Fig 2: Kidney-specific PLAG1 overexpression drives neoplasia. (A) Gross dissection of a Wt1Cre;LSL-PLAG1 mouse and a wild-type littermate at 24 d of age. (B) Survival curve of Wt1Cre;LSL-PLAG1 mice versus Wt1Cre-only and LSL-PLAG1-only littermates. P < 0.05, log-rank test. (C) Relative expression of Igf2 taken from whole kidneys in Wt1Cre;LSL-PLAG1 mice versus LSL-PLAG1-only at postnatal day 8 (P8). Values are presented as mean ± SD from four technical replicates. (*) P < 0.05 by two-sided t-test comparing mean expression levels in each group of kidneys. (D) Kidneys from Wt1Cre;LSL-PLAG1, wild-type littermate, and Six2Cre;LSL-PLAG1 mice at P8. Bar, 500 µm. (E) High-power view of a Wt1Cre;LSL-PLAG1 kidney at P8. (SC) Simple cyst; (AC) atypical cyst; (D) dysplastic nodule; (g) glomerulus.

Fig 3: PLAG1 expression enhances proliferation. (A) Immunoblot of WiT49 with lentivirally overexpressed PLAG1 and the cell density of WiT49 with or without ectopic PLAG1 expression, measured by crystal violet staining. Values are presented as mean ± SD from four technical replicates, (*) P < 0.05. (B) Differentially expressed genes measured by whole-transcriptome sequencing of WiT49 cells with or without ectopic PLAG1 expression in two replicates each. FPKM in parental cells is shown on the X-axis. Comparison of values in PLAG1-overexpressing cells versus parental cells is shown as -log10 of the P-value along the Y-axis. Predicted PLAG1 target genes are highlighted in red. (C) qPCR for IGF2 in PLAG1-overexpressing WiT49. Values are presented as mean ± SD from three technical replicates. (*) P < 0.05. (D) PLAG1 consensus binding sites (GRGGCN6-8GGG) aligned with IGF2 transcripts, marked by the transcription start site (P0–P4). (E) ChIP-qPCR (chromatin immunoprecipitation [ChIP] combined with qPCR) using Flag antibody in WiT49 cells transfected with empty vector versus PLAG1-3xFlag to detect binding to the human IGF2 P3 promoter region. Values are presented as mean ± SD from four technical replicates. (*) P < 0.05. (F) ChIP-qPCR using PLAG1 antibody in whole mouse kidneys at P4 to detect binding to the mouse Igf2 P2 promoter region (which is homologous to the human P3 promoter). Values are presented as mean ± SD from three technical replicates. (*) P < 0.05. (G) IGF2 transcript levels in WiT49 with or without PLAG1 overexpression, as measured by RNA-seq. Values are presented as mean ± SEM, taken from two replicates. (*) P < 0.05. (H) IGF2 transcript levels in Wilms tumors and normal kidneys, segregated by the transcription start site (P0–P4), as measured by RNA-seq.

Fig 4: Human Wilms tumors overexpress PLAG1 through multiple mechanisms, resulting in poorer survival. (A) Genomic aberrations in TARGET Wilms tumors, correlated with expression of PLAG1 by microarray. Copy number gains are shown in red, while copy number losses are shown in blue. (B) PLAG1 expression in tumors with gain of 8q12, where PLAG1 is encoded; loss of 1p, where MIR34A is encoded; or mutations in the miRNA processing pathway by microarray. (*) P < 0.05, unpaired t-test versus “none.” (C) Event-free survival in relapsed favorable histology Wilms tumor patients based on PLAG1 expression (“high” is defined as PLAG1 expression intensity >6). (**) P = 0.01, log-rank test.

Fig 5: Wilms tumors with miRNA processing mutations express high levels of PLAG1. (A) Heat map of genes differentially expressed between Wilms tumors with and without mutations in the miRNA processing pathway (“wild-type” and “mutant,” respectively) in TARGET microarray data (false discovery rate <0.1). The arrowhead marks PLAG1. (B) Log2 fold change of expression for 31 genes overexpressed in the microarray in mutant tumors versus wild-type tumors, as measured by whole-transcriptome sequencing. (*) P < 0.05. (C) PLAG1 levels in individual samples, as measured by whole-transcriptome sequencing (FPKM [fragments per kilobase per million mapped reads]). Horizontal bars mark the geometric mean. (D) PLAG1 levels in individual samples, as measured by quantitative RT–PCR (qRT–PCR). Values shown are mean ± SD from three technical replicates.