Journal: bioRxiv

Article Title: IPMK Deficiency Reduces Skeletal Muscle Oxidative Metabolism and Exercise Capacity

doi: 10.1101/2024.07.28.605526

Figure Lengend Snippet: Primary myoblasts were isolated from Ipmk -loxp mice and treated with either Ad-GFP or Ad-Cre. Then cells were differentiated into myotubes. A . Cell lysates were analyzed by Immunoblotting. B . Cells were pre-incubated in glucose-free buffer and incubated with 25 mM glucose for an indicated period of time. C . Cells were serum starved for 18 hrs and glucose uptake was measured in the presence of 100 nM insulin. D . Cells were serum starved for 18 hrs and were treated with 100 nM insulin for an indicated period of time. Data are mean +/-SEM, n=8. *p<0.05. Representative images from three independent trials for immunoblotting.

Article Snippet: Immunoblotting was conducted with the following antibodies: IPMK from Novus Biologicals and pAMPK, AMPK, pAkt-S473, Akt, nSREBP, Myogenin, GAPDH from Cell Signaling Technology.

Techniques: Isolation, Western Blot, Incubation

Journal: Redox Biology

Article Title: CCL5 via GPX1 activation protects hippocampal memory function after mild traumatic brain injury

doi: 10.1016/j.redox.2021.102067

Figure Lengend Snippet: Memory impairment and recovery in WT and CCL5-KO mice after mTBI. (A) The time table of experimental design for mild TBI induction and memory-cognition function tests – novel object recognition (NOR) and Barnes maze (BM). (B) The preference index of WT and KO mice for new objects in sham and mTBI groups after weight-drop induced mild TBI. Tests were performed on post injury days 4，7，14，and 28. (a，p < 0.05，aa，p < 0.01，aaa，p < 0.001 compared to sham group with t -test; b, p < 0.05, WT 28 days compared to 7 days; *，p < 0.05, WT compared to KO at the same time point with t -test.) (C) The walking path length in WT and KO mice. (ns: no significant difference). Data is shown as mean ± S.E.M (n = 9 for each group). Spatial memory was analyzed by Barnes maze as the latency to reach the shelter box (Primary latency). (D, E) Recall memory was performed after 2 days (short-term, SM), 7 days (long-term, LM) of training and 2 days after weight-drop induced TBI. (F–G) New learning-memory tasks were taken after 4 days and 28 days of injury. Data was analyzed by Two-way ANOVA and presented as mean ± S.E.M. (*, p = 0.0432 compared to KO-TBI 4 dpi BM in 1F by two-way ANOVA; p = 0.0353 in Trial 4 compare to KO-TBI 4 dpi BM in 1F by t -test.) (n = 5, in each group).

Article Snippet: 24hr after TBI, mice were sacrificed, perfused and CCL5 immunostaining was performed with anti-CCL5 antibody (1:100, sc-365826, Santa Cruz biotechnology, Dallas, USA) and donkey anti-rabbit-488 (1:400, Invitrogen, A32790).

Techniques:

Journal: Redox Biology

Article Title: CCL5 via GPX1 activation protects hippocampal memory function after mild traumatic brain injury

doi: 10.1016/j.redox.2021.102067

Figure Lengend Snippet: Neuron damage and neuron numbers in hippocampus 28 days after injury. (A) Representative images of FJC labeling (green) in WT and CCL5-KO hippocampus. (C) Quantitation of FJC positive cells in WT and CCL5 KO mouse sham and mTBI groups. Increased FJC positive neurons were found in both mTBI groups. (n = 3 in each group). (B) Representative images of NeuN labeled (red) neurons in WT and CCL5-KO mouse hippocampus. (D) Quantitation of NeuN positive cells in different groups. NeuN positive neurons were significantly lower in the CCL5-KO mTBI group. (Data was analyzed by t -test and presented as mean ± S.E.M.) (n = 3–5 as showed in figures). Scale bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 24hr after TBI, mice were sacrificed, perfused and CCL5 immunostaining was performed with anti-CCL5 antibody (1:100, sc-365826, Santa Cruz biotechnology, Dallas, USA) and donkey anti-rabbit-488 (1:400, Invitrogen, A32790).

Techniques: Labeling, Quantitation Assay

Journal: Redox Biology

Article Title: CCL5 via GPX1 activation protects hippocampal memory function after mild traumatic brain injury

doi: 10.1016/j.redox.2021.102067

Figure Lengend Snippet: Hypoxyprobe labeled hypoxic cells colocalized with NeuN positive neurons in WT and CCL5-KO mice at 1 day and 7 days post injury (dpi). Cells with hypoxia were labeled by hypoxyprobe (green) and neurons were labeled by NeuN (red) in both WT and CCL5-KO mice at 1 dpi and 7 dpi as well as sham treatment (s). (A–B) Representative images of hypoxylprobe and NeuN labeling in the hippocampal region of WT and CCL5-KO mice. (C–E) Quantitation of NeuN positive cells (NeuN + , C), hypoxyprobe positive cells (hypoxyprobe + , D) and both hypoxyprobe and NeuN positive cells (hypoxyprobe + NeuN + , E) at post injury day-1 and day-7 and sham in both WT and KO mice. (Data was analyzed by t -test and presented as mean ± S.E.M.) (n = 3 in each group). Scale bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 24hr after TBI, mice were sacrificed, perfused and CCL5 immunostaining was performed with anti-CCL5 antibody (1:100, sc-365826, Santa Cruz biotechnology, Dallas, USA) and donkey anti-rabbit-488 (1:400, Invitrogen, A32790).

Techniques: Labeling, Quantitation Assay

Journal: Redox Biology

Article Title: CCL5 via GPX1 activation protects hippocampal memory function after mild traumatic brain injury

doi: 10.1016/j.redox.2021.102067

Figure Lengend Snippet: NADPH and antioxidant activation in WT and CCL5-KO mouse hippocampus after mild TBI. (A) Reactive oxygen species generation induced by brain trauma and related antioxidants in the scavenger pathway. (B) NADPH oxidase activity in mouse hippocampus tissue was measured at 4 and 7 dpi compared to sham group in WT and CCL5-KO mice. (Data was analyzed by t -test and presented as mean ± S.E.M.); (n = 6 in each group). (C–I) The protein levels of antioxidants - SOD1, SOD2 and GPX1 in mouse hippocampus at 4 dpi and 7 dpi and sham groups were analyzed. (C) The representative images of SOD1, SOD2 and GPX1 protein blots in WT mice and CCL5-KO mice. Quantitative results of SOD1 (D, G), SOD2 (E, H) and GPX1 (F, I) in WT mice and CCL5-KO mice. (Data was analyzed by One-way ANOVA and presented as mean ± S.E.M.) (n = 5 in each group). (J–K) The total GSH production and reduced form of GSH were measured and were lower in CCL5-KO mice compared to WT mice. (Data was analyzed by two-way ANOVA in A and t -test in B. Data are presented as mean ± S.E.M.) Quantitative PCR analysis of GPX1 gene expression in WT and CCL5-KO mice control hippocampus (L) and mice with mTBI at 4 dpi and 7 dpi as well as in sham mice (M). (Data was analyzed by t -test and presented as mean ± S.E.M.)

Article Snippet: 24hr after TBI, mice were sacrificed, perfused and CCL5 immunostaining was performed with anti-CCL5 antibody (1:100, sc-365826, Santa Cruz biotechnology, Dallas, USA) and donkey anti-rabbit-488 (1:400, Invitrogen, A32790).

Techniques: Activation Assay, Activity Assay, Real-time Polymerase Chain Reaction, Gene Expression, Control

Journal: Redox Biology

Article Title: CCL5 via GPX1 activation protects hippocampal memory function after mild traumatic brain injury

doi: 10.1016/j.redox.2021.102067

Figure Lengend Snippet: CCL5 effects on cell viability, intracellular oxidative stress and GPX1 activation after H 2 O 2 treatment. (A–B) Primary cortical neurons cultured from WT and CCL5-KO were treated with CCL5 (10 pg/ml, 50 pg/ml, 100 pg/ml and 250 pg/ml) for 30 min before H- 2 O 2 treatment (0, 25, 250 and 500 μM). Cell viability was detected by MTT assay after 24 h (Data was analyzed by two-way ANOVA and presented as mean ± S.E.M.) (n = 5). (C) Representative images of DCFDA (green) and the neuron marker—Tuj-1 (red) labeling in CCL5 KO primary cortical neurons after 24hr treatment. The quantification of DCFDA labeled cells after CCL5 and H 2 O 2 treatment. (Data was analyzed by t -test and presented as mean ± S.E.M.) (n = 3 in each group.) Scale bar = 100 μm in C. (D–E) SHSY5Y cells transfected with EGFP or CCL5 plasmids were treated with 0, 25, 100, 250 μM H 2 O 2 for 24hr. (D) The viability of EGFP and CCL5-expressing cells was measured by MTT assay. (Data was analyzed by t -test and presented as mean ± S.E.M., N = 3–4) (E) Protein blots of GPX1 and GADPH in EGFP and CCL5-expressing SH5Y5 cells with H 2 O 2 treatment. The quantification of GPX1 protein is shown below protein blots. (F) The CCL5 expression levels after transfection with EGFP or CCL5 plasmids. (G) The protein level of GPX1 in primary cultured neurons after treatment with recombinant CCL5. (Data was presented as mean ± S.E.M., N = 3–4.). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 24hr after TBI, mice were sacrificed, perfused and CCL5 immunostaining was performed with anti-CCL5 antibody (1:100, sc-365826, Santa Cruz biotechnology, Dallas, USA) and donkey anti-rabbit-488 (1:400, Invitrogen, A32790).

Techniques: Activation Assay, Cell Culture, MTT Assay, Marker, Labeling, Transfection, Expressing, Recombinant

Journal: Redox Biology

Article Title: CCL5 via GPX1 activation protects hippocampal memory function after mild traumatic brain injury

doi: 10.1016/j.redox.2021.102067

Figure Lengend Snippet: NAC treatment improved memory impairment seen in CCL5 KO mice after mTBI. (A) Experimental design for NAC treatment in CCL5-KO mice after mild TBI induction and the Barnes maze test. (B) The primary latency of KO mice long-term memory recall (LM) before injury, and after injury with a 2 days recall memory test in sham, TBI, and TBI plus NAC treatment (5 mg, 20 mg) after weight-drop-induced mild TBI. (C) New Barnes maze learning and memory training progression in 4 groups 4 days after injury. (D) The primary latency in the short-term recall memory test (SM) between 4 groups. (F) The intracellular oxidative stress in sham, TBI and TBI plus NAC treatment in KO mouse hippocampus. (G) The quantification of hypoxyprobe and NeuN positive cells (hypoxyprobe + NeuN + ) in the different groups. (H) The mRNA expression of GPX1 after TBI in 4 groups.

Article Snippet: 24hr after TBI, mice were sacrificed, perfused and CCL5 immunostaining was performed with anti-CCL5 antibody (1:100, sc-365826, Santa Cruz biotechnology, Dallas, USA) and donkey anti-rabbit-488 (1:400, Invitrogen, A32790).

Techniques: Expressing

Journal: Redox Biology

Article Title: CCL5 via GPX1 activation protects hippocampal memory function after mild traumatic brain injury

doi: 10.1016/j.redox.2021.102067

Figure Lengend Snippet: CCL5 increased GPX1 mRNA transcription reduced cellular ROS and improved memory impairment seen in CCL5 KO mice after mTBI. (A) Experimental design for CCL5 treatment in CCL5-KO mice after mild TBI induction and the Barnes maze test. (B) The primary latency of KO mice before injury, and after injury with a 2 days recall memory test in mice with PBS treatment and CCL5 administration (300 μg/kg) after weight-drop induced mild TBI. (C) New Barnes maze learning and memory training progression in 2 treatment groups (4 days) after injury. (D) The primary latency in the short-term recall memory test (SM) between 2 treatments. (F) The intracellular oxidative stress in TBI sham, PBS and CCL5 treatment in KO mouse hippocampus. (G) The quantification of hypoxyprobe and NeuN positive cells (hypoxyprobe + NeuN + ) in the different groups. (H) The mRNA expression of GPX1 after TBI with CCL5 treatments.

Article Snippet: 24hr after TBI, mice were sacrificed, perfused and CCL5 immunostaining was performed with anti-CCL5 antibody (1:100, sc-365826, Santa Cruz biotechnology, Dallas, USA) and donkey anti-rabbit-488 (1:400, Invitrogen, A32790).

Techniques: Expressing

Journal: Redox Biology

Article Title: CCL5 via GPX1 activation protects hippocampal memory function after mild traumatic brain injury

doi: 10.1016/j.redox.2021.102067

Figure Lengend Snippet: Summary of CCL5 in GPX1 activation and neuron protection after brain injury. (A) Brain injury induces ROS generation in mouse hippocampus which can be scavenged by the SOD and GPX systems and increase neuron survival with the presence of CCL5. (B) Increased H 2 O 2 produces hydroxyl radical (OH⋅) and hydroxy (OH − ) through the Fenton reaction and causes hippocampal neuron death in the absence of CCL5.

Article Snippet: 24hr after TBI, mice were sacrificed, perfused and CCL5 immunostaining was performed with anti-CCL5 antibody (1:100, sc-365826, Santa Cruz biotechnology, Dallas, USA) and donkey anti-rabbit-488 (1:400, Invitrogen, A32790).

Techniques: Activation Assay

Journal: Protein Engineering, Design and Selection

Article Title: Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections

doi: 10.1093/protein/gzab004

Figure Lengend Snippet: Yield and enrichment of 80- and 641-amino acid linkers in a CD276 system. Yeast displaying either pCT-80 or pCT-641 tethered ligands were mixed with nondisplaying yeast and sorted in parallel by adherent cell panning. (A) Enrichment ratio, (B) ligand-displaying yeast yield and (C) nondisplaying yeast yield were quantified. Ligand: AC2. Cell Line: MDA-MB-231. The dotted line (A) indicates the limit of functional enrichment. *P < 0.05 relative to the 80- amino acid linker

Article Snippet: Beads and cells were separately labeled with either mouse anti-EGFR clone ab30 (4 μg/ml) (Abcam), mouse anti-CD276 clone 185504 (4 μg/ml) (Biotechne) or anti-c-Myc clone 9E10 (4 μg/mL) (BioLegend) for 30 min at 4°C.

Techniques: Functional Assay