Supplementary Figure 2: Activation of PERK and IRE-1 pathways in ZIKV infected mouse brains from WT or Ifnr-knockout mice induce ER stress and microcephaly.
From: Stress-induced unfolded protein response contributes to Zika virus–associated microcephaly
a, Intracerebroventricular (ICV) co-injection of ZIKV and PERKi at E12.5 rescues microcephaly of newborn pups. Data are presented as histograms and error bars of mean ± SEM respectively and analyzed by one-way ANOVA followed by Bonferonni post hoc comparison test to compare brain weight (F2,43=59.06, P < 0.0001), normalized cortical length (F2,43=65.85, P < 0.0001), and cortical width (F2,43=74.12, P < 0.0001) of pups after Mock, ZIKV or ZIKV + PERKi as indicated on the x-axes (14, 14, 16 brains from separate litters respectively) a. b-d, Histograms and related immunolabelings showing that ICV ZIKV injection into E12.5 brains increases number of Pax6+ (⋆ P < 0.05, U = 0) but not Tbr2+ (P = 0.150, U = 1.5) cells in E18.5 cortices (n = 7 to 9 animals); the averaged Pax6-intensity remains unchanged (P = 0.9172, U = 20) between conditions (n = 96 to 106 cells from 3 independent embryonic brains) b-c. Immunolabelings showing Dapi (blue), ZIKV (red) Pax6 (green), and Tbr2 (grey) (n = 7 to 9 independent embryonic brains) d. Data are presented as boxplots of median ± first to third quartiles while whiskers extend to maxima and minima with each symbol representing independent embryonic brains (n = 3) as indicated and analyzed by two-sided Mann-Whitney test (b-d). e-f, Western blot analyses of proteins extracted from E15.5 mouse brains ICV injected with Mock or ZIKV at E12.5. Representative immunoblots showing increased phosphorylation of PERK (⋆ P < 0.05, U = 0) and IRE-1in ZIKV-infected brains and quantification of phosphorylated/unphosphorylated protein ratio after normalization of individual samples (n = 2 to 3 independent embryonic brains) with respect to corresponding conditions e. Immunoblot and corresponding scatter-plots showing increased phosphorylation of eIF2a protein (n = 2 to 3 independent embryonic brains, ⋆ P < 0.05, U = 0) f. Data are presented as scatter-plots of means ± SEM with each symbol representing biologically independent samples as indicated on and analyzed by two-sided Mann-Whitney test e-f. g, Corresponding histograms representing fold change of UPR factor transcripts in E14.5 brains from an Ifnr−/− dam intraperitoneally injected with ZIKV at gestational day 9.5. Microdissected cortices were analyzed by qRT-PCR to detect PDI, Atf4, Atf5, Chac1, and Chop; one representative experiment for each condition. Scale bar represents 100 µm d. ⋆⋆⋆ P < 0.001, and ⋆ P < 0.05.
