CemOrange2 and three-color imaging.

Pnhx-2lmp-1::CemOrange2;Pnhx-2GFP::ATZ;Pnhx-2mKate2::lgg-1 transgenic C. elegans expressing sGFP::ATZ (A and B; green; Ex488 nm/Em500-540 nm), LMP-1::CemOrange2 (C and D; pseudocolored blue; Ex545 nm/ Em560-590 nm) and mKate2::LGG-1 (E and F; red; Ex594 nm/Em605-645 nm) were examined by confocal microscopy over >20 z-planes using a 40x PlanApo oil immersion objective (N.A. 1.3). Maximum intensity projections are shown (A, C, E, G). Scale bars = 25 μm. Magnified single XY regions (dashed box) are included to highlight colocalization events (B, D, F, H). Scale bars = 5 μm. mKate2::LGG-1 and LMP-1::CemOrange2 had multiple colocalization events (G and H; magenta merge, white arrowheads). Alpha-1 antitrypsin Z mutation (ATZ), a polymerizing protein which accumulates in the ER and is degraded, in part, by autophagy, show colocalization with mKate2::LGG-1 (G and H; orange merge, yellow arrowheads). Pnhx-2glo-1::CemOrange2;Pvha-6lmp-1::GFP transgenic C. elegans expressing LMP-1::GFP (I and J, green; Ex488 nm/Em500-540 nm) and GLO-1::CemOrange2 (M and N, red; Ex555 nm/Em560-590 nm) were stained with the acidic organelle fluorescent dye, LTDR (K and L, blue; Ex647 nm/Em660-700 nm). Animals were imaged by confocal microscopy over >20 z-planes and maximum intensity projections are shown (I, K, M, O). Scale bars = 25 μm. Magnified single XY regions (dashed box) are included to highlight colocalization events (J, L, N, P). Scale bars = 5 μm. Colocalization events are seen between LMP-1::GFP and LTDR (O and P, cyan merge, arrowheads) but not with GLO-1::CemOrange2.