Fig 1.
General scheme of the secreted dual reporter assay.
The strategy applies to any combination of Gluc reporter and mCherry internal standard. The structures of the specific reporter plasmids used here are indicated with their names in rectangles on the left. The sizes of the different elements are not to scale. SS, signal sequence; TK, thymidine kinase; 5xGal, 5 copies of Gal4 binding site.
Fig 2.
Characteristics of the secreted reporter proteins in tissue culture medium.
(A) Freezing tests. Supernatants of HeLa cells cotransfected with XETG and mCherry were placed on ice and then aliquots were flash-frozen or not in liquid nitrogen before transferring to -80°C; Gluc and mCherry activities of all aliquots were measured in parallel 45 minutes later. Individual bars represent the average of triplicate samples and error bars show the standard deviation. Apparent differences are not statistically significant (p > 0.2). (B) Stability tests of Gluc and mCherry in medium. Supernatants of HCT116 cells cotransfected with XETG and mCherry were collected 40 hours after transfection with PEI and either kept frozen at -80°C or in a tissue culture incubator at 37°C in a fresh plate without cells for another 24 hours. Individual bars represent the average of triplicate samples and error bars show the standard deviation. Activities of the frozen aliquots were set to 100%. Apparent differences are not statistically significant. (C) Activities in serial dilutions. The double-logarithmic graphs show the results of triplicate samples (red dots) with error bars indicating the standard deviation in comparison to the theoretical values of a linear stepwise dilution (green lines); the respective average of the undiluted samples were set to 100%. For mCherry (bottom panel), the samples were the same as for the freezing tests, and they were diluted with Opti-MEM in steps of 5; for Gluc (top panel), supernatants were from HeLa cells cotransfected with XGalG and Gal4.VP16, and they were complemented with 10% fetal calf serum (FCS) before dilution in steps of 5 with Opti-MEM complemented with 10% FCS. Apparent deviations from the predicted values are not statistically significant (p > 0.2).
Fig 3.
(A) Time-course experiment with transfected variant HEK 293T cells. They were cotransfected with equal amounts of the plasmids XETG and mCherry with the calcium-phosphate coprecipitation technique. Individual data points represent the average of triplicate samples (except for the 48 hour time point, which is the average of duplicate samples) and error bars show the standard deviation. All values were standardized to those of the 66 hour time point (arbitrarily set to 1.0). (B) Time-course experiment with transfected HCT116 cells. Exactly the same as in panel A except that cells were transfected with PEI and that all samples are in triplicates.
Fig 4.
Transcriptional activation assays.
The secreted dual reporter assay was used for three reporter systems tested in HeLa cells. Gluc luciferase activities were standardized to mCherry and calculated as % of the maximal activation of each reporter/effector pair set to 100%. Bars represent averages of triplicate samples and error bars the standard deviation. (A) Transcriptional activation of the Gal4 reporter plasmid XGalG by Gal4.VP16. Gal4, the DNA binding domain of Gal4; n.t., medium from non-transfected cells. (B) Transcriptional activation of the GR reporter plasmid XGTG by GR in the presence of 100 nM Dex. "no GR", cells cotransfected with empty expression vector. (C) Transcriptional activation of the ERα reporter plasmid XETG by ERα in the presence of 100 nM E2.
Table 1.
Comparison of secreted and intracellular dual reporter assays.