Normalization of LET-2/EMB-9 ratios and internal aggregates after emb-9 reduction or overexpression and in let-2 or emb-9 glycine substitution mutants. (A) Boxplots show the normalized emb-9 RNAi knockdown and control RNAi (L4440 empty vector) data in LET-2::mNG (C) and EMB-9::mNG (C) animals shown in Fig. 6 B. Colors of the data points indicate different trials. Normalization was performed by dividing the mean BM fluorescence intensity (LET-2::mNG [C] or EMB-9::mNG [C]) measurement from each emb-9 RNAi or control animal by the average of all mean BM fluorescence intensity measurements from control animals for the same trial (see Materials and methods, n > 8 animals for each of three trials, t tests with Welch’s correction, ****P <0.0001). (B) Boxplots show the normalized LET-2::mNG (C) and normalized EMB-9::mNG (C) data for control RNAi (L4440 empty vector) and emb-9 RNAi knockdown animals shown in Fig. 6 C. Colors of the data points indicate different trials. Normalization was performed by dividing the mean BM fluorescence intensity measurement of each LET-2::mNG (C) or EMB-9::mNG (C) animal by the average of all mean BM fluorescence intensity measurements of EMB-9::mNG (C) for the same trial (see Materials and methods, n > 8 animals for each of three trials). (C) Single z-slice images of LET-2::mNG (C) (left) and EMB-9::mNG (C) (right) in day 1 adult worms (72 h after hatch) with and without emb-9 RNAi knockdown. Yellow arrowheads indicate body wall muscle intracellular aggregates. Increased intracellular aggregation was seen in LET-2::mNG (C) emb-9 RNAi-treated worms, and decreased intracellular aggregation was seen in EMB-9::mNG (C) emb-9 RNAi-treated worms (n = 28/28 worms observed for each). The scale bar is 10 μm. (D) Boxplots show the normalized emb-9 overexpression and control (no overexpression) data in LET-2::mNG (C) and EMB-9::mNG (C) animals shown in Fig. 7 B. Colors of the data points indicate different trials. Normalization was performed by dividing the mean BM fluorescence intensity (LET-2::mNG [C] or EMB-9::mNG [C]) measurement of each overexpression or control animal by the average of all mean BM fluorescence intensity measurements of control animals for the same trial (see Materials and methods, n > 5 animals for each of three trials, unpaired t test [left] and a t test with Welch’s correction [right], ****P <0.0001, ns, not significant). (E) Boxplots show the normalized LET-2::mNG (C) and normalized EMB-9::mNG (C) data after emb-9 overexpression and in control (no overexpression) animals shown in Fig. 7 C. Colors of the data points indicate different trials. Normalization was performed by dividing the mean BM fluorescence intensity measurement of each LET-2::mNG (C) or EMB-9::mNG (C) animal by the average of all mean BM fluorescence intensity measurements of EMB-9::mNG (C) for the same trial (see Materials and methods, n > 5 animals for each of three trials). (F) Single z-slice images of LET-2::mNG (C) (left) with and without emb-9::mRuby2 overexpression and EMB-9::mNG (C) (right) with and without emb-9::mNG::mRuby overexpression in day 1 adult worms (72 h after hatch). Yellow arrowheads indicate body wall muscle intracellular aggregates. Increased intracellular aggregation was seen in EMB-9::mNG (C) worms with emb-9::mNG::mRuby overexpression in all body wall muscle segments (n = 11/11 animals observed). The scale bar is 10 μm. (G) Representative sum projections of confocal z-stacks of LET-2::mNG (C) and EMB-9::mNG (C) wild-type animals, LET-2::mNG G1287D and EMB-9::mNG (C) G1173D mutant animals, and animals with a wild-type fluorescently tagged protein paired with an untagged mutant protein: EMB-9 G1173D; LET-2::mNG (C) and EMB-9::mNG (C); LET-2 G1173D. The imaged L1 larvae were grown at 25°C (restrictive temperature for emb-9 and let-2 mutants). Increased intracellular aggregation was seen in animals carrying let-2 or emb-9 glycine mutant alleles (n = 20/20 animals per condition). The scale bar is 10 μm.