Fig. 7: AMPK is activated upon cell fusion and AMPK inhibition impairs both YAP1 cytoplasmic localization and cell-cycle arrest.

a Cell lysates were made from VSV-G- transfected SUM-159 cells before (pre-fusion; 0 min) or 5, 15, and 60 min after washing with fusion buffer. The levels of total (~62 kDa) and phosphorylated (~62 kDa) AMPK were determined by western blot. Vinculin (Vin, ~116 kDa) was used as a loading control. Western blot images are representative of three independent experiments. b The fold change of p-AMPK in lysates at the indicated times after fusion was calculated relative to the pre-fusion cells. All cells were transfected with VSV-G and were then left untreated (black bar), or treated with AMPK or endocytosis inhibitors (compound C, blue bar or PitStop2, white bar). In addition, p-AMPK was quantified in untreated cells that lacked VSV-G (untransfected cells washed with Fusion Buffer but unable to fuse, purple bar). Error bars represent the SEM, n = 3, n = 3, n = 3, and n = 3 independent lysates samples from pre-fused cells and cells isolated 5 (*p = 0.049), 15 (**p = 0.004), and 60 min after fusion, respectively. c, d SUM-159 cells transfected with VSV-G, incubated with or without the AMPK inhibitor compound C or co-transfected with the dominant negative form of the α2 subunit of AMPK (AMPK-DK), were induced to fuse and then fixed at indicated time points and immunostained with an anti-YAP1 antibody (c). The YAP1 nuclear to cytoplasmic ratios were graphed (d). Error bars represent the SEM of cells examined over three independent experiments. For untreated cells (magenta line): n = 44, n = 19, n = 21, and n = 23 cells at 0, 5, 15, and 60 min, respectively. For compound C treated cells (green line): n = 51 (***p < 0.0001), n = 25 (***p < 0.0001), n = 32 (***p < 0.0001), and n = 24 cells (***p < 0.0001) at 0, 5, 15, and 60 min, respectively. For cells transfected with AMPK-DK (black line): n = 38 (***p < 0.0001), n = 26 (ns), n = 23 (***p = 0.0001), and n = 26 cells (***p < 0.0001) at 0, 5, 15, and 60 min, respectively. e Cell lysates from VSV-G transfected cells, either untreated or incubated with compound C, were prepared at the indicated times after fusion. Western blot images are representative of three independent experiments for untreated samples. Total YAP1 (~78 kDa) and YAP1 phosphorylation (S127, phosphorylated by LATS, ~78 kDa) were assayed by western blot. (Tubulin (Tub), 55 kDa) was used as a loading control). f The fold changes of p-YAP1 were calculated in untransfected (-VSV-G, purple bar) cells, and in VSV-G-transfected cells that were untreated (black bar) or incubated with either compound C (blue bar) or PitSop2 (white bar). Error bars represent the SEM, n = 3, n = 3, n = 3, and n = 3 independent lysates samples from pre-fused cells and cells isolated 5, 15 (**p = 0.0084), and 60 (**p = 0.0086) min after fusion, respectively. g Unfused cells treated with AICAR (6 mM) or 2-DG (50 mM) for 1 h were fixed, immunostained with anti-YAP1 antibody and imaged by confocal microscopy to determine YAP1 localization. h The ratio of nuclear to cytoplasmic YAP1 measured in fused cells and control (untreated, black bar) or treated cells is graphed. Error bars represent the SEM of cells examined over three independent experiments, n = 18, n = 18, n = 18, and n = 23 cells for untreated, AICAR treated (**p = 0.008), 2-DG treated (**p = 0.0067), and fused cells (***p < 0.0001), respectively. i, j Nuclear P21 was visualized in compound C treated cells fixed and immunostained 24 h after fusion. j The percentage of P21-positive nuclei in compound C treated cells (white bar) is compared to the untreated non-fused (black bar) and fused cells (blue bar). Error bars represent the SEM, n = 20, n = 25, and n = 30 cells examined over three independent experiments for non-fused, fused, and compound C treated fused cells, respectively. ***p < 0.0001, *p = 0.0119, and *p = 0.0189. k Violin plots depict the fold change in CDKN1A (P21) expression levels in compound C treated (white) and untreated cells measured by qRT-PCR. n = 3 independent experiments (control, black and fused, blue). *p = 0.0412, *p = 0.0138, and **p = 0.0008. ns not significant (p > 0.05). Statistical significance calculations were performed using a two-tailed unpaired Student’s t test. Scale bar size = 10 μm.