Skip to main content
. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Pigment Cell Melanoma Res. 2021 Sep 1;35(1):52–65. doi: 10.1111/pcmr.13013

Figure 6.

Figure 6.

Exogenous ARPC5 expression partially protects against migration or focal adhesion reduction after YAP loss. (A,B) The P3-ARPC5 model utilizes an expression vector expressing physiological levels of ARPC5. The expression vector contains a partial 1569 bp PAX3 promoter driving the expression of an ARPC5 expression cassette. The PAX3 promoter (P3) is active in melanoma cells with low but measurable expression levels (Kubic et al., 2016). Inhibition of YAP in both MOCK and P3-ARPC5 led to undetectable levels (left column graphs in B, siScramble controls (white bars 1,3) versus siYAP (black bars 2,4)) in both mel537 (top row) and A375 (bottom row) cells. However, while ARPC5 levels dropped to undetectable levels in MOCK cells (B, black bar 6) in compared to siScramble control cells (white bars 5,7), the P3-ARPC5 vector partially rescued ARPC5 expression in YAP depleted cells (black bar 8, 32%±13% of control levels in mel537, 60%±14% A375). (C) Exogenous ARPC5 expression partially rescues YAP-dependent reduction in migration in A375 but not mel537 cells. All groups had a significant decrease in migration after YAP inhibition except for A375 cells expressing the P3-ARPC5 vector (insignificant difference from control, p=0.67 marked with ND, with levels of 83.4%±28.8% of control). For cell migration, the percent of wound closure after 24 hours was calculated. (D) Exogenous ARPC5 was able to rescue YAP-dependent focal adhesion reduction partially (mel537) or fully (A375) to control levels. In P3-ARPC5 cells, blocking of YAP did not lead to a significant decrease in focal adhesion numbers, with insignificant differences in comparison to controls (97.2%±33.6% p=0.62 mel537, and 106.5%±27.7% p=0.60 A375). For focal adhesion numbers, methods outlined in Figure 2 are followed. For C and D, white bars indicate siScrambled control and black bars indicate YAP specific siRNA knockdown. Each assay was performed at least in triplicate. Asterisk (*) indicates a p < 0.05 and ND (not detected) a p > 0.05 as determined by 2-tailed t-tests in comparison to siScrambled controls.