Shi-pi-xiao-ji formula suppresses hepatocellular carcinoma by reducing cellular stiffness through upregulation of acetyl-coA acetyltransferase 1

Figure 1 Representative images show the morphology of the vehicle, Shi-pi-xiao-ji-serum, methyl-β-cyclodextrin, and water-soluble cholesterol groups of MHCC97H and HepG2 cells. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

Figure 2 Representative images and statistical results of the colony formation assay in the vehicle, Shi-pi-xiao-ji-serum, methyl-β-cyclodextrin, and water-soluble cholesterol groups of MHCC97H and HepG2 cells. A: Representative images; B: Representative statistical results. The results are expressed as means ± SEM (n = 3). ^aP < 0.05, ^bP < 0.01, ns: P > 0.05. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

Figure 3 Representative images and statistical results of wounding healing and transwell assays. A and B: Representative images and statistical results of the wounding healing assay in the vehicle, Shi-pi-xiao-ji (SPXJ)-serum, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol (Chol) groups of MHCCC97H cells; C and D: Representative images and statistical results of wounding healing assay in the vehicle, SPXJ-serum, MβCD, and Chol groups of HepG2 cells; E and F: Representative images and statistical results of the transwell assay in the vehicle, SPXJ-serum, MβCD, and Chol groups of hepatocellular carcinoma cells. The results are expressed as means ± SEM (n= 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ns: P > 0.05. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

Figure 4 Flow cytometry results show the percentage of apoptotic hepatocellular carcinoma cells in the vehicle, Shi-pi-xiao-ji-serum, methyl-β-cyclodextrin, and water-soluble cholesterol groups of MHCC97H cells and HepG2 cells based on the annexin V/PI double-staining. A and B: Flow cytometry results show the percentage of apoptotic hepatocellular carcinoma (HCC) cells in the vehicle, Shi-pi-xiao-ji (SPXJ)-serum, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol (Chol) groups of MHCC97H cells based on the annexin V/PI double-staining; C and D: Flow cytometry results show the percentage of apoptotic HCC cells in the vehicle, SPXJ-serum, MβCD, and Chol groups of HepG2 cells based on the annexin V/PI double-staining. The results are expressed as means ± SEM (n= 3). ^aP < 0.01, ^bP < 0.001 and ns: P > 0.05. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol; PI: Propidium iodide.

Figure 5 Representative atomic force microscope images and Young’s modulus values. A and B: Representative atomic force microscope (AFM) images (A) and Young’s modulus values (B) of MHCC97H cells in the vehicle, Shi-pi-xiao-ji (SPXJ)-serum, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol (Chol) groups; C and D: Representative AFM images (C) and Young’s modulus values (D) of HepG2 cells in the vehicle, SPXJ-serum, MβCD, and Chol groups. The results are expressed as means ± SEM (n= 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

Figure 6 The total plasma membrane cholesterol levels in the vehicle, Shi-pi-xiao-ji-serum, methyl-β-cyclodextrin, and water-soluble cholesterol groups of MHCC97H and HepG2 cells. The results are expressed as means ± SEM (n= 3). ^cP < 0.001. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

Figure 7 Western blot analysis of acetyl-coA acetyltransferase 1 protein expression levels. A: Representative image shows the expression of acetyl-coA acetyltransferase 1 (ACAT1) protein in vehicle, Shi-pi-xiao-ji (SPXJ)-serum, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol (Chol) groups of MHCC97H and HepG2 cells. Actin was used as loading control; B: Histogram plots show the relative expression levels of ACAT1 protein in the vehicle, SPXJ-serum, MβCD, and Chol groups of MHCC97H and HepG2 cells. The results are expressed as means ± SEM (n= 3). ^aP < 0.01, ns: P > 0.05. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol; ACAT1: Acetyl-coA acetyltransferase 1.

Figure 8 The effects of Shi-pi-xiao-ji-serum on cellular stiffness. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; ACAT1: Acetyl-coA acetyltransferase 1.