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. 2018 Nov 7;1:188. doi: 10.1038/s42003-018-0188-2

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© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2018

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Fig. 4 — APOL1 risk variant activated PKR in human kidney tissue podocytes, in cultured human podocytes, and human APOL1 gene locus transgenic mice (BAC-APOL1 mice). a Kidney tissue from subjects with glomerular disease tissue was assessed for phospho-PKR; signal intensity was calculated, with average intensity in non-risk genotype cases set to 100%. Each dot represents the average signal intensity from one case. b Quantification of weighted colocalization coefficient between phospho-PKR and WT-1 revealed an elevated phospho-PKR in podocytes of risk genotype individuals. c Conditionally immortalized human podocytes, one cell line from each of three APOL1 G0/G0 FSGS patients and two cell lines from each of two APOL1 G1/G2 FSGS subjects, were transfected with APOL1 siRNA or control siRNA. The ratio of phospho-PKR signal to total-PKR was measured from Western blot. Knock down efficiencies were 52.4–75.4% (RNA levels) or 31.1% (protein levels). G1/G2 podocytes manifested increased PKR phosphorylation, which was diminished by APOL1 RNA knock-down (n = 3). Raw values are in Supplementary Table 2. d Quantification of protein synthesis in conditionally immortalized human podocytes after 96 h transfection with APOL1 siRNA or control siRNA. Protein synthesis was reduced in G1/G2 cases compared to G0/G0 cases (n = 6). Raw values are in Supplementary Table 3. e Glomeruli were isolated from BAC-APOL1 transgenic mice using magnetic particles (upper panel: arrows indicated glomeruli, small dots are magnet beads). Bar represents 50 μm. Glomeruli were treated with phosphatase inhibitor with/without PKR inhibitor for 30 min and lysed for Western blot analysis. Phospho-PKR was increased in G1 and G2 glomeruli. f Immunofluorescence staining was visualized using confocal microscopy. Phospho-PKR was visualized as turquoise blue, APOL1 as magenta, and nucleus was as green. The increased number of yellow cells in the BAC-APOL1-G1 and G2 mouse overlay image suggests PKR activation (phosphorylation) in podocytes. Bar represents 20 μm. Quantification of weighted colocalization coefficient between phospho-PKR and APOL1 revealed an elevated phospho-PKR in podocytes of the BAC-APOL1-G1 and G2 mice. Each dot represents a glomerulus. Each horizontal line represents the mean. P values were calculated using a Student one-tailed t-test