Fig 2: Effects of isovitexin combined with MnSOD shRNA or cDNA on carcinogenicity and stemness in HCSLCs or MHCC97H cells. HCSLCs were transduced with Ad-GFP and Ad-shMnSOD, respectively, incubated with or without isovitexin (ISOV; 10 µM). a Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies as a loading control; b and c Formed spheres and colonies (Scale bar, 200 µm); d Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4; e The relative luciferase activity of FOXM1 promoter fragment in HCSLCs transduced with Ad-GFP and Ad-shMnSOD, respectively, in the absence or presence of isovitexin. MHCC97H cells were transduced with Ad-GFP and Ad-MnSOD, respectively, and incubated with or without isovitexin. f Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies as a loading control; g and h Formed spheres and colonies (Scale bar, 200 µm); i Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4; j The relative luciferase activity of FOXM1 promoter fragment in MHCC97H cells transduced with Ad-GFP and Ad-MnSOD, respectively, cultured in the absence or presence of isovitexin

Fig 3: Isovitexin inhibits HCSLC carcinogenicity and stemness via the MnSOD/FoxM1 axis. HCSLCs or those expressing shMnSOD were transduced with Ad-FoxM1. a Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies used as loading control; b and c Formed spheres and colonies (Scale bar, 200 µm); d Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4 in HCSLCs untreated (Mock), and transduced with Ad-shMnSOD and/or Ad-FoxM1. HCSLCs expressing shMnSOD were transduced with Ad-FoxM1 and incubated with or without isovitexin (ISOV; 10 µM). e Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies as a loading control; f and g Formed spheres and colonies (Scale bar, 200 µm); h Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4 in MHCC97H cells transduced with Ad-shMnSOD and/or Ad-FoxM1 and incubated in the absence or presence of ISOV

Fig 4: Effects of isovitexin combined with FoxM1 shRNA or cDNA on carcinogenicity and stemness in HCSLCs or MHCC97H cells. HCSLCs were transduced with Ad-GFP and Ad-shFoxM1, respectively, incubated with or without isovitexin (ISOV; 10 µM). a Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies as a loading control; b and c Formed spheres and colonies (Scale bar, 200 µm); d Immunoblotting for CD133, CD44, ALDH1 (D), Bmi1, Nanog and Oct4 amounts in HCSLCs transduced with Ad-GFP and Ad-shFoxM1, respectively, in the absence or presence of isovitexin. MHCC97H cells were transduced with Ad-GFP and Ad-FoxM1, respectively, and incubated with or without isovitexin. e Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies as a loading control; f and g Formed spheres and colonies (Scale bar, 200 µm); h Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4 in MHCC97H cells transduced with Ad-GFP and Ad-FoxM1, respectively, cultured in the absence or presence of isovitexin

Fig 5: Effects of MnSOD shRNA or cDNA transduction on carcinogenicity and stemness in HCSLCs or MHCC97H cells. HCSLCs were transduced with Ad-GFP or Ad-shMnSOD. 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 The relative luciferase activity of FOXM1 promoter fragment in HCSLCs untreated (Mock), or transduced with Ad-GFP and Ad-sh MnSOD, respectively. MHCC97H cells were transduced with Ad-GFP or Ad-MnSOD. f Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies; ß-actin antibodies were used as a loading control; g and h Formed spheres and colonies (Scale bar, 200 µm); i Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4; j The relative luciferase activity of FOXM1 promoter fragment in MHCC97H cells untreated (Mock), and transduced with Ad-GFP and Ad-MnSOD, respectively