Fig 2: SENP1 knockdown inhibited the acetylation of CCN1 by P300. HAECs were co-transfected with MRTF-A, P300, and shSENP1. (A,B) The expression of CCN1 were examined by qRT-PCR and Western blot. (C) CCN1 transactivation was analyzed using luciferase assay after 24 h transfection of MRTF-A, P300, shENP1, and CCN1 promoter-Luc plasmids into HAECs. (D–F) ChIP assays were performed in HAECs after transfection, and cross-linked chromatin was immunoprecipitated with specific anti-acetyl-h3K9/h3K14 /h4 antibodies and IgG antibody as a negative control. The signs ‘+’ means present and ‘-’ means absent for the conditions indicated in each graph. Quantitation is shown as the means ± SD (p values are as indicated, n = 3. Individual data points were shown as scattered black dots).

Fig 3: A model for the regulation of MRTF-A by SUMOylation. MRTF-A is sequestered in the cytoplasm by SUMOylation in MSCs. When there is a need for differentiation toward angiogenesis, SENP1 is activated by certain regulation factors to induce MRTF-A deSUMOylation, which makes MRTF-A relocate into the nucleus. By cooperating with P300 and SRF on the CArG box, the angiogenic factor CCN1 is transactivated and initiates the angiogenesis process. CXCR4 likely plays a regulatory role on upstream of MRTF-A SUMOylation, but the detailed interactions are yet to be elucidated.

Fig 4: A) Representative images of passage 5 (P5) hMSCs cultured on glass and 1, 6, and 50 kPa hydrogels for 4 days. Scale bars: 50 µm. Following 4 days of culture, quantification of B) cell spread area (µm2), C) cell shape index, a measure of cell circularity, and D) MRTF-A nuclear-to-cytosol ratio was performed. n = 3 hydrogels per group. *** P < 0.001, ** P < 0.01, * P < 0.05.

Fig 5: The correlation between CCN1 transactivation and MRTF-A SUMOylation. (A) The three putative SUMOylation sites of MRTF-A are located within amino acids 471-630. (B) Confirmation of SUMO-binding sites with specific point mutations (K499, K576, and K624) of MRTF-A together with shSENP1. The whole cell lysates were subjected to western immunoblotting with an anti-MRTF-A specific antibody. (C) SUMO-1 specific SUMOylation of MRTF-A was confirmed by transient transfection with conjugation-defective SUMO-1 (?GG) HAECs. (D,E) Increased expression of CCN1 following disruption of SUMOylation by the transient introduction of K499/576/624R mutations examined by qRT-PCR and western blot. (F) Luciferase analysis of CCN1 activation in WT and lysin mutants of MRTF-A. (G,H) Abolishing SUMOylation with conjugation-defective SUMO-1 (?GG) led to increased expression of CCN1 as examined by qRT-PCR and western blot, and transactivation as determined by luciferase assay. The signs ‘+’ means present and ‘-’ means absent for the conditions indicated in each graph. Quantitation is shown as the means ± SD (p values are as indicated, n = 3. Individual data points were shown as scattered black dots).