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. 2023 Jan 22;10(8):2205668. doi: 10.1002/advs.202205668

Figure 4.

Figure 4

CircWDR37 induces PKR homodimerization and phosphorylation. a) Mass spectrometry identified PKR, which was pulled downed from S18 cells lysates by biotin‐labeled in vitro transcripted circWDR37. b) Representative western blots analysis of PKR pulled down by circWDR37. Sense: biotin‐labeled in vitro transcribed circWDR37; Anti‐sense: the corresponding complementary biotinylated RNA. c) Binding of circWDR37 to PKR in HONE1, S18, and HK1 cells were detected by RIP. d) Co‐localization of circWDR37 (red) with PKR proteins (green), respectively, in S18 and HONE1 cells. Scale bar, 20 µm. e) Binding of circWDR37 to the dsRBMs of PKR in HEK293T cells were detected by RIP. Top: a schematic diagram of PKR and its recombinant protein truncation proteins. f) Western blots analysis of the indicated proteins in HONE1 and S18 cells treated with si‐PKR or siSCR. g) Western blots analysis of the indicated proteins in cytoplasmic (cyto) and nuclear (neu) fractions of HONE1 cells transfected with si‐PKR or siSCR. Lamin B1 and α‐tubulin were used as nuclear and cytoplasmic markers, respectively. h) Fluorescence microscopy analysis of HONE1 cells transfected with si‐PKR or siSCR, p65 nuclear fluorescence intensity was determined by ImageJ software. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. i) Western blot analysis of total PKR and phosphorylated PKR (p‐PKR‐T446, p‐PKR‐T451) in HONE1 and S18 cells treated with si‐circWDR37 or siSCR. j) Co‐IP showed the binding of PKR with PACT or TRBP proteins in HONE1 cells treated with si‐circWDR37or siSCR. k) Lysates from HONE1 cells cotransfected with PKR‐Flag and PKR‐HA along with si‐circWDR37 or siSCR were immunoprecipitated with anti‐Flag antibody, the precipitates and whole‐cell lysates were then analyzed by western blots with indicated antibodies. l) A schematic model for the function of circWDR37 in PKR activation. CircWDR37 physically interacts with PKR dsRBMs domain, resulting in homodimerization and autophosphorylation of PKR. Mean (n = 3) ± s.d. (Data were analyzed by (c, e) two‐tailed Student's t‐test, (h) one‐way ANOVA with Scheffe's post‐hoc test). p‐value < 0.05 indicates statistical significance. N.S. indicates no significance.