Table 5. Data and statistical analysis from cytoplasmic sensor replicates.
Top. Data representing biological replicates using transiently transfected cytoplasmic sensor and cytoplasmic cpVenus in HeLa cells. Data are arranged for input into STATA14 statistical software. “0” represents cells treated with 0 nM FK866. “1” represents cells treated with 10 nM FK866. The fl values represent the geometric mean from 488/405 nm fluorescence ratio of the healthy, single HeLa cells measured by cytometry. “experiment”, biological replicate; “fl”, 488/405 nm geometric mean fluorescent value; “treatment”, +/− 10 nM FK866; “sensor”, +/− cytoplasmic NAD+ sensor.
Bottom. Calculated statistical interaction table from STATA14 using REML. The p-value for the ratio of ratios across replicates (bold red) is reported under the statistical interaction “_IsenXtre_1_1”. This represents the fluorescence change following FK866 treatment in the cytoplasmic sensor, relative to changes in cpVenus. Calculating the exponential of the coefficient value, “Coef.”, represents the mean fold-change in fluorescence for the ratio of ratios, across replicates, i.e. e^(0.40) = 1.49, 95% CI (1.38 to 1.60), p<0.001.
The fluorescence change in cytoplasmic cpVenus with treatment across replicates is represented by the “_Itreatment_1” row: e^(coef) = e^(0.00) = 1.00, p=0.982. To calculate the fluorescent change in cytoplasmic sensor independent of cpVenus, combine the “treatment” and “sensor X treatment interaction” using the lincom command (step 50). The combined values (blue) represents the change in the sensor: e^(coef) = e^(0.40) = 1.49, 95% CI (1.42 to 1.57), p<0.001.
| experiment | fl | treatment | sensor |
|---|---|---|---|
| 1 | 9.65 | 0 | 0 |
| 1 | 9.56 | 1 | 0 |
| 1 | 3.06 | 0 | 1 |
| 1 | 4.35 | 1 | 1 |
| 2 | 7.79 | 0 | 0 |
| 2 | 7.46 | 1 | 0 |
| 2 | 2.51 | 0 | 1 |
| 2 | 3.78 | 1 | 1 |
| 3 | 7.33 | 0 | 0 |
| 3 | 7.74 | 1 | 0 |
| 3 | 2.35 | 0 | 1 |
| 3 | 3.62 | 1 | 1 |
| log_fl | Coef. | Std. Err. | z | P> |z| | [95% Conf. | Interval] |
|---|---|---|---|---|---|---|
| _Isensor_1 | −1.14 | 0.03 | −43.51 | 0 | −1.19 | −1.09 |
| _Itreatment_1 | 0.00 | 0.03 | 0.02 | 0.982 | −0.05 | 0.05 |
| _IsenXtre_1_1 | 0.40 | 0.04 | 10.72 | 0 | 0.32 | 0.47 |
| _cons | 2.10 | 0.07 | 28.18 | 0 | 1.96 | 2.25 |
| _IsenXtre_1_1 + _Itreatment_1 | .40 | .0261934 | 15.19 | 0.000 | .35 | .45 |