3.3. Multiplex hextuple luciferase assaying

(A) Simplified schematic of the constitutively expressing luciferase reporter plasmids for all six luciferases (ELuc, FLuc, RedF, NLuc, Renilla/RLuc, and GrRenilla/GRLuc), used to determine the transmission coefficients (see also Fig. 3): (B) Emission spectra of the three D-Luciferin-consuming luciferases used in the multiplex hextuple luciferase assay. Two bandpass emission filters, one measuring between 500 and 530 nm (BP515-30), and a second measuring between 510 and 550 nm (BP530-40), are used to capture select portions of the three emission spectra that will be used for spectral unmixing (see Fig. 11). (C) Emission spectra of the three CTZ-consuming luciferases used in the multiplex hextuple luciferase assay. Two additional bandpass emission filters, one measuring between 370 and 450 nm (BP410-80), and a second measuring between 520 and 620 nm (BP570-100), are used to capture select portions of the emission spectrum that will be used for spectral unmixing (see Fig. 11). (D) Simplified schematic of the experimental setup to determine transmission coefficients for all six luciferases using either total BL emission or bandpass filtered emission over the indicated filters. (E) Formulas to calculate the transmission coefficients (κ) of each D-Luciferin-consuming luciferase over the indicated bandpass emission filters. (F) Formulas to calculate the transmission coefficients (κ) of each CTZ-consuming luciferase over the indicated bandpass emission filters.

(A) Calculation of the transmission coefficients for the three D-Luciferin-consuming luciferases: κELuc₅₁₅, κFLuc₅₁₅, and κRedF₅₁₅ represent the transmission coefficients over the BP515-30 bandpass emission filter (left), while κELuc₅₃₀, κFLuc₅₃₀, and κRedF₅₃₀ represent the transmission coefficients over the BP530-40 bandpass emission filter (right). (B) Calculation of the transmission coefficients for the three CTZ-responsive luciferases, κNLuc₄₁₀, κRenilla/RLuc₄₁₀, and κGrRenilla/GRLuc₄₁₀ represent the transmission coefficients over the BP410-80 bandpass emission filter (left), while κNLuc₅₇₀, κRenilla/RLuc ₅₇₀, and κGrRenilla/GRLuc ₅₇₀ represent the transmission coefficients over the BP570-100 bandpass emission filter (right).

(A) Simultaneous equations being used to solve the D-Luciferin-consuming luciferase contributions in a mixture have three unknown variables corresponding to the amount of light that is specifically emitted by each D-Luciferin-consuming luciferase (ELuc, FLuc, and RedF). (B) Simultaneous equations being used to solve the CTZ-consuming luciferase contributions in a mixture have three unknown variables corresponding to the amount of light that is specifically emitted by each CTZ-consuming luciferase (NLuc, Renilla/RLuc, and GrRenilla/GRLuc). (C) To obtain calculated values for each D-Luciferin-consuming luciferase-linked reporter unit, a matrix inversion of the coefficient matrix (the matrix containing values for all transmission coefficients) obtained using the appropriate bandpass emission filters (see Fig. 10), is multiplied by the value matrix (the matrix containing BL measurements obtained by the microplate reader). (D) Similarly, to obtain calculated values for each CTZ-consuming luciferase-linked reporter unit, a matrix inversion of the coefficient matrix (the matrix containing values for all transmission coefficients) obtained using the appropriate bandpass emission filters (see Fig. 10), is multiplied by the value matrix (the matrix containing BL measurements obtained by the microplate reader).

(A) Simplified schematic of five cellular signaling pathways acting upstream of five transcription factor-binding motifs, each monitored using an orthogonal luciferase: RedF, FLuc, Renilla/RLuc, NLuc and GrRenilla/GRLuc. All perturbations are monitored against a control pathway using the enhanced beetle luciferase reporter (ELuc). (B) The final multi-luciferase plasmid includes five insulated pathway-responsive luciferase transcriptional units and one constitutively expressed luciferase transcriptional unit used as the control for normalization. (C-G) On-target downregulation of a signaling pathway after adding siRNA(s) against (a) key transcription factor(s) binding a transcription factor-binding motif for the NF-κβ (C), TGF-β (D) c-Myc (E), p53 (F), or MAPK/JNK pathway (G). Off-targeting effects are indicated as well. Statistical significance of the fold-change of different genes analyzed by pathways in the multiplex luciferase assay was determined by multiple t-tests using the Holm-Sidak method with alpha = 0.05 (*P <0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, n.s. is non-significant). n=4 for all multiplex hextuple luciferase assays.