent EndoC-βH3 cells untreated or treated with IFNα, measured by qRT-PCR. Mean of n = 3 experimental repeats ± SEM, normalized to untreated non-senescent cells, t test. (L) FACS analysis HLA-I levels in non-senescent and senescent EndoC-βH3 cells, untreated or treated with IFNα for 24 h. (M) Western blot of phospho-STAT1 and total STAT1 levels in non-senescent and senescent EndoC-βH3 cells, untreated or treated with IFNα for 24 h. GAPDH serves as loading control. (N) mRNA levels of indicated functional maturation and interferon-stimulated genes in non-senescent and senescent EndoC-βH3 cells expressing a control vector or a p16 overexpression vector, measured by qRT-PCR. Mean of n = 3 experimental repeats ± SEM, normalized to control non-senescent cells, t test.