Fig. 3.

ER stress-rescuing effects of MANF and sulfatide in C. elegans. a Schematic of experimental workflow to test rescuing effects of human MANF from conditioned cell culture PBS. b Exemplar GFP fluorescence images showing the rescue of the hsp-4p::GFP phenotype in the manf-1 null tm3603 mutant by incubation with purified MANF secreted from conditioned media. MANF alone rescued only mildly compared with fuller rescue by MANF plus exogenous sulfatide addition (500 µM in DMSO; sulfatide alone had no rescue), with indicated penetrance of rescue (N ≥ 100 for each genotype). c Exemplar western blot of Hs-MANF in C. elegans manf-1 mutants after incubating with Hs-MANF, showing sulfatide dependency of endocytosis and rescue of hsp-4::GFP levels by endocytosed Hs-MANF. d Normalized levels of Hs-MANF::V5 immunoblot signals (from three biological replicates of N ≥ 100 animals) showing block of Hs-MANF endocytosis in temperature-sensitive endocytosis mutant dyn-1(ts) cultured from 25 °C but not at the permissive temperature, 20 °C. e Exemplar immunofluorescence images of C. elegans tissues stained with anti-His (green) and DAPI (blue) indicating the uptaken His::MANF with sulfatide. Scale bar, 15 µm