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. Author manuscript; available in PMC: 2024 Feb 25.
Published in final edited form as: Toxicol Appl Pharmacol. 2022 Apr 26;444:116032. doi: 10.1016/j.taap.2022.116032

Figure 4: Combined effects of retinoic acid pathway modulators on phenotypic profiles.

Figure 4:

U-2 OS cells were pre-treated with a single concentration of the RAR antagonists CD 2665 (10 μM), ER 50891 (1 μM), the RXR antagonist UVI 3003 (10 μM) or the RA synthesis inhibitor citral (100 μM) for 1 h, prior to treatment with the test chemicals listed in Table 2 (e.g. RAR/RXR agonists, RA metabolism inhibitor, reference chemicals, negative control) in concentration-response for an additional 24 h. Results are displayed as the average of four biological replicates, with the exception of treatments with ER 50891, which was interfering with cell segmentation in one biological replicate. (A) Phenotypic profiles for reference chemicals, and ATRA. The columns represent the 1300 features, arranged within the fluorescent channel, as indicated by the color key on top. Profiles are arranged in rows for individual treatments, with increasing chemical concentration from top to bottom within each horizontal section of the heatmap. The black arrows highlight two groups of features that are affected by ATRA treatment, but not affected when cells were pretreated with RAR antagonists. (B) Concentration-response curves for test chemicals pre-treated with the different modulators. Global Mahalanobis distance of each well was calculated relative to the mean of wells (n=24 per biological replicate) of the corresponding pre-treatment (in absence of the test chemical). In this graph, the Mahalanobis distances of the pre-treatment wells is subtracted, so that all curves start at 0. The stars indicate the phenotype altering concentration (PAC, i.e. the concentration at which the signal exceeded 1 * nMAD of the noise). (C) Overview of the PACs for the curves displayed in (B). The gray boxes indicate the tested concentration range. The error bar indicates the lower and upper bound (95% confidence interval) of the potency estimates. The pre-treatments were spread across two plates per biological replicate, hence there are two values for pre-treatment with DMSO. Sequential treatments that did not result in a PAC are displayed as open circles ½ an order of magnitude above the highest tested concentration. Abbreviations: AGP: actin, golgi, plasma membrane; ER: endoplasmic reticulum; Mito: mitochondria; PAC: phenotype altering concentration; Pos: position (features not associated with a particular channel).