Figure 3. KDEL Receptors Regulate the Secretion of GLuc-ASARTDL.

(A) A human RNAi library was screened for effects of gene knockdown on Tg-induced GLuc-ASARTDL secretion. The plot shows the effect of each gene on cell-viability versus Tg-induced GLuc-ASARTDL secretion compared to control siRNA for each gene in the library. Positive hits are colored for the GLuc signal effect (maximal GLuc is green) and sized by seed-corrected Z score (maximum Z score is the largest sphere).

(B) KDEL receptor mRNA levels estimated with real-time RT-qPCR in SH-SY5Y cells transfected with siRNAs specific for KDELR1, KDELR2, or KDELR3 (n = 3; *p < 0.05, ****p < 0.0001 versus control siRNA; two-way ANOVA and Tukey’s test).

(C) GLuc activity in media under normal conditions (pre-Tg), and after an 8 hr incubation in 100 nM Tg (post-Tg) following RNA interference of the KDEL receptors in SH-SY5Y cells stably expressing GLuc-ASARTDL (n = 9; ****p < 0.0001 versus control siRNA; two-way ANOVA and Tukey’s multiple comparison test).

(D–F) Correlation of extracellular GLuc activity with mRNA expression of (D) KDELR1, (E) KDELR2, and (F) KDELR3 following transfection with 5–20 nM siRNAs.

(G) SH-SY5Y cells transiently transfected with GLuc-ASARTDL and transduced with lentiviral vectors expressing KDEL receptors at different MOIs were exposed to 200 nM Tg or vehicle for 24 hr. Fold change in GLuc secretion is indicated (n = 6; one-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001 KDEL receptor versus control).

(H) GLuc activity in the media from SH-SY5Y cells stably expressing GLuc-Untagged after transfection with KDEL receptor siRNAs (n = 6).

(I) SH-SY5Y cells overexpressing KDEL receptors at different MOI and transiently transfected with GLuc-Untagged were exposed to 200 nM Tg or vehicle for 24 hr. Fold change in GLuc secretion is indicated (n = 6).

See also Figures S4 and S5.