Journal: Aging Cell

Article Title: AAV‐Mediated nuclear localized PGC1α4 delivery in muscle ameliorates sarcopenia and aging‐associated metabolic dysfunctions

doi: 10.1111/acel.13961

Figure Lengend Snippet: Ectopic expression of NLS‐PGC1α4 increased myotube sizes in vitro. (a, b) Construction strategy of NLS‐PGC1α4‐GFP plasmid, GFP fluorescence and immunoblotting analysis using GFP antibody assessing the localization of NLS‐PGC1α4‐GFP in C2C12 myoblasts or myotubes transfected with GFP fused NLS‐PGC1α4. Scale bar = 100 μm. (c) H&E staining showing fiber sizes of C2C12 myotubes treated with adenoviral delivery system of Ad‐GFP or Ad‐NLS‐PGC1α4‐GFP (short for Ad‐NLS‐PGC1α4) for 48 h and relative fold of myotube diameter were quantified shown as histogram. n = 3 per group. Scale bar = 100 μm. (d, e) Real time PCR and protein quantification analysis showed Mstn and Igf1 expression of C2C12 myotubes treated with Ad‐GFP and Ad‐NLS‐PGC1α4 for 48 h. n = 4 per group. Data are presented as mean ± SEM and * p < 0.05, ** p < 0.01 compared to control group. Scale bar represents 100um.

Article Snippet: For cocultured and neutralizing antibody assays, mouse IGF1 (4 μg/mL) neutralizing antibodies (R&D Systems, AF791) or IgG (Beyotime, A7028) were added into cultured media consists of conditioned medium and differentiation medium mixed with 1:1 ratio (vol./vol.).

Techniques: Expressing, In Vitro, Plasmid Preparation, Fluorescence, Western Blot, Transfection, Staining, Real-time Polymerase Chain Reaction, Control

Journal: Aging Cell

Article Title: AAV‐Mediated nuclear localized PGC1α4 delivery in muscle ameliorates sarcopenia and aging‐associated metabolic dysfunctions

doi: 10.1111/acel.13961

Figure Lengend Snippet: AAV‐mediated NLS‐PGC1α4 ameliorates aging‐induced dysfunction in muscles. (a–g) Phenotypical and molecular analysis of muscles from aged mice (18 months‐old) administrated locally in GAS with AAV delivery system of AAV‐GFP or AAV‐NLS‐PGC1α4‐GFP (short for AAV‐NLS‐PGC1α4) for 8 weeks. n = 6 per group. (a) Increase of percentage of lean mass; (b) grip strength; (c) weights of GAS muscles; (d) representative H&E staining and percentage fibers with central nuclei; (e) quantifications of muscle fiber sizes distribution; (f) mRNA expression levels of Igf1 , Mstn , Atrogin‐1 and MuRF‐1 ; (g) protein levels and quantification of IGF1, MSTN, MAFbx and MuRF‐1 in GAS muscle. Data are presented as mean ± SEM and * p < 0.05, ** p < 0.01 compared to control group. Scale bar represents 100um.

Article Snippet: For cocultured and neutralizing antibody assays, mouse IGF1 (4 μg/mL) neutralizing antibodies (R&D Systems, AF791) or IgG (Beyotime, A7028) were added into cultured media consists of conditioned medium and differentiation medium mixed with 1:1 ratio (vol./vol.).

Techniques: Muscles, Staining, Expressing, Control

Journal: Aging Cell

Article Title: AAV‐Mediated nuclear localized PGC1α4 delivery in muscle ameliorates sarcopenia and aging‐associated metabolic dysfunctions

doi: 10.1111/acel.13961

Figure Lengend Snippet: IGF1 and METRNL mediates beneficial effects of NLS‐PGC1α4 on muscle and adipose tissue functionality in aged mice. (a–d) Analysis of aged mice locally administrated with AAV‐GFP or AAV‐NLS‐PGC1α4 in GAS. n = 6 per group. (a) Volcano plot showing the differential genes in gastrocnemius muscle between AAV‐NLS‐PGC1α4 and AAV‐GFP group mice; (b) The serum METRNL levels; (c, d) The protein levels of pmTOR, tmTOR, pP70S6K, P70S6K, pAKT308, pAKT473, tAKT, β‐actin and quantifications in QU and TA muscles. (e) IGF1 protein contents in culture medium from myotubes treated with Ad‐GFP or Ad‐NLS‐PGC1α4; (f, g) Representative MYHC immunofluorescence staining and relative diameter of myotubes treated with culture medium from Ad‐GFP or Ad‐NLS‐PGC1α4 with or without Anti‐IGF1 neutralizing antibody treated myotubes. (h, i) The mRNA levels of Metrnl and the protein levels and quantification of PGC1α4, METRNL and β‐actin in GAS muscles; (j) METRNL protein contents in serum of aged mice locally administrated with AAV‐GFP or AAV‐NLS‐PGC1α4‐GFP in GAS; (k, l) The mRNA levels of Il4 , Il13 , M2 macrophage genes Arg1 , Mrc1 , Clec10a , Retnla (k) and thermogenic genes Ucp1, Cidea, Elovl3 (l) in iWAT from aged mice locally administrated with AAV‐GFP or AAV‐NLS‐PGC1α4‐GFP in GAS and followed by intraperitoneal injection (i.p.) of Anti‐Metrnl neutralizing antibody every other day for 2 weeks.

Article Snippet: For cocultured and neutralizing antibody assays, mouse IGF1 (4 μg/mL) neutralizing antibodies (R&D Systems, AF791) or IgG (Beyotime, A7028) were added into cultured media consists of conditioned medium and differentiation medium mixed with 1:1 ratio (vol./vol.).

Techniques: Muscles, Immunofluorescence, Staining, Injection

Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) qRT/PCR showing that mGSRR cells display increased Cdh2 and decreased Cdh1 mRNA expression compared with mGS. Two-tailed Student’s t test. (B) Western blot showing expression of Slug, Snail1, and Zeb1 are gradually increased upon repeated irradiation in mGS cells. (C) Western blot showing that Snail overexpression induces elevation of N-cad, Olig2, and Zeb1, and suppression of Tuj1 in mGS cells. (D) Wnt/β-catenin transcriptional activity is suppressed in mGSRR and mGS with Snail1 overexpression (OE) compared with mGS cells. ***P < 0.001, Tukey’s HSD test. (E) Clonogenic survival assay shows mGS Snail1 OE cells have a higher survival rate than mGS cells. Two-tailed Student’s t test. *P < 0.05, **P < 0.01. (F) Western blot showing increased N-cad, β-catenin, Slug, and Zeb1 expression 2 days after mouse recombinant IGF1 (100 ng/mL), but not TGF-β1 (10 ng/mL) treatment in mGS cells. (G) Western blot showing IGF1 overexpression increases N-cad, β-catenin, Zeb1, and IGF1R expression in mGS cells. (H) Survival curves for mice implanted with 1000 cells (GS with IGF1 expression vector) and subjected to whole-brain irradiation (2 Gy/day, days 3 to 7, 10 Gy total). (I) Left: schematic showing experimental design for clonogenic survival assay with repeated irradiation. Single mGS cells seeded in agarose medium were exposed to repeated irradiation (5 doses of 4 Gy, every 3 days) with or without drug rescue. IGF1R (AEW541 0.5 μM; PPP 0.2 μM) and TGF-β1 (LY2157299 10 μM, SB431542 10 μM) inhibitors were used. Right: quantification of percentage of surviving colonies shows that IGF1R inhibitors selectively decreased survival rate. Drugs alone had no effect on colony formation (data not shown). ***P < 0.001, Dunnett’s test. (J) Mice implanted orthotopically with mGSRR cells had a survival benefit after whole-brain irradiation (2 Gy × 5 days) with adjuvant PPP (15 mg/kg, i.p. twice a day from day 3–7) in contrast to vehicle control, only IR or PPP alone (8 mice/group; log-rank test). All blots show representative images (n = 3 or more).

Article Snippet: Cells were treated with recombinant mouse IGF1 (100 ng/mL, R&D Systems, #791-MG), recombinant mouse TGF-β1 (10 ng/mL, R&D Systems, #7666-MB), SB216763 (1–5 μM, Selleckchem, S1075), or CHIR99021 (1–5 μM, Selleckchem, S1263).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Irradiation, Over Expression, Activity Assay, Clonogenic Cell Survival Assay, Recombinant, Plasmid Preparation, Adjuvant