Fig 1: The APP/FASN modulation impacted the tumorigenicity of CSCs in vitro. (A: The mRNA expression of APP and FASN in CSCs treated with cerulenin at different concentrations (0 μM, 10 μM, and 20 μM) was measured by qPCR. ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; two-way ANOVA. B: the qPCR results of CSCs with APP knocking down through four different lentiviruses, labeled as shAPP #1, shAPP #2, shAPP #3, and shAPP #4. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; ns, not statistically significant; one-way ANOVA. C: soft agar assay. CTRL: The cells were not treated. shAPP #1/shAPP #2: The cells with APP knockdown are referred to as shAPP #1 and shAPP #2 (n = 3). D: cell colonies with more than 20 cells were counted. shAPP #1/shAPP #2: The cells with APP knockdown are referred to as shAPP #1 and shAPP #2. ∗, P < 0.05; ∗∗, P < 0.01; one-way ANOVA. E: tumor sphere formation assay. CTRL: The cells were not treated. shAPP #1/shAPP #2: the cells with APP knocking down (n = 3). F: spheres with at least a diameter of 100 μm were calculated. ∗, P < 0.05; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; one-way ANOVA.
Fig 2: The impact of APP knockdown and its potential downstream pathways. A: transwell invasion assay. CTRL: The cells were not treated. shAPP #1/shAPP #2: the cells with APP knocking down (n = 3). B: the invasion capacity of the cells with APP depletion was measured. ∗, P < 0.05; ∗∗∗∗, P < 0.0001; one-way ANOVA. C: the mRNA expression of APP-related markers in CSCs treated with cerulenin 20 μM, shAPP#1, and shAPP#2 was assessed by qPCR. ∗, P < 0.05; ∗∗∗∗, P < 0.0001; ns, not statistically significant; two-way ANOVA. D: correlation analysis of APP and its downstream markers from TCGA and GTEx databases.
Fig 3: The APP rescue experiment in cerulenin-treated CSCs. A: Tumor Sphere assay. CTRL: The cells were not treated. +Cerulenin: The cells received a 20 μM concentration of cerulenin treatment (n = 3). +50, 100, 200 ng APP: The cells received a 50, 100, 200 ng of APP treatment (n = 3). B: Tumorigenicity of the cells treated with cerulenin was rescued when additional APP was added. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ns, not statistically significant; one-way ANOVA. C: Cell migration assay. CTRL: The cells were not treated. +Cerulenin: The Huh7 CSCs received a 20 μM concentration of cerulenin treatment and the HepG2/C3A CSCs received a 10 μM concentration of cerulenin treatment (n = 3). +50, 100, 200 ng APP: The cells received a 50, 100, 200 ng of APP treatment (n = 3). D: The migration capacity of the cells treated with cerulenin was rescued when additional APP was added. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; one-way ANOVA. E: Transwell invasion assay. CTRL: The cells were not treated. +Cerulenin: The cells received a 20 μM concentration of cerulenin treatment (n = 3). +50, 100, 200 ng APP: The cells received a 50, 100, 200 ng of APP treatment (n = 3). F: The invasion capacity of the cells treated with cerulenin was rescued when additional APP was added. ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; ns, not statistically significant; one-way ANOVA.
Fig 4: The altered metabolic patterns in liver CSCs. A: The mRNA expression of FA metabolism and glycolysis of CSCs with and without cerulenin treatment was measured by qPCR. Cerulenin was treated at a concentration of 20 μM. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; Student t test. B: the DEG analysis was presented as volcano plots, and the APP gene was listed in both of the plots. Left: Comparison of RNA expression between CSCs and parental cells. Right: A comparison of RNA expression between cerulenin-treated and untreated CSCs. The DEG Analysis Parameter Set was established using the DEG Method. DESeq2. The P-value threshold was set at 0.05, and the fold change threshold was 1.5. C: RNA-sequencing sample heatmap. Left: Huh7 CSCs without treatment versus Huh7 parental cells. Right: Huh7 CSCs with cerulenin treatment at 20 μM versus Huh7 CSCs without treatment. Huh7_PT: Huh7 parental cells. Huh7_TCSC: Huh7 CSCs without treatment. Cerulenin_1/2/3: Huh7 CSCs with cerulenin treatment at a concentration of 20 μM. D: The top 50 genes, such as APP, according to the PPI network analysis were listed. E: DEG pathway dotplot. KEGG Analysis Parameter Set Analysis with gene set range:10–500. The adjusted P-value threshold was 0.05, and the fold change threshold was 1.2.
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