Journal: Cancer Communications

Article Title: Activation of Piezo1 contributes to matrix stiffness‐induced angiogenesis in hepatocellular carcinoma

doi: 10.1002/cac2.12364

Figure Lengend Snippet: Analysis of differentially expressed pro‐angiogenic and anti‐angiogenic factors between shPiezo1‐CM and Scramble‐CM. (A) Piezo1‐knockdown human HCC cell lines were verified by Western blotting. (B) Representative images of tube formation assay showed the effect of shPiezo1‐CM on HUVEC tube formation. (C) Representative images of wound healing assay showed the effect of shPiezo1‐CM on HUVEC migration. (D) Screening of differentially expressed pro‐angiogenic and anti‐angiogenic factors using a human angiogenesis array between Scramble‐CM and shPiezo1‐CM (upper panel). Fold change analysis of differentially expressed pro‐angiogenic and anti‐angiogenic factors between Scramble‐CM and shPiezo1‐CM (bottom panel). (E) Western blotting assay of three differentially expressed pro‐angiogenic factors (VEGF, IGFBP2, CXCL16) in wild‐type, shCtrl‐transfected, and shPiezo1‐transfected MHCC97H cells cultured on high‐stiffness substrate. (F) Western blotting assay of VEGF, IGFBP2 and CXCL16 in wild‐type, shCtrl‐transfected, and shITGB1‐transfected MHCC97H cells grown on high‐stiffness substrate. The protein expression, number of branches, capillary length, and wound‐healed area were quantified using ImageJ software. * P < 0.05, ** P <0.01, *** P < 0.001. Abbreviations: HCC, hepatocellular carcinoma; WT, wild type; Ctrl, control; CM, conditioned medium; HUVEC, human vascular endothelial; VEGF, vascular endothelial growth factor; IGFBP2, insulin like growth factor binding proteins; CXCL16, CXC chemokine ligand 16; H, high stiffness (16 kPa); ITGB1, integrin β1.

Article Snippet: The primary antibodies were diluted as follows: LOX (1:100, Abcam, Cambs, Cambridge, UK), COL1 (1:100, Affinity, Cincinnati, OH, USA), Piezo1 (1:50, Abcam), CD31 (1:100, Abcam), hypoxia inducible factor‐1α (HIF‐1α) (1:100, Abcam), VEGFA (1:100, Proteintech, Wuhan, Hubei, China), insulin‐like growth factor binding protein 2 (IGFBP2) (1:200, BOSTER, Wuhan, Hubei, China), CXC chemokine ligand 16 (CXCL16) (1:200, Proteintech).

Techniques: Knockdown, Western Blot, Tube Formation Assay, Wound Healing Assay, Migration, Transfection, Cell Culture, Expressing, Software, Control, Binding Assay

Journal: Cancer Communications

Article Title: Activation of Piezo1 contributes to matrix stiffness‐induced angiogenesis in hepatocellular carcinoma

doi: 10.1002/cac2.12364

Figure Lengend Snippet: Piezo1 activation induced by high‐stiffness stimulation regulates the expression of HIF‐1α ubiquitination and its downstream pro‐angiogenic factors in HCC cells. (A) The expression of HIF‐1α, VHL, VEGF, IGFBP2 and CXCL16 in MHCC97H cells grown on different stiffness substrates. (B) MG132 intervention increased the expression of HIF‐1α and its downstream target genes in MHCC97H cells cultured on low‐stiffness substrate. (C) Effects of Yoda1 on the expression of HIF‐1α, VHL, VEGF, IGFBP2 and CXCL16 in MHCC97H cells grown on low‐stiffness substrate. (D) Effects of GsMTx4 on the expression of HIF‐1α, VHL, VEGF, IGFBP2 and CXCL16 in MHCC97H cells grown on high‐stiffness substrate. (E) Effects of Piezo1 knockdown, Yoda1 intervention and MG132 intervention on the expression of target proteins in MHCC97H cells grown on high‐stiffness substrate. (F) Effects of integrin β1 knockdown and Yoda1 intervention on the expression of target proteins in HCC cells grown on high‐stiffness substrate. (G) Co‐IP assay demonstrates an endogenous interaction between VHL protein and HIF‐1α protein. (H) HIF‐1α ubiquitination level was increased in shPiezo1‐ or shITGB1‐transfected MHCC97H cells compared with that in shCtrl‐transfected MHCC97H cells. (I) Representative IHC staining images of HIF‐1α and VEGF in orthotopic HCC tumors in WT, shCtrl, shITGB1 #1 and shPiezo1 #1 groups with high liver stiffness background. The protein expression area was quantified using ImageJ software. *** P < 0.001. Abbreviations: L, low stiffness (6 kPa); M, medium stiffness (10 kPa); H, high stiffness (16 kPa); HIF‐1α, hypoxia‐inducible factor 1α; VEGF, vascular endothelial growth factor; IGFBP2, insulin like growth factor binding proteins; CXCL16, CXC chemokine ligand 16; VHL, von hippel‐lindau; WT, wild type; Ctrl, control; ITGB1, integrin β1; IHC, immunohistochemical; Co‐IP, co‐immunoprecipitation.

Article Snippet: The primary antibodies were diluted as follows: LOX (1:100, Abcam, Cambs, Cambridge, UK), COL1 (1:100, Affinity, Cincinnati, OH, USA), Piezo1 (1:50, Abcam), CD31 (1:100, Abcam), hypoxia inducible factor‐1α (HIF‐1α) (1:100, Abcam), VEGFA (1:100, Proteintech, Wuhan, Hubei, China), insulin‐like growth factor binding protein 2 (IGFBP2) (1:200, BOSTER, Wuhan, Hubei, China), CXC chemokine ligand 16 (CXCL16) (1:200, Proteintech).

Techniques: Activation Assay, Expressing, Ubiquitin Proteomics, Cell Culture, Knockdown, Co-Immunoprecipitation Assay, Transfection, Immunohistochemistry, Software, Binding Assay, Control, Immunohistochemical staining, Immunoprecipitation

Journal: Cancer Communications

Article Title: Activation of Piezo1 contributes to matrix stiffness‐induced angiogenesis in hepatocellular carcinoma

doi: 10.1002/cac2.12364

Figure Lengend Snippet: The mechanism by which activation of Piezo1 contributes to matrix stiffness‐induced angiogenesis in HCC. Matrix stiffness, as an initiator, enhances Piezo1 activation and Ca 2+ influx, and then suppresses HIF‐1α ubiquitination, ultimately promoting pro‐angiogenic factor expression to accelerate HCC angiogenesis. Besides, a positive feedback regulation loop as stiff matrix/integrin β1/miR‐625‐5p /Piezo1 and COL1/stiffer matrix mediates matrix stiffness‐caused Piezo1 upregulation. Abbreviations: HIF‐1α, hypoxia‐inducible factor 1α; VEGF, vascular endothelial growth factor; IGFBP2, insulin like growth factor binding proteins; CXCL16, CXC chemokine ligand 16; COL1, collagen1; UB, ubiquitin.

Article Snippet: The primary antibodies were diluted as follows: LOX (1:100, Abcam, Cambs, Cambridge, UK), COL1 (1:100, Affinity, Cincinnati, OH, USA), Piezo1 (1:50, Abcam), CD31 (1:100, Abcam), hypoxia inducible factor‐1α (HIF‐1α) (1:100, Abcam), VEGFA (1:100, Proteintech, Wuhan, Hubei, China), insulin‐like growth factor binding protein 2 (IGFBP2) (1:200, BOSTER, Wuhan, Hubei, China), CXC chemokine ligand 16 (CXCL16) (1:200, Proteintech).

Techniques: Activation Assay, Ubiquitin Proteomics, Expressing, Binding Assay