Fig. 6: Inhibition of ASAH1 results in the activation of DUSP5 and inhibition of the MAPK pathway.

A Venn diagram showing overlap among the most differentially expressed genes (upregulated and downregulated) in MDA-MB-231 cells expressing two different ASAH1 shRNAs as compared with nonspecific (NS) shRNA. B Heatmaps showing the top upregulated and downregulated genes in MDA-MB-231 expressing NS or ASAH1 shRNA. C Heatmaps showing the expression of dual-specificity phosphatases (DUSPs) in MDA-MB-231 expressing NS or ASAH1 shRNA. D, E Quantitative reverse-transcriptase–polymerase chain reaction (qRT-PCR) was used to measure the mRNA levels of DUSPs identified by RNA sequencing in MDA-MB-231 expressing NS or ASAH1 shRNA in TNBC cells. Actin mRNA was used as the internal control. F Indicated proteins were measured in MDA-MB-231 expressing NS or ASAH1 shRNA via immunoblotting. ACTINB was used as a loading control. G Indicated proteins were measured in MDA-MB-468 expressing NS or ASAH1 shRNA via immunoblotting. ACTINB was used as the loading control. H Indicated proteins were measured in MDA-MB-231 after treatment with DMSO or different concentrations of carmofur for 72 h via immunoblotting. ACTINB was used as the loading control. I MDA-MB-231 cells expressing NS shRNA with or without DUSP5 cDNA were analyzed for the shown protein by immunoblotting. ACTINB was used as the loading control. J MDA-MB-231 cells expressing ASAH1 shRNA with or without DUSP5 shRNA were analyzed for the shown protein by immunoblotting. ACTINB was used as the loading control. K MDA-MB-231 cells expressing NS shRNA with or without DUSP5 cDNA and MDA-MB-231 cells expressing ASAH1 shRNA with or without DUSP5 shRNA were analyzed using soft-agar assay. Representative images are shown. Scale bar, 500 μm. L Colony sizes plotted for the experiment shown in (K). Data represent the mean ± standard error for three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant.