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. 2023 Jul 17;120(30):e2220296120. doi: 10.1073/pnas.2220296120

Fig. 3.

Fig. 3.

circASH2 impairs the cytoskeleton assembly in HCC. (A) Trypsin digestion assay on 4 HCC cell lines with circASH2 overexpression [linearASH2 (580 bp) as a negative control] or knockdown. Cells without (−) or with (+) trypsinization were fix-stained with crystal violet (0.1%, R.T., 5 min) and photographed. (Scale bars, 1 cm.) (B) Cytoskeleton formation in Huh7 cells with circASH2 knockdown and HA22T cells with circASH2 overexpression was shown by phalloidin staining of F-actin (red, 1:200, R.T., 10 min) and immunofluorescence of paxillin (green, 1:200, R.T., 1 h), while DAPI was used to stain the nucleus. (Scale bar, 10 μm.) (C and D) In vitro 3D invasion assays were performed using 4 HCC cell lines with circASH2 overexpression or knockdown. The representative pictures are shown (C), and the diameter of the sphere was measured and analyzed (D, *P < 0.05, ***P < 0.001, unpaired Student’s t test or one-way ANOVA test). (Scale bar, 100 μm.) (E) Real-time capture of 4 HCC cell lines (circASH2 overexpression or knockdown) migration was performed on the xCELLigence System and Real-time Cell Analysis (RTCA) Dual Purpose (DP) Instrument over 48 h. Data were processed by RTCA Software 2.0 (***P < 0.001, two-way ANOVA test). (F and G) Transwell assays with 4 HCC cell lines (circASH2 overexpression or knockdown). Representative fields of the porous membranes are shown (F, scale bar, 100 μm), and cell numbers per field are quantified (G, means ± SD, **P < 0.01, and ***P < 0.001, unpaired Student’s t test or one-way ANOVA test). (H) Trypsin digestion assay on Huh7 cells with circASH2 knockdown (ASO-circASH2). Cells without (−) or with (+) trypsinization were fix-stained with crystal violet and photographed. (Scale bars, 1 cm.) (I) Transwell migration assays with circASH2 knockdown (ASO-circASH2) Huh7 cells. Representative fields of the porous membranes are shown. (Scale bar, 100 μm.) (J) 3D invasive ability of circASH2 knockdown (ASO-circASH2) Huh7 cells was measured, and the representative pictures are shown. (Scale bar, 100 μm.) (K) Cytoskeleton formation in Huh7 cells with circASH2 knockdown (ASO-circASH2) was shown by phalloidin staining of F-actin (red) and immunofluorescence of paxillin (green), while DAPI was used to stain the nucleus. (Scale bar, 10 μm.)