Fig. 2

SOCE is constitutively active and enhanced in ACM C-MSC. Expression of STIM1 (A) and ORAI1 (B) in total RNA extracts of C-MSC from HC donors and ACM patients. GAPDH was used as a house-keeping gene and qRT-PCR data are presented as the genes threshold cycles (Ct) with respect to the housekeeping gene GAPDH (ΔCt) (n = 6 biological replicates; Two-tailed Student’s t-tests). C Representative images of WB analysis of proteins extracted from HC and ACM C-MSC cultured in GM, hybridized with anti-STIM1 and anti-ORAI1 antibodies. Immunostaining of the housekeeping H3 is shown for normalization. D–E Densitometric analysis of STIM1 (n = 8 biological replicates) and ORAI1 (n = 8 biological replicates) levels, normalized on H3 (Two-tailed Student’s t-tests). F Representative tracings of resting Ca²⁺ entry in C-MSC from HC donors and ACM patients evaluated by using the Mn²⁺-quenching technique. The measurements were performed on unstimulated cells cultured in GM. (G) Quenching rate of Fura-2/AM fluorescence induced by Mn²⁺ addition in unstimulated (BASE) C-MSC from HC donors and ACM patients (n = 182 cells HC vs. n = 129 ACM; Two-tailed Student’s t-tests). H Representative tracings of resting Ca²⁺ entry in C-MSC ACM patients evaluated by using the Mn²⁺-quenching technique. The measurements were performed on cells cultured in GM and maintained in the absence (BASE) of presence of BTP-2 (20 μM, 20 min) or PYR6 (10 μM, 10 min). I Quenching rate of Fura-2/AM fluorescence in unstimulated (BASE) or treated C-MSC from ACM patients (n = 280 cells BASE vs. n = 115 BTP-2 vs. n = 82 PYR6; one-way ANOVA test). Data information: mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.0001