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. 2024 Aug 20;15:7132. doi: 10.1038/s41467-024-51488-2

Fig. 4. The Mdm2/MdmX complex regulates the expression and subcellular localization of Sprouty4.

Fig. 4

HT1080 p53KO cells were transfected with siRNAs against Mdm2 or siCtrl for 24 h; or treated with 7 µM MEL23 or DMSO (vehicle) for 24 h. A Volcano plots show all proteins identified by mass spectrometry. Colored dots represent significantly differentially expressed proteins that were downregulated (blue dots) and upregulated (red dots) in each condition shown at the left of the plot. Gray dots represent non-significant changes. MEL23-treated cells were compared to DMSO-treated cells. Cells transfected with siMdm2#1 or #2 were compared to siCtrl-transfected cells, n = 3 samples. Black arrows point to the location of Spry4 in each Volcano plot. B Spry4 expression in HT1080 p53KO cells in response to Mdm2 knockdown or treatment with MEL23 for 24 h by immunoblotting. β-actin and α-tubulin were used as loading control for immunoblot, n = 6 samples. C Co-immunoprecipitation of Mdm2 and Spry4 in the presence or absence of MG132. β-actin was used as a loading control. Mdm2 was pulled down by using either a mix of antibodies against Mdm2 (4B11, 3G5, and 2A9) that recognize different domains within the protein, this condition was called “mix”, or by using a single monoclonal antibody D1V2Z, this condition was called “DIV”, n = 3 samples. D Quantification of Spry4, Mdm2, and MdmX protein levels after treatment with MG132 (or vehicle, DMSO) for 4 h, n = 3 samples. E Spry4 mRNA levels in response to Mdm2 knockdown (n = 5 samples) or treatment with MEL23 for 24 h (n = 4 samples). RPL32 was used as housekeeping. F Localization of Spry4 in HT1080 p53KO cells in response to Mdm2 knockdown. Immunofluorescence showed Spry4 staining (green), the cell surface was outlined by phalloidin staining (orange), and nuclei (blue) were detected by DAPI staining of DNA, n = 3 groups. Graphs shown in (D, E) represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.