Journal: Cancer Communications

Article Title: P300/CBP‐associated factor (PCAF)‐mediated acetylation of Fascin at lysine 471 inhibits its actin‐bundling activity and tumor metastasis in esophageal cancer

doi: 10.1002/cac2.12221

Figure Lengend Snippet: Fascin interacts with PCAF in ESCC cells. A, The interaction between Fascin and PCAF was investigated by immunoprecipitation assay using anti‐Fascin and anti‐PCAF antibodies in HEK293T and KYSE140 cells, respectively. B, Immunofluorescence analysis of the localization of endogenous Fascin and PCAF in ESCC KYSE150 cells. Endogenous Fascin (green) and PCAF (red) were stained with specific antibodies, and nuclei were stained with DAPI (blue). C, Fascin directly interacted with PCAF in in vitro GST‐pull down assays using GST‐PCAF and GST‐Fascin. D, The N‐terminal domain, acyltransferase domain (HAT), and C‐terminal BROMO domains of PCAF were fused to GST and purified for GST‐pull down assays with His‐fused Fascin (His‐Fascin). E, The GST‐fused truncated forms of Fascin were purified for GST‐pull down assays with His‐fused PCAF (His‐PCAF). The purified GST fusion proteins were examined with Coomassie brilliant blue staining, and the pulled‐down His proteins were examined by Western blotting. Arrows indicate proteins with correct molecular weights. Abbreviations: PCAF: P300/CBP‐associated factor; ESCC: esophageal squamous cell carcinoma; IP: immunoprecipitation; IB: immunoblot; GAPDH: glyceraldehyde‐3‐phosphate dehydrogenase; DAPI: 4’,6‐diamidino‐2‐phenylindole; CBB: Coomassie brilliant blue; GST: Glutathione S‐transferase; HAT: Histone acyltransferase; BROMO: bromodomain

Article Snippet: The denatured proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western blotting with anti‐Fascin rabbit polyclonal antibody (PA513696; Life Technologies).

Techniques: Immunoprecipitation, Immunofluorescence, Staining, In Vitro, Purification, Western Blot

Journal: Cancer Communications

Article Title: P300/CBP‐associated factor (PCAF)‐mediated acetylation of Fascin at lysine 471 inhibits its actin‐bundling activity and tumor metastasis in esophageal cancer

doi: 10.1002/cac2.12221

Figure Lengend Snippet: Acetylation of Fascin‐K471 by PCAF impairs cancer cell migration and tumor metastasis. A, Western blotting after restoration of GFP‐vector (Vector), Fascin‐GFP (WT), Fascin K471R ‐GFP (K471R), and Fascin K471Q ‐GFP (K471Q) expression in stable Fascin‐knockdown KYSE150 and SHEEC cells. Endogenous Fascin and Fascin‐GFP were detected using anti‐Fascin and GFP‐tag antibodies, respectively. β‐actin was used as a loading control. B and C, The migration of Fascin‐knockdown KYSE150 (B) and SHEEC (C) cells restored GFP‐vector (Vector), Fascin‐GFP (WT), Fascin K471R ‐GFP (K471R), and Fascin K471Q ‐GFP (K471Q) was investigated by using the xCELLigence Real‐Time Cell Analysis DP instrument. Left panel, real‐time detection of impedance response/cell index curves determined over 42 h (B) and 49 h (C). Right panel, the slope of the cell profiles. The experiments were performed independently in triplicate. Data are presented as the mean ± standard deviation (SD). The Student's t ‐test was used for statistical analysis. D, Representative mice inoculated with cells in (B) (upper images), and bioluminescent images (lower images) at 32 days after injection in the left footpad. The invasion sites on the thigh are magnified. E, Representative excised primary tumors (from the footpad to the heel of the foot; upper images) and invaded tumors (from the heel of the foot to the inguinal lymph node; lower images) of mice in (D). Primary and invaded tumor weights were compared among the four groups. Data are presented as the mean ± SD of 8 mice in each group. The Student's t ‐test was used for statistical analysis. F, Representative H&E and IHC staining for GFP was used to detect the primary tumor (upper images) and representative IHC staining of cytokeratin (CK) in the inguinal lymph nodes (lower images). G, Number of mice with and without lymph node metastases after 32 days of inoculation. * P < 0.05. Abbreviations: PCAF: P300/CBP‐associated factor; GFP: Green fluorescent protein; sh: short hairpin; NC: negative control; HE: hematoxylin‐eosin; IHC: immunohistochemistry; LN: lymph node; CK: cytokeratin; SD: standard deviation

Article Snippet: The denatured proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western blotting with anti‐Fascin rabbit polyclonal antibody (PA513696; Life Technologies).

Techniques: Migration, Western Blot, Plasmid Preparation, Expressing, Standard Deviation, Injection, Immunohistochemistry, Negative Control