Supplementary Figure 3: α-cell transcriptome analysis and gene expression changes after β-cell loss. | Nature Cell Biology

Supplementary Figure 3: α-cell transcriptome analysis and gene expression changes after β-cell loss.

From: Pancreatic islet-autonomous insulin and smoothened-mediated signalling modulate identity changes of glucagon+ α-cells

Supplementary Figure 3

(a) PCA plot of all samples analyzed by RNA-Seq. Native β-cells (‘β’, n = 5 mice) show a different profile compared to α-cell groups including native α-cells (‘α’, n = 6 mice), α-cells 1 month after DT-induced β-cell ablation (‘αDT’, n = 3 mice), α-cells overexpressing Pdx1 (‘αPdx1OE’, n = 3 mice), and α-cells overexpressing Pdx1 combined with β-cell ablation (‘αPdx1OE+DT’, n = 5 mice). (b) Heat map showing scaled expression (blue, high; white, low) of α- / β-cell-enriched genes differentially expressed in αDT, αPdx1OE, αPdx1OE+DT conditions compared to native α-cells. (c) Gene expression changes (log2 value) in αPdx1OE+DT (top), αPdx1OE (middle) and αDT (bottom) compared to native α-cells. DEGs indicated by red-colored background show induced β-cell signature upon the conditions, while DEGs indicated by green-colored background show refractory responses. * FDR < 0.05. n = 6 mice in α, n = 5 mice in αPdx1OE+DT, n = 3 mice in αPdx1OE, and n = 3 mice in αDT. (d) Transgenes required for constitutive labeling α-cells with the reporter Venus and experimental design for α-cell labeling and purification after β-cell loss. (e) RT-qPCR analyses for different components of the insulin/IGF1-R signaling pathway. Components of the pathway are downregulated in α-cells 5 days post-DT. Center indicates the mean. qPCR were performed in triplicates and data processing and statistical analyses were performed using the web-based software ‘RT2 Profiler PCR Array Data Analysis version 3.5’, from Qiagen. Quantification of the gene expression changes were performed using the 2−ΔΔCt method. P-values were calculated using a Student’s t-test (two-tail distribution and equal variances between the two samples) on the 2−ΔCt values. Genes with P<0.05 are shown. n = 3 mice. qPCR was performed once. (f) Experimental design for DT-mediated β-cell loss, islet isolation and qPCR. Igf1 mRNA is upregulated in islets after β-cell loss, likely as an attempt to sustain insulin signaling. Data shown as mean +/− sem; n = 3 mice with 3 technical replicates each. Center indicates the mean. Two-tailed unpaired t-test P = 0.0086. (g) Gene set enrichment analysis (GSEA) from RNA-Seq showing enrichment of the insulin signaling pathway components PKC (left) and PI3K (right) in α-cells after DT-mediated β-cell ablation (αDT) compared to native α-cells (α). P-values were calculated by an empirical phenotype-based permutation test. n = 6 mice in α, n = 3 mice in αDT. NES: normalized enrichment score. See Supplementary Table 2 as source data.

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