Fig 1: Intratumoral NK-92 cells up-regulate SESN2 and SESN3 expression(A) Representative flow cytometry plots showing CD45+CD56+ NK-92 cells in the spleens, peritoneal cavity and tumor xenografts. NK-92 cells were adoptively transferred into tumor-bearing mice through intraperitoneal injection and were then isolated from indicated tissues based on CD45 and CD56 expression. (B) SESN1, SESN2 and SESN3 protein levels in NK-92 populations isolated from different tissues. C: in vitro cultured NK-92 cells. Sp-NK: NK-92 cells isolated from spleens. Pe-NK: NK-92 cells isolated from peritoneal cavity. T-NK C56hi: intratumoral NK-92 cells expressing high CD56. T-NK CD56dim: intratumoral NK-92 cells expressing dim CD56. (C) Statistics for (B). N= 5 per group. *, p<0.05; ***, p<0.001.
Fig 2: Circulating miR-16-5p is derived from the atrophic-limbs and hearts of sarcopenic mice and interferes with restoration of LV dysfunction in I/R mice. a, qRT-PCR analysis of the expression of miR-16-5p in the brain, liver, limbs, heart, lung, aorta, pancreas, stomach, bone marrow, kidney, and prostate of mice subjected [TS (+)] or not [TS (-)] tail-suspension (all; n = 5). The ratio of miR-16-5p expression in TS (+) mice was calculated compared that in TS (-) mice (standardized to the expression level of 1). Light gray bar; TS (-), Dark gray bar; TS (+). (b) Absolute changes in the LVEF on days 8 in miR-16-5p mimic (n = 6) and control-miR mimic (n = 6), *p < 0.05. (c) ?LVEF (Day 8–Day 1) in the 2 groups of mice. Left bar; control-miR mimic, Right bar; miR-16-5p mimic. (d) Schematic image of the proposed mechanism. Cardio-repair disturbance in the context of sarcopenia is mediated by exosomal-miR-16-5p secreted from the atrophic limbs and hearts. miR-16-5p directly interferes with the transcription of SESN1, and then activates mTOR signaling, which in turn induces cell apoptosis.
Fig 3: Suppression of the autophagy in NRVMs via the miR-16-5p-mediated transcriptional suppression of SESN1. (a) Target/binding site of SESN1 and miR-16-5p, and Sesn1 mutation sequence. (b) Luciferase assay; SESN1 contains a target gene of mir-16-5p. Left side; Sesn1-3'UTR-wild type, Right side; Sesn1-3'UTR-mutant, Gray bar; miR-16-5p mimic (n = 4), Light gray bar; miR-mimic control (n = 4), (c) Representative blots of SESN1, phosphorylated-mTOR, mTOR, and ß-actin protein expression in NRVMs. Left side; normoxia. Center; hypoxia. Right side; hypoxia with miR-16-5p mimic transfection. (d) Fold change of SESN1 activation in NRVMs under hypoxia with (n = 14) and without miR-16-5p mimic transfection (n = 12); the baseline refers to NRVMs under normoxia (n = 13). (e) Fold change of mTOR-phosphorylation in NRVMs under hypoxia with (n = 6) and without miR-16-5p mimic transfection (n = 5); the baseline refers to NRVMs under normoxia (n = 6). Left bar; normoxia. Center bar; hypoxia. Right bar; hypoxia with miR-16-5p transfection. (f) Representative images of autophagosomes in NRVMs (All; n = 4) [monodansylcadaverine (MDC); green]; the nuclei were stained with DAPI [blue]. Left side; normoxia. Center; hypoxia. Right side; hypoxia with miR-16-5p transfection. The upper panel is a low magnitude image. The lower panel is an expanded image of autophagosomes and DAPI-positive cells. Yellow arrow; representative autophagosome-positive cells. Scale bar = 50 µm. (g) The ratio of autophagosome positive NRVMs (All; n = 4). Left bar; normoxia. Center bar; hypoxia. Right bar; hypoxia with miR-16-5p transfection. The percentage of autophagosome positive NRVMs in all NRVMs is represented. Western blotting (h) and analysis of the protein expression of LC3B-II (i) in NRVMs (normo n = 3, the others; n = 4; respectively) transfected with miR-16-5p mimic was also evaluated. Left bar; normoxia. Center bar; hypoxia. Right bar; hypoxia with miR-16-5p mimic transfection.
Supplier Page from Abcam for Anti-SESN1 antibody [EPR1930(2)]