Fig. 3: Gene expression analysis of TSC2^-/-, TSC2^+/−, and TSC2^+/+ renal organoids.

a Volcano plots showing the distribution of all differentially expressed genes (DEGs) in TSC2^−/− renal organoids compared to TSC2^+/+ (left) and TSC2^+/− (right) renal organoids (FDR < 0.05). Each dot represents a unique gene; red denotes log₂ (fold change) >2, upregulated genes in TSC2^−/−; blue denotes log₂ (fold change) <-2, downregulated in TSC2^−/−. Selected statistically significant upregulated and downregulated genes (NCBI/Entrez names) are indicated, as determined by a two-sided Chi-Square test. b Principal Component Analysis (PCA) of RNA-Seq data from renal organoids of the three genotypes, n = 3 samples for each genotype, five organoids per sample. c Heatmap showing hierarchical clustering of three different genotypes of kidney organoids using the top 3000 most variable genes. Color scale representative of gene expression level: red denotes log₂ ≤ 3, blue denotes log₂ ≥ -3. d Representative enrichment plots corresponding to gene set enrichment analysis (GSEA) for pairwise comparison of TSC2 ^−/− vs. TSC2^+/−. e Venn diagrams indicating 187 common differentially expressed genes, including signature AML markers, in TSC2^−/− vs. TSC2^+/+ renal organoids and kidney AML vs. normal kidney. f Comparative mRNA expression levels for AML hallmark genes in TSC2^−/−, TSC2^+/+, and TSC2^+/− renal organoids (n = 3 each) compared to human kidney AML (n = 28) and human kidney (n = 8). P values for individual comparisons done using a two-sided Mann–Whitney U test are indicated. Gene expression is shown in FPKM values. g Comparative ENO2 mRNA expression levels in TSC2^+/+ and TSC2^+/−, TSC2^−/− renal organoids (n = 3 each). P values for the indicated individual comparisons done using two-tailed Student’s t test are shown. Gene expression is shown in FPKM values. h, i Box-and-whisker plot showing minimum value, first quartile, median, third quartile and maximum value for ENO2 content (g) and for enolase activity (h) in whole extracts of TSC2^+/+ and TSC2^−/− renal organoids. P value for the 2-tailed Student’s t test comparing TSC2^−/− versus TSC2^+/+ is shown. n = 4 independent experiments, containing three organoids each.