Fig 2: Isovitexin inhibits carcinogenicity and stemness in HCSLCs. a Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies used as a loading control; b and c Formed spheres and colonies (Scale bar, 200 µm); d Immunoblotting for CD133, CD44 and ALDH1, Bmi1, Nanog and Oct4; e CD133+ cell population in MHCC97H cells and HCSLCs. f Decreased the amounts of MnSOD and FoxM1 in HCSLCs from MHCC97H cells following treatment with isovitexin (ISOV: 5.0, 10.0 and 20.0 µM). Isovitexin induced reduction of spheroid formation (g, Scale bar, 200 µm), colony formation (h, Scale bar, 200 µm), and protein amounts of CD133, CD44, ALDH1 and Bmi1, Nanog and Oct4 (i), CD133+ cell population (j) in HCSLCs

Fig 3: Flow cytometry analysis of ABCG2 and ALDH1 positive cell population following cisplatin treatment. Treatment with cisplatin was for 72 h at a concentration of 10 µM. a Representative flow cytometric histograms of ABCG2 and ALDH1 expression. Samples were analyzed after exposure to primary antibodies, and an Alexafluor-488 conjugated secondary antibody. Total cell count is depicted on the y-axis. Left: Isotype control antibody alone (light grey), OSC2 (untreated) cells (heavy black), and OSC2 treated (light black). Right: Scr = control (heavy black), shDSPP silenced cells (light black), shMMP20 silenced cells (light grey) and shDM combined DSPP-MMP20 silenced cells (heavy grey). All histograms show the distribution of fluorescence intensity (x-axis), reported as arithmetic means. b Quantification of ABCG2 and ALDH1 positive OSC2 cell population, presented as percentage (%) of total cell number. Statistically significant lower percentages (*p < 0.05) of ABCG2 and ALDH1 positive cell were noted in cisplatin-treated compared with untreated OSC2 cells, as well as in shDSPP, shMMP20 and shDM cells compared to scramble control (Scr) cells, respectively. Experiments were performed in triplicate

Fig 4: Western blot (WB) analyses of OCSC markers in DSPP-MMP20 silenced OSC2 cells (a). Normalization of ALDH1, ABCG2, BMI1, Podoplanin (PDPN), CD44, CD133 and LGR4 protein levels was with ß-actin . Y- (vertical) axis of histograms represent expression level for each protein relative to ß-actin (b). OSC2 = parent (untreated) cells; shC = control (scramble sequence) cells; shD = DSPP silenced cells; shM = MMP20 silenced cells; shDM = combined DSPP-MMP20 silenced cells. *p < 0.05 compared with ShC. Experiments were performed in triplicate

Fig 5: The MnSOD/FoxM1 axis is a novel target for isovitexin-associated inhibition of carcinogenicity and stemness in HCSLCs from HepG2 and SMMC-7721 cells a Immunoblotting for MnSOD and FoxM1 expression levels in HepG2 and SMMC-7721 cells and respective HCSLCs, with ß-actin antibodies as a loading control; b and c Sphere and colony formation rates in HepG2 and SMMC-7721 cells and respective HCSLCs. d Immunoblotting for determining CK133, CD44 and ALDH1 expression level in HepG2 and SMMC-7721 cells and respective HCSLCs, with ß-actin as an internal control. Data are mean ± SD (n = 3); P < 0.05 indicated statistical significance. * HepG2 cells. #SMMC-7721 cells. e Immunoblotting for assessing MnSOD and FoxM1 expression levels in HCSLCs from HepG2 and SMMC-7721 cells incubated with or without isovitexin (ISOV; 10 µM). Data are mean ± SD (n = 3); P < 0.05 indicated statistical significance. * Vehicle control in HCSLCs from HepG2 cells. #Vehicle control in HCSLCs from SMMC-7721 cells. f Schematic diagram of the mechanism underlying isovitexin inhibits HCSLC carcinogenicity and stemness via the MnSOD/FoxM1 axis. Isovitexin effectually inhibited carcinogenicity and stemness in HCSLCs by downregulating FoxM1 likely through preventing MnSOD overexpression induced mitochondrial H2O2-mediated an increased binding of E2F1 and Sp1 onto FoxM1 promoter