Figure S1.

Multiple Dual Fluorescent Reporters for Determining Gene Effects on Cell Shape, Migration, and Proliferation, Related to Figure 1

(A) Several different constructs (1-6) were assembled and tested for the simultaneous expression of Mb- and H2B-tagged marker proteins and a given gene of interest.

(B) Immunostaining of stable cell lines expressing constructs 1-3 with three different antibodies (anti-GFP, anti-DsRed, and anti-Kate2).

(C) Live imaging (see also Movie S1) with 3 laser lines (458, 488, and 546nm) and 5 different detector settings to capture the chromatin and membrane of all cells containing constructs 4-6 (see, a), allowing simultaneous visualization of the effect of different genes on cell division and migration in the same acquisition field. Legend: Mb - Tag that localizes proteins to the cell membrane. 2A – 21-aminoacid viral peptide sequence cleaved at the C-terminal position, allowing equimolar and independent localization of two flanking proteins coded in the same ORF. Au1, V5, HA, His - short epitope tags that can be detected by specific antibodies. H2B - Histone 2B protein tag that localizes proteins to the chromatin/cell nucleus. pA - polyadenylation transcription stop signal.

(D) Brainbow 2.1 expression in endothelial cells of the mouse retina. This mouse line allows the induction after Cre recombination of up to 4 FPs (nGFP, YFP, MbCFP, and RFP) that are excited and emit signals at different wavelengths. Confocal scanning of the endogenous protein fluorescence signals after tissue fixation can only provide high resolution of the strongest signals. Immunostaining with antibodies to GFP greatly improves signal intensity, but detects nGFP, YFP, and MbCFP simultaneously, compromising their proper distinction in the same tissue.