Journal: Science advances

Article Title: Bcl-xL overexpression in T cells preserves muscle mitochondrial structure and function and prevents frailty in old mice.

doi: 10.1126/sciadv.adr1378

Figure Lengend Snippet: Fig. 1. Bcl-xL overexpression alters T cell populations in Lckpr-bcl-xL mice across aging. (A) Total splenic cell counts and (B) T cell count obtained from the spleens of C57BL6/J and Lckpr-bcl-xL mice of all age groups (n = 15 to 20). (C) CD3+hi and CD3+lo levels of splenic T cells from C57BL6/J and Lckpr-bcl-xL mice of all age groups (n = 15). mo, months. (D) Distribution of T helper and cytotoxic T cells, (E) distribution of naïve/memory/effector within T helper cells, and (F) distribution of naïve/ memory/effector within cytotoxic T cells in C57BL6/J and Lckpr-bcl-xL mice of all age groups (n = 8 to 10). Data are presented as the means ± SD.

Article Snippet: 293T cells were cotransfected with the Bcl- xL expression vector pCDH- puro- BCL- XL (plasmid no. 46972) or control vector pLKO.1- puro (plasmid no. 8453) (both from Addgene) along with the lentiviral packing plasmids ENV VSV- G, REV, and pMDLg/ pRRR (III generation packing), as described before (61).

Techniques: Over Expression, Cell Counting

Journal: Science advances

Article Title: Bcl-xL overexpression in T cells preserves muscle mitochondrial structure and function and prevents frailty in old mice.

doi: 10.1126/sciadv.adr1378

Figure Lengend Snippet: Fig. 2. Bcl-xL improves metabolic performance, induces apoptosis resistance of T cells, and modulates cytokine production. (A) Proton leak, (B) basal respiration, and (C) NMOC of splenic T cells from C57BL6/J and Lckpr-bcl-xL mice of all age groups (n = 8 to 12). (D) Lipid peroxidation of splenic T cells from C57BL6/J and Lckpr-bcl-xL mice of all age groups (n = 5 to 9). h, hours. (E) Intrinsic apoptosis (caspase 9) of isolated T cells and (F) stress-induced extrinsic apoptosis (annexin V) of T cells cultured in vitro during 48 hours under control and dexamethasone (100 nM) or upon serum removal (n = 5 to 6). a.u., arbitrary units. (G) Cytokine production (IL-1β, IL-6, TNFα, and IFN-γ) by cultured T cells upon 24 hours of stimulation with PMA/ionomycin (n = 4 to 5). Data are presented as the means ± SD.

Article Snippet: 293T cells were cotransfected with the Bcl- xL expression vector pCDH- puro- BCL- XL (plasmid no. 46972) or control vector pLKO.1- puro (plasmid no. 8453) (both from Addgene) along with the lentiviral packing plasmids ENV VSV- G, REV, and pMDLg/ pRRR (III generation packing), as described before (61).

Techniques: Isolation, Cell Culture, In Vitro, Control

Journal: Science advances

Article Title: Bcl-xL overexpression in T cells preserves muscle mitochondrial structure and function and prevents frailty in old mice.

doi: 10.1126/sciadv.adr1378

Figure Lengend Snippet: Fig. 3. Bcl-xL boosts macroautophagy under starvation conditions. (A) Bcl-xL lysosomal degradation in MEFs maintained in the presence or absence of serum during 24 hours (n = 6 independent experiments). (B) Bcl-xL effect on macroautophagy measured in NIH3T3 mouse fibroblasts stably expressing Bcl-xL or the pLKO1 control vector and transfected with the macroautophagy reporter (mCherry-GFP-LC3). Cells were maintained in serum-supplemented (+) or serum-deprived (−) culture media. Yellow puncta (mCherry+/GFP+) represent autophagosomes, and red puncta (mCherry+/GFP−) represent autolysosomes (n = 9 fields with n > 15 cells). (C to E) Immuno- fluorescence for LAMP1 (C), LC3-II (D), and LysoTracker staining (E) in NIH3T3 cells stably expressing Bcl-xL or the pLKO1 vector control and cultured in the presence (+) or absence (−) of serum (n = 10 to 15 fields with n > 10 cells). (F and G) LC3-II levels of splenic T cells from (F) old and (G) very old C57BL6/J and Lckpr-bcl-xL mice cultured in the presence or absence of serum (n = 3 to 4). Data are presented as the means ± SD.

Article Snippet: 293T cells were cotransfected with the Bcl- xL expression vector pCDH- puro- BCL- XL (plasmid no. 46972) or control vector pLKO.1- puro (plasmid no. 8453) (both from Addgene) along with the lentiviral packing plasmids ENV VSV- G, REV, and pMDLg/ pRRR (III generation packing), as described before (61).

Techniques: Stable Transfection, Expressing, Control, Plasmid Preparation, Transfection, Fluorescence, Staining, Cell Culture

Journal: Science advances

Article Title: Bcl-xL overexpression in T cells preserves muscle mitochondrial structure and function and prevents frailty in old mice.

doi: 10.1126/sciadv.adr1378

Figure Lengend Snippet: Fig. 4. Bcl-xL overexpression modulates Treg distribution and promotes Treg infiltration in skeletal muscle to reduce IFN-γ inflammation. Percentage of FoxP3+ Treg cells in (A) whole blood and in (B) spleens from C57BL6/J and Lckpr-bcl-xL mice of all age groups (n = 4 to 5). (C) Representative images of CD3+, FoxP3+, and DAPI staining in muscle samples from C57BL6/J and Lckpr-bcl-xL mice of all age groups. Quantification of (D) infiltrated CD3+ cells and (E) FoxP3+ cells and (F) ratio of FoxP3+/CD3+ (n = 3 fields with n > 50 cells). (G) IFN-γ levels measured in plasma from C57BL6/J and Lckpr-bcl-xL mice (n = 6). Data are presented as the means ± SD.

Article Snippet: 293T cells were cotransfected with the Bcl- xL expression vector pCDH- puro- BCL- XL (plasmid no. 46972) or control vector pLKO.1- puro (plasmid no. 8453) (both from Addgene) along with the lentiviral packing plasmids ENV VSV- G, REV, and pMDLg/ pRRR (III generation packing), as described before (61).

Techniques: Over Expression, Staining, Clinical Proteomics

Journal: Science advances

Article Title: Bcl-xL overexpression in T cells preserves muscle mitochondrial structure and function and prevents frailty in old mice.

doi: 10.1126/sciadv.adr1378

Figure Lengend Snippet: Fig. 5. Mice overexpressing Bcl-xL in T cells display improved muscle regeneration following BaCl2 injury. (A) Diagram of the study design. (B) Representative im- ages of hematoxylin and eosin staining and quantification of the mean muscle fiber diameter from C57BL6/J and Lckpr-bcl-xL mice treated with PBS or BaCl2 (n = 5 fields with n > 100 fibers). (C) Representative images of DAPI and α-tubulin staining and quantification of central nuclei in muscle fibers from C57BL6/J and Lckpr-bcl-xL mice treated with PBS or BaCl2 (n = 5 fields with n > 100 fibers). Nascent myotubes with central nuclei are highlighted in red. Mature myotubes with central nuclei are indicated with red arrows. Data are presented as the means ± SD. Abbreviation: dpi, days postinjection.

Article Snippet: 293T cells were cotransfected with the Bcl- xL expression vector pCDH- puro- BCL- XL (plasmid no. 46972) or control vector pLKO.1- puro (plasmid no. 8453) (both from Addgene) along with the lentiviral packing plasmids ENV VSV- G, REV, and pMDLg/ pRRR (III generation packing), as described before (61).

Techniques: Staining

Journal: Science advances

Article Title: Bcl-xL overexpression in T cells preserves muscle mitochondrial structure and function and prevents frailty in old mice.

doi: 10.1126/sciadv.adr1378

Figure Lengend Snippet: Fig. 6. Mice overexpressing Bcl-xL in T cells maintain the muscle mitochondrial structure and function and prevent oxidative damage during aging. (A) Repre- sentative electron microscopy images of muscle mitochondria from C57BL6/J and Lckpr-bcl-xL mice at the indicated age ranges. (B) Quantification of the mitochondrial structure as of mitochondrial outer membrane integrity and crista density (n = 7 fields with n > 20 mitochondria). (C) TFAM protein levels (n = 7) and mitochondrial count in muscle samples of C57BL6/J and Lckpr-bcl-xL mice of all age groups (n = 3 to 4 fields with n > 200 mitochondria). (D) Muscle mitochondrial bioenergetics of adult and old C57BL6/J and Lckpr-bcl-xL mice (n = 5). (E) MDA levels and (F) protein carbonylation levels of muscle samples of C57BL6/J and Lckpr-bcl-xL mice of all age groups (n = 7). Data are presented as the means ± SD.

Article Snippet: 293T cells were cotransfected with the Bcl- xL expression vector pCDH- puro- BCL- XL (plasmid no. 46972) or control vector pLKO.1- puro (plasmid no. 8453) (both from Addgene) along with the lentiviral packing plasmids ENV VSV- G, REV, and pMDLg/ pRRR (III generation packing), as described before (61).

Techniques: Electron Microscopy, Membrane

Journal: Science advances

Article Title: Bcl-xL overexpression in T cells preserves muscle mitochondrial structure and function and prevents frailty in old mice.

doi: 10.1126/sciadv.adr1378

Figure Lengend Snippet: Fig. 7. Mice overexpressing Bcl-xL in T cells display increased grip strength and motor coordination, making them less frail. (A) Grip strength loss, (B) motor coor- dination loss, and (C) exercise endurance loss of 8 to 10 (n = 135)–, 17 to 20 (n = 116)–, and >24 (n = 34)–month-old C57BL6/J and Lckpr-bcl-xL mice. Reference values are C57BL6/J and Lckpr-bcl-xL mice of 2 to 5 months old (n = 135). There were no differences in the absolute values of grip strength, motor coordination, or exercise endurance across the two young animal groups. (D) Prevalence of frailty in >24-month-old C57BL6/J and Lckpr-bcl-xL mice. C57BL6/J nonfrail (n = 9), prefrail (n = 6), and frail (n = 2); Lckpr-bcl-xL nonfrail (n = 17), prefrail (n = 1), and frail (n = 0). Data are presented as mosaic plots, and P = 0.01244 was calculated with Fisher’s exact test. (E) Valencia frailty score in >24-month-old C57BL6/J (n = 17) and Lckpr-bcl-xL (n = 18) mice. (F) Representative images of hematoxylin and eosin staining of muscle fibers from C57BL6/J and Lckpr-bcl-xL mice of all age groups. (G) Quantification of the mean cross-sectional area of muscle fibers (n = 4 fields with n > 100 fibers) and (H) total protein content (n = 7) in the muscle of C57BL6/J and Lckpr-bcl-xL mice of all age groups. Data are presented as the means ± SD.

Article Snippet: 293T cells were cotransfected with the Bcl- xL expression vector pCDH- puro- BCL- XL (plasmid no. 46972) or control vector pLKO.1- puro (plasmid no. 8453) (both from Addgene) along with the lentiviral packing plasmids ENV VSV- G, REV, and pMDLg/ pRRR (III generation packing), as described before (61).

Techniques: Staining

Journal: Science advances

Article Title: Bcl-xL overexpression in T cells preserves muscle mitochondrial structure and function and prevents frailty in old mice.

doi: 10.1126/sciadv.adr1378

Figure Lengend Snippet: Fig. 8. Mice overexpressing Bcl-xL in T cells are more metabolically competent at old ages. (A) Body weight gain of 8 to 10 (n = 135)–, 17 to 20 (n = 116)–, and >24 (n = 34)–month-old C57BL6/J and Lckpr-bcl-xL mice. Reference values are C57BL6/J and Lckpr-bcl-xL mice of 2 to 5 months old (n = 135). (B) Spontaneous activity measured as horizontal activity in C57BL6/J (n = 6) and Lckpr-bcl-xL (n = 9) mice over 42 hours at the indicated ages. (C) Food and drink consumption in C57BL6/J and Lckpr-bcl-xL mice at the indicated ages (n = 5 to 7). (D) Regression curves for total body mass, fat mass, and lean mass of C57BL6/J and Lckpr-bcl-xL mice during aging (n = 15 to 20 per group). Glucose tolerance test: glycemia evolution and glycemia area under the curve (AUC) analysis in C57BL6/J and Lckpr-bcl-xL mice of (E) 6 to 14 months (n = 10) and (F) 15 to 23 months (n = 9). Data are presented as the means ± SD. (G) Rendition of the findings reported.

Article Snippet: 293T cells were cotransfected with the Bcl- xL expression vector pCDH- puro- BCL- XL (plasmid no. 46972) or control vector pLKO.1- puro (plasmid no. 8453) (both from Addgene) along with the lentiviral packing plasmids ENV VSV- G, REV, and pMDLg/ pRRR (III generation packing), as described before (61).

Techniques: Metabolic Labelling, Activity Assay