Skip to main content
. 2022 Jun 14;25(7):104589. doi: 10.1016/j.isci.2022.104589

Figure 5.

Figure 5

Myogenic Tead1 inhibits myogenic-endothelial cross-talk

(A-C) Co-culture of GFP expressing C166 ECs with myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs for 3days

(B) Representative images of C166-GFP EC and C2C12 co-cultures at 6days, with DAPI counter-stain. Scale bar, 100 μm.

(C) Quantification of the total GFP + cell area relative to the total image area. (n = 24 cell culture replicates per group).

(D and E) Culture of C166 ECs in presence of conditioned medium harvested from myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs. Conditioned media were collected from myotube cultures and applied daily to C166 EC cultures for 3 days.

(E) Quantification of the number of C166 ECs per well in conditioned medium from control and Tead1 siRNA treated C2C12 myotubes. (n = 49 wells cell culture replicates per group).

(F-I) Co-culture of GFP expressing C166 ECs with myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs in presence of Aplnr siRNAs or Aplnr inhibitor (ML221).

(G) Aplnr expression after the transfection of siRNAs targeted Aplnr compared to si-scrambled control (n = 16 per group).

(H) Quantification of the total GFP + cell area relative to total image area after siAplnr k/d (n = 24 per group).

(I) Quantification of the total GFP + cell area relative to total image area after 10μM ML221 (Aplnr inhibitor) compared to DMSO control (n = 24 per group).

(J–M) Analysis of MCK-Tead1 overexpressing mice following cardiotoxin-induced injury in TA muscle compared to WT controls. (K) Expression of Pecam1, Icam1, and Tek (Tie2) mRNA by RT-qPCR in tibialis anterior muscles isolated from adult myofiber-specific Tead1-overexpressing mice (MCK-OE-Tead1) compared to WT C57BL6 controls. (n = 4 mice per group). (L) Representative images of CD31 immunostaining in CTX-injured TA muscles at 3, 7 days.p.i. Scale bar, 50 μm. (M) Quantification of CD31+ endothelial cells at non-injured, 3 and 7 days.p.i. in regenerating regions (n = three to four TA muscles per group).

All graphs are reported as mean ± SEM and p values are reported from two-tailed, unpaired t-test between the conditions.