podDKO mice develop a renal phenotype by 24 weeks

A. and B. Body weight (A) and blood glucose (B) are not significantly different in podDKO mice at 24 weeks compared with littermate controls.

C. Urinary Albumin Creatinine ratio (uACR) is significantly increased in podDKO mice at 24 weeks. Unpaired t-test, **p<0.01, n=7-8 mice per group.

D. Images and quantification of PAS staining shows tubular protein casts (indicated by arrows) and glomerulosclerosis in podDKO mice. Scale bar=25 μm. Unpaired t-test *p<0.05.

E. Masson’s trichrome staining shows increased fibrosis (blue staining) in podDKO mice at 24 weeks.

Scale bar=25 μm

F. Transmission Electron Micrograph (TEM) images of glomerular filtration barrier (GFB) (scale bar upper panel=5 μm, lower panel=500 nm) show ultrastructural damage to the GFB in podDKO mice with significantly increased foot process width. Unpaired t test **p<0.01

G. Immunofluorescent staining and quantification of WT1 in podDKO and Cre negative control mice at 24 weeks of age shows significant reduction in % podocytes per glomerulus in podDKO mice. Nuclei counterstained with DAPI. Scale bar=100 μm. Unpaired t-test ****p<0.0001, ≥8 glomeruli analysed per mouse, 3 mice per group. Scale bar=100 μm.

Simultaneous knockout of podocyte IR and IGF1R in vitro is highly detrimental

A. Western blot shows >80% reduction of IR and IGF1R protein in ciDKO cells. t-test, ****p<0.00001, n=18.

B. Phosphorylation of AKT and p44/42MAPK in response to acute insulin stimulation at 10 nM and 100 nM for 10 minutes was significantly reduced in ciDKO podocytes. One way ANOVA, **p<0.01, *p<0.05, n=3.

C. Phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 and 100 ng/ml for 10 minutes was reduced in ciDKO podocytes. One way ANOVA, *p<0.05, n=3.

D. Fewer than 50% of ciDKO cells survive 7 days after gene excision. t-test, ****p<0.0001, n=3-4 independent experiments.

Proteomic analysis of ciDKO podocytes reveals downregulation of spliceosomal proteins

A. Schematic outlining workflow for proteomic analysis.

B. Heat map showing hierarchical clustering of ciDKO vs wild-type podocyte proteomes (decreased protein expression in green, increased expression in red) and the GO and KEGG terms enriched in four major clusters.

C. and D. KEGG enriched terms in STRING. Downregulated pathways (green) are associated with higher enrichment scores in comparison to upregulated pathways (red). Enrichment scores are computed by STRING using the Kolmogorov-Smirnoff test. KEGG term “Spliceosome” (highlighted border) is associated with a high enrichment score and the highest false discovery rate across all terms.

E. Western blots show significantly reduced levels of EIF4A, SF3B4 and PTBP2 in ciDKO podocytes compared with wild-type cells. Unpaired t-test, ***p<0.001, **p<0.01, *p<0.05, n=3 independent experiments.

Exposure of cultured podocytes, but not glomerular endothelial cells, to the spliceosome inhibitor pladienolide B results in dose dependent cell death

A. Annotated KEGG pathway showing a majority of spliceosome proteins are significantly downregulated (green) in ciDKO podocytes.

B-D. Pladienolide B exposure for 48 hours in HeLa cells (B), glomerular endothelial cells (GenC) (C) and podocytes (D). One way ANOVA, ****p<0.0001, **p<0.01, *p<0.05, n=3 independent experiments.

E. Bright field images of wild-type podocytes exposed to the indicated concentrations of pladienolide

B. Scale bar=100 μm

Loss of podocyte IR and IGF1R is associated with increases in intron retention and unproductive transcript expression

A. Schematic outlining long read RNA sequencing workflow

B. An overview of the alternative splicing events quantified by FLAIR. Boxes represent exons (blue=constitutive exons; yellow=alternative exons), lines represent introns.

C. Boxplot showing the fraction of transcripts with intron retention events in ciDKO and wild-type podocytes. t-test, **p<0.01, n=4 for each experimental condition.

D. Box plot of the fraction of productivity events in the ciDKO and wild-type transcriptomes shows a higher proportion of unproductive transcripts in ciDKO podocytes. NGO=no start codon; NST=start codon but no stop codon; PRO=productive transcripts; PTC=premature termination codon ie. unproductive transcripts. t-test, ***p<0.001, **p<0.01, *p< 0.05, n=4 for each experimental condition.

E. Proportional stacked bar graph of productivity events in the ciDKO and wild-type transcriptomes. NGO=no start codon; NST=start codon but no stop codon; PRO=productive transcripts; PTC=premature termination codon ie. unproductive transcripts.

Compound knockdown of the IR and IGF1R alters the podocyte transcriptome

A. UCSC genome browser tracks of transcripts annotated to Fn1. Red lines indicate the number of reads in each sample containing the EDA and EDB exons.

B. Boxplot showing differential expression of an Fn1 transcript containing EDA/EDB exons in ciDKO podocytes.

C. Exon/intron structure of Fn1 transcript differentially expressed in IGF1R DKO vs wild-type podocytes. Expression of an EDA and EDB exon-containing transcript (not normally expressed in mature podocytes and associated with fibrosis) is increased in IGF1R DKO podocytes.

D. qPCR using primers specific to the Fn1 EDB exon shows an increase in the expression of fibrosis associated EDB exon-containing transcripts in ciDKO podocytes. t-test, **p<0.001, n=4 independent experiments.

E. qPCR shows decreased expression of Hcfc1r1 mRNA in ciDKO podocytes. t-test, ****p<0.00001, n=4 independent experiments.