Journal: Journal of Translational Medicine

Article Title: Secretion of miRNA-326-3p by senescent adipose exacerbates myocardial metabolism in diabetic mice

doi: 10.1186/s12967-022-03484-7

Figure Lengend Snippet: Removal of epididymal adipose tissue alleviates diastolic dysfunction in STZ mice. A Representative images of senescence associated beta-galactosidase (SA-β-gal) staining of EAT. B Detection of SA-β-gal positive cells from Con- or STZ-EAT (n = 3 per group). C Relative gene expression of senescence associated genes in EAT from Con- or STZ-mice (n = 3 per group). D Experimental design of EAT removal surgery. E Representative of doppler and tissue doppler echocardiography and evaluation of diastolic function (n = 6 per group). F Sarcomere length tracing of isolated murine cardiomyocytes using an Ionoptix HTS system. Evaluation of G myocardial contraction, H fraction shortening and I calcium handling in Langendorff-isolated adult mouse ventricular myocytes (AMVMs) (n = 5 per group). J Representative micrographs of heart tissue sections examined by transmission electron micrographs. Data are presented as the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to Con group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared to STZ + EATr group

Article Snippet: Briefly, adipose tissue chunks were collected in PBS, fixed with fixative solution (Solarbio) for 15 min. Then, the adipose tissue chunks were washed 3 times in PBS and placed in SA-β-gal activity solution (Solarbio).

Techniques: Staining, Expressing, Isolation, Transmission Assay

Journal: Journal of Translational Medicine

Article Title: Secretion of miRNA-326-3p by senescent adipose exacerbates myocardial metabolism in diabetic mice

doi: 10.1186/s12967-022-03484-7

Figure Lengend Snippet: LEVs isolated from senescent EAT in diabetic mice drives contractile and mitochondrial dysfunction in NMVMs. A Representative transmission electron micrograph of isolated LEVs. B Representative micrograph of EAT derived LEVs (PKH67-labeled, green) co-cultured with NMVMs. C Contractile velocity of untreated (UT) or NMVMs treated with Con-EAT LEVs or STZ-EAT LEVs (n = 6 per group). D Representative micrograph and E quantification of untreated (UT) and NMVMs treated with Con-EAT LEVs or STZ-EAT LEVs stained for mitochondrial membrane potential (TMRM), mitochondrial superoxide (MitoSox), and DAPI (n = 5 per group). F Real-time oxygen consumption rates (OCR) were evaluated for untreated (UT) or NMVMs treated with Con-EAT LEVs or STZ-EAT LEVs; basal and maximal respiration rates are shown (n = 8–9 per group). G Percentage of SA-β-gal positive cells in STZ-EAT cultured ex vivo in presence of Seno or Veh (n = 3 per group). H Relative gene expression of senescence associated genes in STZ-EAT cultured ex vivo in presence of Seno or Veh. (n = 3 per group). I Contractile velocity, J mitochondrial membrane potential and mitochondrial superoxide relative intensity fluorescence of NMVMs treated with LEVs from STZ-EAT cultured ex vivo in presence of Seno or Veh (n = 5–6 per group). (K) Real-time oxygen consumption rates (OCR) were evaluated for NMVMs treated with LEVs from STZ-EAT cultured ex vivo in presence of Seno or Veh, basal and maximal respiration rates are shown (n = 8–9 per group). Data are presented as the mean ± SEM; *P < 0.05, ***P < 0.001, ****P < 0.0001 compared to UT group; ## P < 0.01, ### P < 0.001, #### P < 0.0001 compared to Con-EAT LEVs group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 compared to Veh-STZ-EAT LEVs group

Article Snippet: Briefly, adipose tissue chunks were collected in PBS, fixed with fixative solution (Solarbio) for 15 min. Then, the adipose tissue chunks were washed 3 times in PBS and placed in SA-β-gal activity solution (Solarbio).

Techniques: Isolation, Transmission Assay, Derivative Assay, Labeling, Cell Culture, Staining, Ex Vivo, Expressing, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Secretion of miRNA-326-3p by senescent adipose exacerbates myocardial metabolism in diabetic mice

doi: 10.1186/s12967-022-03484-7

Figure Lengend Snippet: Senolytic treatment alleviates cardiac function in STZ mice. A Experiment design for senolytic treatment in vivo. B Representative images and C quantification of (SA-β-gal) staining of EAT isolated from Veh or Seno treated STZ-mice (n = 3 per group). D Relative gene expression of senescence associated genes in Veh or Seno treated STZ-EAT (n = 3 per group). E Representative of doppler and tissue doppler echocardiography and evaluation of diastolic function (n = 6 per group). F Contractile function and G calcium handling of AMVMs isolated from Veh or Seno treated STZ-mice were evaluated (n = 5 per group). H Representative transmission electron micrographs of Veh or Seno treated STZ-mice hearts. Relative miRNA-326-3p expression in I blood and J AMVMs from Veh or Seno treated STZ-mice (n = 4–5 per group). K Protein levels of Rictor, p-AKT, AKT and Gapdh in AMVMs isolated from Veh or Seno treated STZ-mice evaluated by immunoblotting (n = 3). Data are presented as the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to Veh group

Article Snippet: Briefly, adipose tissue chunks were collected in PBS, fixed with fixative solution (Solarbio) for 15 min. Then, the adipose tissue chunks were washed 3 times in PBS and placed in SA-β-gal activity solution (Solarbio).

Techniques: In Vivo, Staining, Isolation, Expressing, Transmission Assay, Western Blot

Journal: bioRxiv

Article Title: Senescent Schwann cells induced by aging and chronic denervation impair axonal regeneration after peripheral nerve injury

doi: 10.1101/2022.12.07.519441

Figure Lengend Snippet: (A) Representative IF images of sciatic nerves 7 days after reconnection surgery (7dpi) in aged, adult and chronically denervated (42dpi) (SCG10, green). Scale bar, 500uM. (B) Axonal density and distance comparison 7 days after reconnection surgery (7dpi) in aged, adult and chronically denervated (42dpi) animals and IF of reconnected nerves. (C, E) Brightfield and fluorescence confocal acquisition of β-galactosidase activity on longitudinal sections of contralateral non-injured and chronically denervated (42dpi) sciatic nerves of adult and aged animals after b-galactosidase assay. (B-gal: magenta, DAPI: blue). (n=5 animals per group, p<0.05 by Student’s t-test compared between conditions; error bars indicate S.D.) (D) c-Jun activation in the nucleus of SCs on l ongitudinal c ryostat sections of adult and aged mice sciatic nerves in nondamaged animals (ctrl), 7 and 42 dpi (n=5 animals per group, p<0.05 by Student’s t-test compared between conditions; error bars indicate S.D.). Scale bar, 100 μM. (F-H) qRT-PCR and fluorescence quantification of p16INK4a in contralateral noninjured and chronically denervated (42dpi) sciatic nerves of adult and aged. Scale bar 50 μM. (I-J) Fluorescence confocal acquisition of β-galactosidase activity on longitudinal sections of injured (7dpi) sciatic nerves of adult WT and c-Jun OE animals after b-galactosidase assay. Scale bar 50 μM. (B-gal: magenta, DAPI: blue). (K-L) qRT-PCR and fluorescence quantification of p16INK4a in chronically denervated (42dpi) sciatic nerves of adult WT and c-Jun OE animals. Scale bar 50 μM. (n=5 animals per group, p<0.05 by Student’s t-test compared between conditions; error bars indicate S.D.).

Article Snippet: Cells were fixed on 4% PFA and senescence determination was made by measuring SA-β-gal activity (KAA02 Merck) and IF against the markers of senescence: p16, p21, histone y-H2AX, HMGB1, lamin-b1 and P19arf in parallel to S100 SCs marker (Table 4).

Techniques: Fluorescence, Activity Assay, Activation Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Senescent Schwann cells induced by aging and chronic denervation impair axonal regeneration after peripheral nerve injury

doi: 10.1101/2022.12.07.519441

Figure Lengend Snippet: (A-B) Confocal microscopy and graph comparison of β-galactosidase activity in adult chronically denervated (42dpi) or aged (19dpi) animals treated with ABT-263 compared with vehicle treatment, (β-gal: magenta, DAPI: blue). (n=3-5 animals per group, p<0.05 by Student’s t-test compared between conditions; error bars indicate S.D. Scale bar, 100 μM. (C-H) Representative IF confocal images and graph comparison of p16INK4a (green), γ-H2AX (red) and c-Jun (red) positive SCs (SOX10= magenta or green) on longitudinal cryostat sections of adult chronically denervated (42dpi) or aged (19dpi) animals, treated with ABT-263 compared with vehicle treatment. (DAPI= blue) (n=3-5 animals per group, p<0.05 by Student’s t-test compared between conditions; error bars indicate S.D.). Scale bars, 50 μM.

Article Snippet: Cells were fixed on 4% PFA and senescence determination was made by measuring SA-β-gal activity (KAA02 Merck) and IF against the markers of senescence: p16, p21, histone y-H2AX, HMGB1, lamin-b1 and P19arf in parallel to S100 SCs marker (Table 4).

Techniques: Confocal Microscopy, Activity Assay