Fig. 1

Validation of assays using molecular compounds. A Representative IFC images of the mitochondrial staining (NAO and TMRM). The top two images show NAO/TMRM staining before and after FCCP treatment. The boxplot shows the intensity in pixels of the TMRM staining within the Object mask, which was used to quantify membrane potential. The bottom two images show the NAO/TMRM staining before and after ethidium bromide treatment. The boxplot shows the intensity in pixels of the NAO staining for both conditions, which was used to quantify mitochondrial mass. B Representative IFC images of the autophagy staining (LC3). When autophagy is induced with bafilomycin, the autophagosomes become visible. The boxplot shows the number of autophagosomes per cell (Spot Count) using the Spot mask (Bright, 10, 0, 2). C Representative IFC images of a cell with a healthy and intact Golgi and a cell with fragmented Golgi after the addition of nocodazole. The boxplot shows the area of the Golgi staining after applying a 70% intensity threshold. During nocodazole treatment, the surface area of the Golgi system becomes larger, indicating that the Golgi system is fragmented. To quantify fragmentation, both surface area as well as minor axis intensity were used (Additional FIle 1: Figure S3). D Representative IFC images of the ATF6 staining. When ER stress is triggered, ATF6 translocates to the nucleus. The boxplot shows the similarity score between the nuclear staining and the ATF6 staining, which was used as a measure to quantify ER stress. E Representative IFC images of the NF-κβ staining (p65). Upon NF-κβ activation, p65 translocates to the nucleus. The boxplot shows the similarity between the nuclear staining and the p65 staining, which was used as a measure to quantify NF-κβ translocation. All boxplots represent one single experiment, and one treated and one untreated condition. The length and concentration of drug treatment are described in the “ Methods” section. The boxplot upper and lower hinge reflect the 25 and 75% percentiles and the black line reflects the median. The upper and lower whisker extend to 1.5 * IQR. Data beyond the end of the whiskers are plotted as large black circles. All statistics were calculated using nonlinear mixed effect models. p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001****. F Boxplots showing the natural variation of assays in healthy controls and the percentage values of the healthy controls treated with the molecular compounds from Fig. 1A–E. For the healthy control range, the median value of the designated feature for each assay was compared to the mean all other healthy controls taken along in the same run and converted to percentages. For the compounds, the median value of the feature for the compound-treated healthy control was divided by the median value for the untreated control and converted to percentages. The compound treated-control values are indicated as colored dots. The boxplot upper and lower hinge reflect the 25 and 75% percentiles and the black line reflects the median. The upper and lower whisker extend to 1.5 * IQR