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. 2021 Jul 15;8(18):2102079. doi: 10.1002/advs.202102079

Figure 3.

Figure 3

TRIM21 regulates Src protein level to mediate GABAAR signaling. Western blot analysis of TRIM21 expression in HCT116 cells treated with a) propofol, b) muscimol, or d) bicuculine. c) qRT‐PCR analysis of TRIM21 mRNA expression in HCT116 cells treated with propofol or control condition (N = 3, mean ± SD, Student's t‐test, P > 0.5). e) Western blot analysis of Src and p‐Src expression in HCT116 cells with or without overexpression of TRIM21. GAPDH is a loading control. f) qRT‐PCR analysis of Src mRNA expression in HCT116 cells with or without overexpression of TRIM21 (N = 3, mean ± SD, Student's t‐test, P > 0.5). g) Fluorescent micrographs of adhesion between the HUVEC monolayer and the HCT116 cells (green) with or without overexpression of TRIM21 and Src (scale bar = 100 µm). h) Quantification of the HCT116 cells adhesion to the HUVECs monolayer (N = 6, mean ± SD, one‐way ANOVA test with post‐hoc Bonferroni test, F = 11.04, P = 0.001). i) F‐actin labeling of membrane protrusions in HCT116 cells with or without overexpression of TRIM21 and Src (scale bar = 100 µm). Arrows indicate extensions of cell membrane protrusions. j) Immunofluorescence staining of Talin and Vinculin in HCT116 cells with or without overexpression of TRIM21 and Src (scale bar = 50 µm). Arrows indicate focal adhesions. k) Quantification of the membrane‐localized Talin and Vinculin in focal adhesion (N = 4, mean ± SD, one‐way ANOVA test with post‐hoc Bonferroni test, F = 25.24, P < 0.001). l) Western blot analysis of Src and phosphorylated Src (p‐Src) expression in HCT116 cells with or without overexpression of TRIM21 and following propofol or control condition. GAPDH is a loading control. m) Fluorescent micrographs of the adhesion between the HUVEC monolayer and the HCT116 cells (green) with or without overexpression of TRIM21 and treated with propofol or control condition (scale bar = 100 µm). n) Fluorescent micrographs showing adhesion of muscimol‐treated HCT116 cells (green) with or without TRIM21 overexpression to the HUVEC monolayer (scale bar = 100 µm). o) Quantification of panel (n) (N = 6, mean ± SD, Student's t‐test, P = 0.002). p) F‐actin labeling of membrane protrusions in muscimol‐treated HCT116 cells with or without TRIM21 overexpression (scale bar = 100 µm). Arrows indicate extensions of cell membrane protrusions. q) Immunofluorescence staining of Talin and Vinculin in muscimol‐treated HCT116 cells with or without TRIM21 overexpression (scale bar = 50 µm). Arrows indicate focal adhesions. Ctrl, control. *P < 0.05, ***P < 0.001, # P < 0.05, and ## P < 0.01. Ctrl, control; HUVEC, human umbilical vein endothelial cell; SD, standard deviation; p‐Src, phosphorylated Src.