Rapamycin treatment affects the inflammatory phase of the cardiac regeneration process. Zebrafish were treated with rapamycin via intraperitoneal injection two days before the amputation procedure and every second day afterwards (a). The effect of rapamycin treatment was verified by the accumulation of Beclin-1 and Lc3 after 9 doses. Results were obtained from at least three biological replicates. Signal levels were normalized first to GAPDH-loading controls and then compared to the control treatment. (fc: fold change). Full-length blots representative of the results can be found in the Supplementary Information (Supp. Fig. 3). Upon amputation, rapamycin-treated zebrafish showed an increased number of apoptotic TUNEL+-nuclei in the affected ventricle area (c), as well as an increased presence of mpx+-neutrophils (d). Also, mpeg1+-macrophage recruitment at 5 dpa was affected by the treatment, as shown by their decreased presence of the affected area (e, % area covered by macrophages). Altogether, the results suggest an alteration in the inflammatory mechanisms triggered upon apex amputation. The results obtained from the quantification of all fluorescent signals were normalized to the area of the regenerating tissue considered, which was delimited by the amputation plane on the ventricle (white discontinuous line). Scale bars represent 50 μm. TUNEL+: Control N ≥ 14, Rapamycin N ≥ 13; mpx+: Control N ≥ 14, Rapamycin N ≥ 16; mpeg1+: Control N ≥ 8, Rapamycin N ≥ 9. *p ≤ 0.05, **p ≤ 0.001.
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