Fig 2: Identification ofSpred1 and Spry1 as direct miR-132/212 targets by luciferase assay and in cultured HUVECs. (A) The position of predicted miR-132 and miR212 targets of Spred1 by Targetscan and mutant form of 3'UTR as indicated in red. (B) Luciferase assay of Spred1-3'UTR reporter in response to transfection with indicated scramble or microRNA mimics at final concentration of 25 nM. N = 3, values in the graph are shown as mean ± SEM, **P < 0.01; ***P < 0.001. (C) Luciferase activity of wildtype and mutant Spred1-3'UTR in response to different dose of miR-132, N = 6 for WT; N = 3 for mutant. Values in the graph are shown as mean ± SEM *P < 0.05. (D) The position of predicted miR-132 and miR212 targets of Spred1 by Targetscan and mutant form of 3'UTR as indicated in red. (E) Luciferase assay of Spred1-3'UTR reporter in response to transfection with indicated scramble or microRNA mimics at final concentration of 25 nM. N = 3, values in the graph are shown as mean ± SEM; **P < 0.05; ***P < 0.001. (F) Luciferase activity of wildtype and mutant Spred1-3'UTR in response to different final concentration of miR-132 mimics transfection, N = 6 for WT; N = 3 for mutants. Values in the graph are shown as mean ± SEM, *P < 0.05. (G) RASA1 expression after miR-132, miR212 overexpression and inhibition in HUVECs. The number above indicates the relative expression compared with sham normalized by ß-Tubulin expression. (H) SPRED1 and SPRY1 expressions after miR-132, miR212 overexpression and inhibition in HUVECs. The number above indicates the relative expression compared with sham normalized by ß-Tubulin expression.

Fig 3: miR-132/212 modulate Ras-MAPK signalling by suppressing Rasa1, Spred1 and Spry1 in HUVECs. (A) Experimental setting for quantitative measure of active ERK1/2 using Bio-plex phosopho-ERK1/2 assay. (B) A working model for miR-132/212 in modulation of Ras-MAPK pathway. (C) Quantification of Spred1, Spry1 or Rasa1 expression level after siRNA transfection against Spred1, Spry1 or Rasa1 in HUVECs, Values in the graph are shown as mean ± SEM, *** P < 0.001 n = 3. (D) Quantification of phosophorylated ERK1/2 level by Bio-plex pro phosopho-ERK1/2 set. Note the sustain ERK1/2 phosphorylation is prolonged after miR-132, 212 transfection or siRNA against Spred1, Spry1, Rasa1 or combinations of the three. (E) Modelling the decay of phosophorylated ERK1/2 level from D.

Fig 4: Ergothioneine effect on satellite cell markers 2 h after exercise. Satellite cell markers in soleus and gastrocnemius samples. (A) PAX7 protein expression. (B) MyoD protein expression. (C) Myf-5 protein expression. (D) Myogenin protein expression. (E) Spry-1 protein expression. (F) Representative Western Blots; *p < 0.05; **p < 0.01 vs. Ctrl.

Fig 5: Knockdown targets of miR132 and miR212 Rasa1, Spred1 and Spry1 mimics effect of miR-132 and miR-212 in HUVECs pericytes neovascularization assay. (A) Representative image from HUVECs and pericytes co-culture assay after transfection with siRNAs against Spred1, Spry1, Rasa1 and combination of these three siRNA. (B) Quantification of the number of tubules, junctions and total tubule length in the HUVECs and pericytes co-culture assay after transfection with siRNAs against Spred1, Spry1 and Rasa1, Values in the graph are shown as mean ± SEM, *P < 0.05; **P < 0.01; n = 3.