(
a) Luciferase reporter assays of NFATc1 (pGL3-NFAT), YAP1 (8XGTIIC) and CTNNB1 (SUPERTOPFLASH) transcription activities in HEK293T cells. N
= 3, data are shown as means ± SD. *p<0.05, **p<0.01, ***p<0.001, two-tailed unpaired Student’s
t-test and one-way ANOVA followed by Tukey’s multiple comparisons tests when ANOVA was significant (
Figure 9—figure supplement 1—source data 1). Expression one of NFATc1, YAP1 and CTNNB1 led to activation of the other two. The only exception is that YAP1 overexpression reducd NFATc1 activties. (
b, d) Immunoprecipitation (IP) assays of HEK293T cell lysates with the indicated expression constructs. (
b) Yoda1 treatment (40 μM, 4 hr) promoted complex formation of NFATc1 and YAP1, which was inhibited by CsA or Gd
3+ treatment. (
c) Western blotting assay of HEK 293 T cells transfected with HA-tagged YAP1 and S-tagged PPP3CA as indicated. DMSO (vehicle) or Yoda1 (40 μM) treatment was performed 24 hr after transfection for 4 hr. Arrow indicates endogenous PPP3CA. (
d) Expression of NFATc1, YAP1 and CTNNB1 together promoted their complex formation. (
e) Western blotting analyses of
Piezo1/2 deficient BMSCs treated with Yoda1. Yoda1 treatment failed to reduce phosphorylation of Ctnnb1 and Yap1. Ppp3ca: Catalytic subunit of CaN. (
f) Western blotting analyses of wild-type primary BMSCs treated by Gd
3+. Increased Nfatc1 phosphorylation (blue arrow), as well as increased phosphorylation of Yap1 and Ctnnb1 was observed (
Figure 9—figure supplement 1—source data 1).