Fig. 2: ASAH1 knockdown inhibits TNBC tumor growth and metastasis. | Cell Death & Disease

Fig. 2: ASAH1 knockdown inhibits TNBC tumor growth and metastasis.

From: ASAH1 facilitates TNBC by DUSP5 suppression-driven activation of MAP kinase pathway and represents a therapeutic vulnerability

Fig. 2

A TNBC MDA-MB-231 and MDA-MB-468 cells expressing either nonspecific (NS) small hairpin RNA (shRNA) or ASAH1 shRNAs were analyzed by performing a quantitative reverse-transcriptase–polymerase chain reaction (RT-qPCR), and immunoblotting to analyze ASAH1 mRNA and protein expressions. The ASAH1 mRNA expression level in ASAH1 shRNA-expressing cells relative to NS shRNA-expressing cells is presented (left). The ASAH1 protein expression level in ASAH1 shRNA-expressing cells relative to NS shRNA-expressing cells is presented (right). ACTINB was used as the loading control. B The ceramide level was measured in the indicated TNBC MDA-MB-231 and MDA-MB-468 cells expressing either NS small hairpin RNA (shRNA) or ASAH1 shRNAs using the human ceramide antibody (ceramide-Ab) ELISA Kit (cat. no. MBS3804520). The relative ceramide level in each condition is shown. C TNBC MDA-MB-231 and MDA-MB-468 cells expressing either NS small hairpin RNA (shRNA) or ASAH1 shRNAs were analyzed for their abilities to grow in soft-agar assay. Representative images are shown with a scale bar of 500 µm. D Relative colony sizes for the images are shown in (C). E MDA-MB-231 cells expressing either NS small hairpin RNA (shRNA) or ASAH1 shRNAs were subcutaneously injected into the flanks of female NSG mice (n = 3). The average tumor volume was assessed weekly and plotted. F Representative tumor images at the experimental endpoint is shown. G Indicated TNBCs MDA-MB-231 and MDA-MB-468 cells expressing either NS small hairpin RNA (shRNA) or ASAH1 shRNAs were analyzed for their invasive ability using Matrigel-based invasion assays. Representative images are shown with a scale bar of 200 µm. H Quantitation of the data presented in panel G and relative invasion percentage in each condition is plotted. I Migration was analyzed in a wound-healing assay for TNBC MDA-MB-231 and MDA-MB-468 cells expressing either NS small hairpin RNA (shRNA) or ASAH1 shRNAs. Representative images are shown with a scale bar of 200 µm. J Quantitation of the data presented in (I) and relative migration percentage in each condition is plotted. Data represent the mean ± standard error for three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant.

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