Journal: Communications Biology
Article Title: APOL1 risk allele RNA contributes to renal toxicity by activating protein kinase R
Figure Lengend Snippet: APOL1 risk variants activated PKR and downstream signals. a HEK293FT cells over-expressing APOL1 were harvested for detection of APOL1 and eIF2α protein by Western blotting. Renal risk variants G1 and G2 increased phospho-eIF2α compared to the G0 allele. b HEK293FT cells over-expressing APOL1 were harvested to identify which of the four eIF2 kinases (HRI, PKR, PERK, GCN2, respectively) were activated. The G0 variant showed minimally increased phospho-PKR, indicating activation, while renal risk variants G1 and G2 manifested substantially increased phospho-PKR. c HEK293FT cells over-expressing APOL1 were treated with a PKR inhibitor or vehicle control, then harvested for detection of PKR and eIF2α. The APOL1 renal risk variants increased phospho-PKR and phospho-eIF2α compared to the G0 allele, effects that were abolished or greatly diminished by PKR inhibition. d Transfected HEK293FT cells expressing APOL1 RNA encoded by the G1 and G2 variants increased interferon α and interferon β RNA expression compared to the G0 variant, as measured by qRT-PCR. e Total protein synthesis was analyzed in transfected HEK293FT cells expressing APOL1 RNA with the G0, G1, and G2 variants. Translation initiation was measured using the L-azidohomoalanine protein synthesis assay. Quantification of protein synthesis, with the APOL1 risk variants measured against the G0 transfected cells (set to 100%) and puromycin exposure (set to 0%). Both risk variants G1 and G2 reduced protein synthesis by HEK293FT cells. f Quantification of cell viability of stable HEK293 cell line expressing APOL1. The cells were lysed and incubated with CellTiter-Glo to measure the amount of ATP in the cells. HEK293 cells with the G1 and G2 variants manifested decreased cell viability compared with the G0 variants. Each dot is calculated from one well. Each horizontal line represents mean. All results are presented as ratio of controls, normalized to 100% and P values were calculated using a Student one-tailed t -test. Welch correction was used for panel ( d )
Article Snippet: Samples were analyzed by PCR (RT) or quantitative RT-PCR (qRT-PCR) using Power SYBR Green PCR master mix (ThermoFisher).
Techniques: Expressing, Western Blot, Variant Assay, Activation Assay, Inhibition, Transfection, RNA Expression, Quantitative RT-PCR, Incubation, One-tailed Test