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. 2016 Aug 16;291(41):21350–21362. doi: 10.1074/jbc.M116.732479

FIGURE 7.

FIGURE 7.

Effect of AGAP1 and Kif2A on cell spreading. A and B, reduced expression of endogenous AGAP1 or Kif2A and replacement with FLAG-AGAP1 and Kif2A-GFP. Hela cells treated with siRNA targeting noncoding regions on either AGAP1 or Kif2A and transfected with either empty plasmid or plasmids for expression of FLAG-AGAP1, Arf GAP1-HA, Kif2A-GFP, or Kif2C-GFP, as indicated, were plated on fibronectin-coated cover glass for 20 min. The area of rhodamine-phalloidin-stained cells was determined for at least 40 cells per condition using ImageJ version 1.46r. Results shown are the mean ± S.E. from three independent experiments. Differences were tested by ANOVA followed by multiple comparison post-tests using the program PrismTM. A, control Si versus AGAP1 Si + empty vector, p < 0.001; AGAP1 Si + empty vector versus AGAP1 Si + FLAG-AGAP1, p < 0.001; AGAP1 Si + empty vector versus AGAP1 Si + ArfGAP1-HA, p < 0.05. B, control Si + empty vector versus Kif2A Si + empty vector, p < 0.001; Kif2A Si + empty vector versus Kif2A Si + Kif2A GFP, p < 0.005; Kif2A Si + empty vector versus Kif2A Si + Kif2C-GFP, not significant. C, effect of expression of AGAP1 mutants on spreading of cells with reduced endogenous AGAP1. Hela cells treated with siRNA targeting the noncoding region of AGAP1 and transfected with plasmids for expression of the indicated fragments of AGAP1 or mutants of AGAP1 were plated on fibronectin-coated coverslips for 20 min, and cell surface area was determined as in A. Control Si + empty vector versus AGAP1 Si + empty vector, p < 0.001; AGAP1 Si + empty vector versus AGAP1 Si + FLAG-GLDPH, not significant; AGAP1 Si + empty vector versus AGAP1 Si + FLAG-PZA, + [R599K]AGAP1, + [E125Q]AGAP1, p < 0.001. AGAP1Si + PZA versus AGAP1Si + FLAG-[R599K]AGAP1, p < 0.001. D, effect of expression of FLAG-[K474D,K475D]AGAP1 on spreading of cells with reduced endogenous AGAP1 expression. The experiment is similar to that described in C except that FLAG-[K474D,K475D] AGAP1 was the only mutant examined. Control Si + empty vector versus AGAP1 Si + empty vector, p < 0.001; AGAP1 Si + empty vector versus AGAP1 Si + FLAG-AGAP1, p < 0.001; AGAP1 Si + empty vector versus AGAP1 Si + FLAG-[K474D,K475D]AGAP1, not significant. E, effect of overexpression of AGAP1 and GLDPH on spreading of cells. Spreading of cells transfected with empty vector (pCI) or plasmids for expression of FLAG-AGAP1 or FLAG-GLDPH was determined as in A. Empty vector versus FLAG-GLDPH, p < 0.001; empty vector versus FLAG-AGAP1, p < 0.01.