Journal: PLoS ONE

Article Title: Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

doi: 10.1371/journal.pone.0231064

Figure Lengend Snippet: Characteristics of sIBM patients.

Article Snippet: The levels of GDF15 and FGF21 were measured using a Quantikine Human GDF15 ELISA Kit and Quantikine Human FGF21 ELISA Kit (R&D Systems, Minneapolis, MN), respectively.

Techniques: Biomarker Discovery

Journal: PLoS ONE

Article Title: Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

doi: 10.1371/journal.pone.0231064

Figure Lengend Snippet: A. Pathological images of the muscle tissue from sIBM patient (patient 4). (a) Hematoxylin-eosin staining of muscle biopsy tissue from a patient with sIBM revealed endomysial inflammatory infiltrates invading nonnecrotic fibers (arrow). (b) Modified Gomori trichrome staining of muscle biopsy tissue from a patient with sIBM revealed rimmed vacuoles (arrow). ( c ) Modified Gomori trichrome staining of muscle biopsy tissue from a patient with sIBM revealed ragged-red fibers (arrow). (d) The absence of cytochrome c oxidase staining in muscle biopsy tissue from a patient with sIBM (arrow). B. Pathological image of muscle tissue from an sIBM patient (patient 4). (a) Abnormal mitochondria with concentric cristae were observed by electron microscopy. Scale bar = 5 μm. (b) Enlarged mitochondria with abnormal cristae and dense granules. Scale bar = 5 μm. C. Measurement of the serum levels of GDF15 and FGF21 in sIBM patients and controls. The data represent the mean ± SEM. * p < 0.05 ( p = 0.015, nonparametric Wilcoxon rank sum test; n = 9 patients and n = 4 controls). D. Representative immunostaining of sIBM patient tissue and normal myoblasts with desmin and MF20. Scale bars = 50 μm. E. Bioenergetic assay of normal and sIBM patient myoblasts (sIBM1) in comparison with the OCR. The data represent the mean ± SD. ** p < 0.01, *** p < 0.001 (unpaired two-tailed Student’s t -test versus normal myoblasts). F. Bioenergetic assay of normal and sIBM patient myoblasts (sIBM1) in comparison with the ECAR. The data represent the mean ± SD.

Article Snippet: The levels of GDF15 and FGF21 were measured using a Quantikine Human GDF15 ELISA Kit and Quantikine Human FGF21 ELISA Kit (R&D Systems, Minneapolis, MN), respectively.

Techniques: Staining, Modification, Electron Microscopy, Immunostaining, Comparison, Two Tailed Test

Journal: PLoS ONE

Article Title: Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

doi: 10.1371/journal.pone.0231064

Figure Lengend Snippet: A. Sensitivity of normal control myoblasts to BSO. The data represent the mean ± SE. *** p < 0.001 (two-way ANOVA and Tukey-Kramer test versus DDW). B. Sensitivity of sIBM patient myoblasts to BSO, as measured by the cell viability assay and the level of LDH in the culture medium. The data represent the mean ± SEM. * p < 0.05, *** p < 0.001 (two-way ANOVA and Tukey-Kramer test versus BSO + DDW). The red square indicates a significant increase or decreased compared with DDW. C. Cell protective effect of MA-5 on sIBM myoblasts, as measured by the cell viability assay and the level of LDH in the culture medium. The data represent the mean ± SEM. # p < 0.05, ### p < 0.001 (two-way ANOVA and Tukey post hoc test versus control); ** p < 0.01, *** p < 0.001 (two-way ANOVA and Tukey-Kramer test versus BSO + DMSO). The red square indicates a significant increase or decrease compared with DMSO. D. Measurement of sIBM patient myoblasts by the cell viability assay (left panel) after 72-h DMSO application as a control or 72-h 10 μM MA-5 treatment under oxidative stress conditions induced by 24-h BSO treatment. The levels of GDF15 (middle panel) in the medium of cultured myoblasts were measured in the same manner. The levels of FGF21 (right panel) in the medium of cultured myoblasts were measured in the same manner. The data represent the mean ± SE. ## p < 0.01, ### p < 0.001 (two-way ANOVA and Tukey-Kramer test versus DMSO); ** p < 0.01, *** p < 0.001 (two-way ANOVA and Tukey-Kramer test versus BSO + DMSO).

Article Snippet: The levels of GDF15 and FGF21 were measured using a Quantikine Human GDF15 ELISA Kit and Quantikine Human FGF21 ELISA Kit (R&D Systems, Minneapolis, MN), respectively.

Techniques: Control, Viability Assay, Cell Culture

Journal: Chinese Medicine

Article Title: Effects of Bushen Tianjing Recipe in a rat model of tripterygium glycoside-induced premature ovarian failure

doi: 10.1186/s13020-017-0131-3

Figure Lengend Snippet: Representative immunohistochemistry images ( a ) and quantitative analysis ( b , c , d ) of Bcl-2, Bax, and caspase 3 in histological sections from all experimental groups. Immunostaining ( brown ) are indicated by black arrows . For each animal, ten random high power fields (HPFs) from five sections were used for quantitative analysis. Each dot in b and c represents the median value across these HPFs. Bars and error bars are medians and quartiles, respectively. All statistical analyses were performed using the nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test. NS not significant. Magnification ×100

Article Snippet: IHC kits for Bax (Catalog No.: BA0315-1), Bcl-2 (Catalog No.: BA0412), and caspase-3 (active form, Catalog No.: BA2142) were provided by Wuhan Boster Bio-engineering Co., Ltd. (Wuhan, China).

Techniques: Immunohistochemistry, Immunostaining

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: a, Outline of the studies. b, mRNA levels of Cdkn2a, Cdkn1a, Il6, Mmp3 and Lmnb1, normalized to Actb and Tub mRNA (n = 6 for each group). c, Representative SA-β-gal staining (SA-β-gal+ SnCs in arrows; n = 3 per group/three images per n), EdU (green, EdU negative non-proliferating SnCs in arrows; n = 3 for each group/5–6 images per n) and Hoechst labelled nuclei (blue). Scale bar, 100 μm. d–g, Quantification of the percentage of SA-β-gal+ cells (d), EdU+ cells (e), Lamin B1 levels, with around 1,000 cells (f) and HMGB1+ cells (n = 3 per group; at least 100 cells were counted) in MDFs 3 or 6 days after culturing in young or old mouse serum (g). h, Bioluminescence from p16-3MR-expressing cells in non-senescent MDFs cultured in serum from either young or old mice for 3 (n = 9 for each group) or 6 days (n = 6 for each group). i, Representative SA-β-gal staining (left; + cells are marked with arrows; n = 3 for each group/5–6 images per n) and percentage of SA-β-gal+ cells (right). j, Gene expression of senescence and SASP markers in human renal epithelial cells cultured with young or old human plasma for 3 or 6 days (d3 or d6; n = 8 per group). k, IL-6 level secreted by human renal epithelial cells treated with young or old human plasma for 6 days (n = 8 per group). l, Pearson correlation of secreted IL-6 levels by human renal epithelial cells treated with young or old human plasma and IL-6, MMP-3 and HMGB1 levels in human plasma (Pearson correlation coefficient and P are shown in the figures). Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using a two-tailed t-test with a Welch’s correction (b,d–i,k) (*P < 0.05; **P < 0.01) and two-way ANOVA followed by two-stage step-up method by Benjamini, Krieger and Yekutieli, FDR < 0.05 (*q < 0.05; ** q < 0.01) (j). Rel, relative. Scale bars, 100 μm.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Staining, Expressing, Cell Culture, Two Tailed Test

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: (a) mRNA levels for Cdkn2a and Cdkn1a and SASP factors Il6, Mmp3 and Laminb1, normalized to Actb mRNA, determined by RT–PCR (n = 8 for young or old serum treatment for 3 days; n = 4 for young + old (50/50) serum treatment for 3 days). (b) Representative EdU (green; EdU negative non-proliferating SnCs in arrows), HMGB1 (red; SnCs marked by HMGB1 nuclear loss with arrows), Hoechst labelled nuclei (blue) visualized by fluorescence microscopy (3–6 images per n) and SA-β-gal staining (3–7 images per n) and (c) quantification of EdU-positive SnCs in MDFs 3 days after culturing in young, old or young + old (50/50) mouse serum (n = 4 for each group). (d) Bioluminescence from 3MR-expressing cells (Renilla luciferase assay) in non-senescent MDFs from cultured in young (4-month-old), old (32-month-old) or young+old (50/50) mouse serum for 6 days (A.U.) (n = 4 for young or old mouse serum; n = 6 for young+old mouse serum). Data are means ± s.e.m. of biologically independent samples. Statistical significance was tested using one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons with *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars, 100 μm. Rel, relative.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Reverse Transcription Polymerase Chain Reaction, Fluorescence, Microscopy, Staining, Expressing, Luciferase, Cell Culture

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: a, Experimental setup for heterochronic blood exchange. b, Luminescence images of young p16-3MR mice receiving young blood from C57BL/6J mice (YY) and young p16-3MR mice receiving old blood from C57BL/6J mice (YO) 14 days after blood exchange (left) and quantification of the luminescence (right) (in arbitrary units, a.u.) (n = 7, 4, 3, 4 or 4 mice). c, Ratio of circulating SASP proteins (>1.5-fold) of old mouse blood (n = 7) normalized to young mouse blood (n = 3) measured by antibody array. d, Gene expression of the senescence and SASP markers in lung, hippocampus, heart, skeletal muscles (gastrocnemius (GA) and tibialis anterior (TA)), kidney and liver (n = 6 for YY; n = 7 for YO). Whisker plots represent the 10th and 90th percentiles and the line corresponds to the median. e, Representative images of SA-β-gal staining in skeletal muscle (n = 4 for YY and n = 5 for YO; 3–6 images per mouse) and quantification of SA-β-gal+ cells per area of YY and YO mice. Scale bar, 50 μm. f, Representative images of SA-β-gal staining (n = 8 mice for each group/7–10 images per mouse) and HMGB1 immunohistochemistry (n = 5 for YY; n = 7 for YO; 6–10 images per mouse) and quantification of SA-β-gal+ cells per area (n = 8 per group) of kidney sections of YY and YO mice. Scale bars, 100 μm. g, Representative SA-β-gal staining in liver (n = 9 for YY; n = 8 for YO; 7–10 images per mouse) and quantification of SA-β-gal+ cells per area in YY and YO mice and γ-H2AX foci in a hepatocyte (n = 6 per group; 5–8 images per mouse). this experiment was performed three independent times. Scale bar, 100 μm. Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using a two-tailed t-test with a Welch’s correction (b,e–g) with *P < 0.05; **P < 0.01 and multiple Mann–Whitney tests with a two-stage linear step-up procedure by Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05 (c,d).

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Ab Array, Expressing, Muscles, Whisker Assay, Staining, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: (a) Representative luminescence images of young p16-3MR mice (3-month-old) receiving blood (22-month-old) from old C57BL/6J mice treated with vehicle (YO+Veh) or ABT263 (YO+ABT) 14 days after blood exchange (left) and quantification of the luminescence (right) (A.U.) (n = 4 mice for YO+Veh; n = 3 mice for YO+ABT). Each data point represents an individual mouse. (b) Representative EdU (green; EdU negative non-proliferating SnCs with arrows), HMGB1 (red; SnCs marked by nuclear loss with arrows), and Hoechst labeled nuclei (blue) visualized by immunostaining (n = 4 for each group / 5–8 images per n) and (c) SA-β-gal staining in MDFs cultured in Veh- or ABT-treated old mouse serum for 3 days (n = 4 for veh-treated old mice serum treated; n = 3 for ABT-treated old mouse serum; 3–6 images per n). Quantification of (d) EdU + and (e) SA-β-gal + MDFs. (f) mRNA levels for Cdkn2a and Cdkn1a and SASP factors Il6 and Mmp 3 days after culturing in Veh- or ABT-treated old mouse serum, determined by RT–PCR (n = 4 for each group). Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using two-tailed Student’s t test (a, d-e) (exact P value was shown in the figures) and multiple t tests with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05 (f). Scale bars are shown in each image. Rel, relative.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: In Vivo, Labeling, Immunostaining, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: (a) Relative protein expression ratio (< 0.7-fold) of SASP proteins in plasma from DQ-treated C57BL/6J old mice (DQ; n = 4) normalized to vehicle treated C57BL/6J old mice (Veh; n = 3), measured by antibody array. Each data point represents an individual mouse. Additional SA-β-gal images of (b) kidney and (c) liver in young C57BL/6J mice receiving old C57BL/6J mice treated with Veh (YO+Veh) or DQ (YO+DQ). (d) Representative EdU (green; EdU negative non-proliferating SnCs in arrows), HMGB1 (red; SnCs marked by HMGB1 nuclear loss in arrows), and Hoechst labeled nuclei (blue) visualized by fluorescence microscopy (n = 4 for each group / at least 7 images per n) and (e) SA-β-gal staining in MDFs cultured in Veh- or DQ-treated old mice serum for 3 days (n = 4 for veh-treated old mice serum treated; n = 5 for DQ-treated old mice serum / at least 6 images per n). Quantification of (f) EdU + (n = 4 for Veh-treated old mice serum treated; n = 3 for DQ-treated old mice serum treated) and (g) SA-β-gal + (n = 4 per group) MDFs. (h) mRNA levels for Cdkn2a and Cdkn1a and SASP factors Il6 and Mmp3 3 days after culturing in Veh- or DQ-treated old mice serum, determined by RT–PCR (n = 4 for Veh-treated old mice serum treated; n = 5 for DQ-treated old mice serum treated). Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using multiple t test with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05; **q < 0.01 (a, h) and two-tailed Student’s t test (f-g) with *, P < 0.05. Scale bars are shown in each image.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Expressing, Ab Array, Labeling, Fluorescence, Microscopy, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: (a) Additional SA-β-gal images of kidney (left) and liver (right) in young C57BL/6J mice receiving old C57BL/6 blood treated with vehicle (YO+Veh) or ABT263 (YO+ABT). (b) HMGB1 immunohistochemistry (brown staining of HMGB1 re-localized to cytoplasm of kidney cells with arrows) (n = 5 per group; 10–15 images per mice). (c) Immunohistochemical staining for KIM-1 (n = 6 for YO+Veh; n = 4 for YO+ABT; 4–5 images per mice) and LTL (n = 5 per group; 5–7 images per mice) on kidney tissues and (d) quantification of KIM-1 + area (%). (e) Serum concentration of KIM-1 (n = 6 per group), (f) blood urea nitrogen (n = 12 for YO+Veh; n = 9 for YO+ABT) and creatine (n = 4 per group). (g) Representative images of Sirius Red (n = 6 for YO+Veh; n = 5 for YO+ABT; 10–15 images per mice) and Masson Trichrom staining and desmin immunohistochemistry (n = 6 mice for YO+Veh; n = 5 mice for YO+ABT; 10–15 images per mice) in livers. Arrows indicate collagen deposition. (h) Quantifications of fibrotic area, as % of area occupied by Sirius Red stain, and desmin + area (n = 6 for YO+Veh; n = 5 for YO+ABT). (i) Quantification of mRNAs encoding Col1a1, Col3a1, Col4a1 and Col4a2 in the liver (n = 6 per group). (j) Oil Red O + area (%) indicated as adiposity index (n = 6 per group; 5–9 images per mice). (k) Serum analyses for ALT (n = 8 for YO+Veh; n = 9 for YO+ABT) and bilirubin (n = 9 per group). All data are expressed as means± s.e.m. of biologically independent samples. A two-tailed t test with a Welch’s correction (d-f, h, j-k; *, P < 0.05) and multiple t test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05; **q < 0.01 (i). Scale bars, 100 μm. Rel, relative.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Immunohistochemistry, Staining, Immunohistochemical staining, Concentration Assay, Two Tailed Test

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: a, Study design used in b–g. b, Venn diagram showing the overlap between up-regulated circulating SASP proteins identified in old versus young mice (>1.5-fold) and down-regulated proteins in vehicle (Veh)-treated versus DQ-treated old mice (<0.7-fold). c, List of overlapping proteins in b. d,e, Senescence/SASP-associated gene expression in kidney (d) and liver (e) of young mice receiving old blood treated with Veh (YO+Veh; n = 4) or DQ (YO+DQ; n = 6). f,g, Representative SA-β-gal staining (n = 5 for YO+Veh; n = 7 for YO+DQ; 10–15 images per mouse) and quantification of SA-β-gal+ cells per area in kidney (f) and liver (g) (n = 5 for YO+Veh; n = 8 for YO+DQ; 7–10 images per mouse). h, Study design used in i–p. i, Venn diagram showing the overlap between up-regulated circulating SASP proteins in old versus young mice (>1.5-fold) and down-regulated proteins in Veh-treated versus ABT263-treated old mice (<0.7-fold). j, List of overlapping proteins in i. k–l, Senescence/SASP-associated gene expression in kidney and liver of young mice receiving old blood treated with Veh (YO+Veh) or ABT263 (YO+ABT) (n = 6 per group). m,n, Representative images of SA-β-gal staining in kidney (n = 8 for YO+Veh; n = 7 for YO+ABT; 6–10 images per mouse) and quantification of SA-β-gal+ cells per area (m) and HMGB1+ tubular cells (n). Scale bar, 500 μm. o,p, Images of SA-β-gal staining in liver (n = 5 for YO+Veh; n = 6 for YO+ABT; ten images per mouse) and quantification of SA-β-gal+ cells per area (o), γ-H2AX foci (n = 5 per group; 6–8 images per mouse) and TUNEL+ hepatocytes (n = 4 for YO+Veh; n = 5 for YO+ABT; 5–9 images per mouse) (p). Scale bar, 100 μm. Two independent experiments were performed. Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using multiple Mann–Whitney tests with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05 (d,e,k,l) and a two-tailed t-test with a Welch’s correction (f,g,m–p) with *P < 0.05; **P < 0.01; ***P < 0.001. Rel, relative.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Expressing, Staining, TUNEL Assay, MANN-WHITNEY, Two Tailed Test

Journal: The FASEB Journal

Article Title: Resveratrol‐enhanced SIRT1‐mediated osteogenesis in porous endplates attenuates low back pain and anxiety behaviors

doi: 10.1096/fj.202002524R

Figure Lengend Snippet: Resveratrol attenuates inflammation and sensory innervation in endplates. A‐C, ELISA analysis of the PGE2, IL‐1β, and RT‐PCR analysis of expression of TNFα in the lumbar endplates lysates from mice in the sham, vehicle and 40 mg/kg/d resveratrol treated groups at 4 and 8 weeks post‐LSI surgery. D‐E, Representative images of the immunofluorescence and quantitative analysis of COX2+ cells (green) in endplates from mice in the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar: 100 μm. F‐G, Representative images of the immunofluorescence and quantitative analysis of CGRP+ sensory fibers (green) in the endplates from mice in the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar: 50 μm. H‐I, Representative images of the immunofluorescence and quantitative analysis of the CGRP+ sensory neurons (green) in L 2‐4 DRGs from mice in the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar: 100 μm. N ≥ 6 per group. * P < .05, ** P < .01. (Student’s t test)

Article Snippet: The concentrations of PGE2 and IL‐1β in the L 4‐5 endplates and the concentration of BDNF in the hippocampus were measured by a PGE2 ELISA kit (514010, Cayman Chemical, Ann Arbor, MI, USA), IL‐1β ELISA kit (BMS6002, Thermo Fisher, Waltham, MA, USA), and BDNF ELISA kit (dy248, R&D System, Minneapolis, MN), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence

Journal: The FASEB Journal

Article Title: Resveratrol‐enhanced SIRT1‐mediated osteogenesis in porous endplates attenuates low back pain and anxiety behaviors

doi: 10.1096/fj.202002524R

Figure Lengend Snippet: Resveratrol promotes osteogenesis in the porous endplates after LSI surgery. A‐B, Representative immunofluorescence images of the Osterix staining in the L 4‐5 endplates and the quantitative analysis of the number of osterix+ cells per section from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar, 20 μm. C‐D, Representative images of the calcein double‐labeling of porous endplates with quantification of the mineral apposition rate from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery, distance between white arrowheads were measured. E‐F, Representative images of Trap staining of the endplates and quantitative analysis of the osteoclast number per section from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar, 50 μm. G‐H, The RT‐PCR analysis of Slit3 and Runx2 expression in the L 4‐5 endplates from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. I, The quantitative ELISA evaluation of Netrin‐1 concentration in the L 4‐5 endplates from the sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. N ≥ 6 per group. * P < .05, ** P < .01. (Student’s t test)

Article Snippet: The concentrations of PGE2 and IL‐1β in the L 4‐5 endplates and the concentration of BDNF in the hippocampus were measured by a PGE2 ELISA kit (514010, Cayman Chemical, Ann Arbor, MI, USA), IL‐1β ELISA kit (BMS6002, Thermo Fisher, Waltham, MA, USA), and BDNF ELISA kit (dy248, R&D System, Minneapolis, MN), respectively.

Techniques: Immunofluorescence, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The FASEB Journal

Article Title: Resveratrol‐enhanced SIRT1‐mediated osteogenesis in porous endplates attenuates low back pain and anxiety behaviors

doi: 10.1096/fj.202002524R

Figure Lengend Snippet: Resveratrol alleviates hippocampal inflammation and enhances pCREB activation, BDNF expression in the hippocampus. A‐C, The RT‐PCR analysis of the expression of the inflammatory mediators TNFα (A), IL‐1β (B), and IL‐6 (C) in the hippocampus of resveratrol‐treated and control mice at 8 weeks post‐LSI surgery. D, Representative images of the pCREB immunostaining at the dentate gyrus of the hippocampus from sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar, 200 μm. E‐F, Representative images of the BDNF immunostaining at the dentate gyrus of the hippocampus and the quantitative ELISA analysis of BDNF in the hippocampus from sham, vehicle, and resveratrol groups at 8 weeks post‐LSI surgery. Scale bar, 200 μm. * P < .05. (Student’s t test)

Article Snippet: The concentrations of PGE2 and IL‐1β in the L 4‐5 endplates and the concentration of BDNF in the hippocampus were measured by a PGE2 ELISA kit (514010, Cayman Chemical, Ann Arbor, MI, USA), IL‐1β ELISA kit (BMS6002, Thermo Fisher, Waltham, MA, USA), and BDNF ELISA kit (dy248, R&D System, Minneapolis, MN), respectively.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Activation of innate immune cGAS-STING pathway contributes to Alzheimer’s pathogenesis in 5×FAD mice

doi: 10.1101/2022.10.30.514314

Figure Lengend Snippet: a , Nest construction score for 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. Age-matched Cgas +/+ and Cgas −/− mice were used as control. Mean ± SD; n = 12; **, p < 0.01, ***, p < 0.001, one-way ANOVA with Bonferroni’s post hoc test. b-e , Quantification of the indicated Aβ40 ( b ) and Aβ42 ( c ) in TBS fraction, and Aβ40 ( d ) and Aβ42 ( e ) in guanidine fraction of cortical tissues in 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice by ELISA. Mean ± SD; n = 6; n . s ., not significant; *, p < 0.05, **, p < 0.01, Student’s t -test. f , Thioflavin S staining of hippocampal tissues in 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. Scale bar, 50 μm. g , Quantification of Thioflavin S-labeled amyloid core numbers per mm 2 in f . Mean ± SD; n = 6; ***, p < 0.001, Student’s t -test. h , Immunostaining of Aβ and Iba1 in hippocampal dentate gyrus (DG) of 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. Scale bar, 40 μm. i , Quantification of Aβ plaque-associated Iba1 + microglia (indicated by white arrow) in h . Mean ± SD; n = 6; **, p < 0.01, Student’s t -test. j , Western immunoblotting analysis of the expression of the indicated proteins involved in cGAS-SITNG pathway of cortical tissues in 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. n = 3. k , Quantification of the expression of p-STING), p-TBK1, p-p65, and p-IRF3 relative to β-actin in j . Mean ± SD; n = 3; *, p < 0.05, ***, p < 0.001, Student’s t -test. l , Transcriptional analysis of a panel of A1 astrocyte-inducing, disease-associated microglial markers, and interferon (IFN)-stimulated genes in hippocampal tissues of 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice compared to WT mice. n = 4.

Article Snippet: The secreted cytokines from primary neural cells after oligomeric Aβ42 treatment were quantified by mouse cytokine ELISA kits, including mouse IL-6 ELISA kit (E-EL-M0044c, Elabscience), mouse TNF-α ELISA kit (E-MSEL-M0002, Elabscience), mouse IL-1α ELISA kit (E-EL-M3059, Elabscience), and moue IFN-β ELISA kit (42410-1, PBL Assay Science) accordingly to the manufacture’s instruction.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Immunostaining, Western Blot, Expressing

Journal: bioRxiv

Article Title: Activation of innate immune cGAS-STING pathway contributes to Alzheimer’s pathogenesis in 5×FAD mice

doi: 10.1101/2022.10.30.514314

Figure Lengend Snippet: a , Schematic diagram showing primary cultured microglia treated with oligomeric Aβ42 (5 μM) for 24 h, followed by immunoblotting and ELISA analysis. b , Immunoblotting analysis of the expression of the indicated proteins involved in cGAS-SITNG pathway of primary WT microglia 24 h after the Aβ treatment. The non-treatment of the Aβ was used as control. n = 3. c , Quantification of the expression of p-TBK1), p-p65, and p-IRF3 relative to β-actin in b . Mean ± SD; n = 3; n . s ., not significant; *, p < 0.05, Student’s t -test. d-f , ELISA analysis for IL-6 ( d ), TNF-α ( e ) and IL-1α ( f ) in the supernatants of primary Cgas +/+ or Cgas −/− microglia 24 h after Aβ42 treatment. The non-treatment of Aβ42 was used as control. Mean ± SD; n = 4; *, p < 0.05, **, p < 0.01, ***, p < 0.001, one-way ANOVA with Bonferroni’s post hoc test. g , Transcriptional analysis for activated astrocytic genes in 6-month-old Cgas +/+ ;5×FAD ( n = 5) and Cgas −/− ;5×FAD ( n = 5) mice. A1-specific: genes activated only by LPS; A2-specific: genes activated only by ischaemia; Pan-reactive: genes activated by either LPS or ischaemia. h , GFAP + astrocytic staining of cortical region in 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. Scale bar, 50 μm. i , Quantification of percentage of GFAP + area in h . Mean ± SD; n = 6; **, p < 0.01, Student’s t -test. j , Schematic diagram showing WT, Cgas +/+ , and Cgas −/− primary microglia treated with oligomeric Aβ42 (5 μM) for 24 h, the resulted microglia conditioned medium (WT MCM, Cgas +/+ MCM, and Cgas −/− MCM, respectively) individually added to Cgas +/+ primary astrocytes for inoculation for 24 h, and the concentrated astrocyte conditioned medium (WT ACM, Cgas +/+ ACM, and Cgas −/− ACM, respectively)) individually inoculated with Cgas +/+ primary neurons treated in parallel with oligomeric Aβ (750 nM) for 24 h. Primary neurons were pre-treated with adenovirus-associated virus (AAV) expressing a GFP with CaMKII promotor. k , Fluorescent signals in primary neuron cultures treated with oligomeric Aβ, together with WT, Cgas +/+ , or Cgas −/− ACM. Scale bar, 50 μm. l, m , Percentage of GFP-labeled primary neurons ( l ) and numbers of primary neurons ( m ) in k . Mean ± SD; n = 4; n . s ., not significant; *, p < 0.05, **, p < 0.01, one-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: The secreted cytokines from primary neural cells after oligomeric Aβ42 treatment were quantified by mouse cytokine ELISA kits, including mouse IL-6 ELISA kit (E-EL-M0044c, Elabscience), mouse TNF-α ELISA kit (E-MSEL-M0002, Elabscience), mouse IL-1α ELISA kit (E-EL-M3059, Elabscience), and moue IFN-β ELISA kit (42410-1, PBL Assay Science) accordingly to the manufacture’s instruction.

Techniques: Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Control, Staining, Virus, Labeling

Journal: bioRxiv

Article Title: Activation of innate immune cGAS-STING pathway contributes to Alzheimer’s pathogenesis in 5×FAD mice

doi: 10.1101/2022.10.30.514314

Figure Lengend Snippet: a , Schematic diagram showing Cgas +/+ primary microglia treated with oligomeric Aβ42 (5 μM) for 24 h, together with cGAS inhibitor RU.521 (50 μM) or STING inhibitor H-151 (15 μM), the resulted microglia conditioned medium (MCM) added to Cgas +/+ primary astrocytes for inoculation for 24 h, and the concentrated astrocyte conditioned medium (ACM) inoculated with cGAS +/+ primary neurons treated in parallel with oligomeric Aβ (750 nM) for 24 h. Primary neurons were pre-treated with adenovirus-associated virus (AAV) expression a GFP with CaMKII promotor. b , Fluorescent signals in primary neuron cultures treated with oligomeric Aβ42, together with the RU.521 or H-151 ACM. Scale bar, 50 μm. c, d , Percentage of GFP-labeled primary neurons ( c ) and numbers of primary neurons ( d ) in b . Mean ± SD; n = 4; n . s ., not significant; *, p < 0.05, **, p < 0.01, one-way ANOVA with Bonferroni’s post hoc test. e , Schematic diagram of STING inhibitor H-151 treatment on 3-month-old 5×FAD mice (see Methods). f-i , Quantification of the indicated Aβ40 ( f ) and Aβ42 ( g ) in TBS fraction, and Aβ40 ( h ) and Aβ42 ( i ) in guanidine fraction of cortical tissues in H-151-treated 5-month-old 5×FAD mice by ELISA. Mean ± SD; n = 6; n . s ., not significant; *, p < 0.05, **, p < 0.01, Student’s t -test. j , Thioflavin S staining of DG and cortical tissues in H-151-treated 5-month-old 5×FAD mice. Scale bar, 50 μm. k, l , Quantification of Thioflavin S-labeled amyloid core numbers per mm 2 in DG ( k ) and cortical ( l ) regions in j . Mean ± SD; n = 6; **, p < 0.01, Student’s t -test. m, n , Quantification of percentage of Thioflavin S-labeled Aβ plaque area in DG ( m ) and cortical ( n ) regions in j . Mean ± SD; n = 6; **, p < 0.01, Student’s t -test. o , Immunostaining of Aβ (6E10), Iba1 + microglial and GFAP + astrocytes of DG region in H-151-treated 5-month-old 5×FAD mice. Scale bar, 50 μm. p-r , Quantification of Aβ plaque numbers per mm 2 ( p ) and percentage of Iba1 + ( q ) and GFAP + ( r ) area in o . Mean ± SD; n = 6; **, p < 0.01, ***, p < 0.001, Student’s t -test.

Article Snippet: The secreted cytokines from primary neural cells after oligomeric Aβ42 treatment were quantified by mouse cytokine ELISA kits, including mouse IL-6 ELISA kit (E-EL-M0044c, Elabscience), mouse TNF-α ELISA kit (E-MSEL-M0002, Elabscience), mouse IL-1α ELISA kit (E-EL-M3059, Elabscience), and moue IFN-β ELISA kit (42410-1, PBL Assay Science) accordingly to the manufacture’s instruction.

Techniques: Virus, Expressing, Labeling, Enzyme-linked Immunosorbent Assay, Staining, Immunostaining

Journal: bioRxiv

Article Title: Vascular endothelial growth factor-D improves lung vascular integrity during acute lung injury

doi: 10.1101/2024.12.16.628787

Figure Lengend Snippet: A) NICHES analysis of AFs, mesothelial cells, pericytes, gCap, and aCap in human microvascular niche yields a quantitative cell-cell signaling atlas of AF-aCap, AF-gCap, Mesothelial-aCap, Mesothelial-gCap, Pericyte-aCap, and Pericyte-gCap visualized by low-dimensional UMAP embedding. B) FeaturePlot of top differentially expressed L/R pairs ANGPT1 - TEK , SLIT2 - ROBO4 , VEGFD - KDR , BMP5-BMPR2 , ADM - RAMP2 , WNT5A - FZD6 , PTN - RACK1 , OXT - EDNRB , and ADCYAP1 - VIPR1 from NICHES analysis C) Top 10 differentially expressed L/R pairs between different cell-cell interaction signals processed through NICHES and plotted in DoHeatmap. D) Immunostaining of representative L/R pairs from AF to microvascular endothelial cells, including SLIT2 - ROBO4 , ADM - RAMP2 , BMP5 - BMPR2 , and VEGFD - VEGFR2 in human lung section. ITGA8 is a marker for AF; PRX is a marker for microvascular endothelial cells. Scale bar 20 μm.

Article Snippet: The protein levels of SLIT2, ADM, BMP5, and VEGFD in samples harvested from different timepoints were determined using corresponding ELISA kits: SLIT2 (# NB030384), ADM (# NBP2-78738), BMP5 (# NBP2-69996), VEGFD (# NBP2-78890), all from Novus Biologicals.

Techniques: Immunostaining, Marker

Journal: bioRxiv

Article Title: Vascular endothelial growth factor-D improves lung vascular integrity during acute lung injury

doi: 10.1101/2024.12.16.628787

Figure Lengend Snippet: A) C57Bl/6 mice were injected with LPS for 18 hours, followed by a collection of bronchioalveolar lavage (BAL) fluid. The expression level of VEGF-D was measured in the BAL fluid in mice with or without LPS challenge. B) Mice were injected with LPS, followed by treatment with VEGF-D (225 μg/kg). The BAL fluid and histological samples were collected after 18 hours of treatment. C) The FITC-albumin probe was injected retro-orbitally 16 hours after the LPS challenge. The amount of probe in BAL fluid was quantified after another 2 hours. The FITC-albumin levels in BAL fluid in the control and experimental groups were determined. D) The BAL fluid was spun down at 5,000 g for 10 minutes and resuspended in 300 μL of 1XPBS. The number of cells in the BAL from different conditions was determined using a hemacytometer. n = 12 – 14 per group. E) LPS and LPS+VEGFD lungs were excised, fixed in 4% paraformaldehyde, embedded in paraffin, and used for histochemical analysis after haematoxylin and eosin staining. Images are representative of 6–9 lung specimens for each condition. Scale bar 150 μm. F) Lung pathological index was performed by a blinded reviewer from 1 to 4, where 4 was the worst pathology. The score was used for both peribronchial and alveolar inflammation. The average of two scores was taken to obtain a range between 1 (no inflammation or pathology) and 4 (maximum inflammation and pathology). The lung pathological index was calculated in LPS and LPS+VEGFD conditions. G-I) The expression level of TNF-α and IL-6 were measured in BAL fluid from LPS and LPS+VEGFD conditions using corresponding ELISA kits. Mice without LPS challenge were used as control. N = 12 - 14 per treatment group. Data presented as mean ± SEM. * and ** indicate p<0.05 and p<0.01, respectively.

Article Snippet: The protein levels of SLIT2, ADM, BMP5, and VEGFD in samples harvested from different timepoints were determined using corresponding ELISA kits: SLIT2 (# NB030384), ADM (# NBP2-78738), BMP5 (# NBP2-69996), VEGFD (# NBP2-78890), all from Novus Biologicals.

Techniques: Injection, Expressing, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reduced Plasma Kallistatin Is Associated With the Severity of Coronary Artery Disease, and Kallistatin Treatment Attenuates Atherosclerotic Plaque Formation in Mice

doi: 10.1161/JAHA.118.009562

Figure Lengend Snippet: Kallistatin inhibited low‐shear stress–induced carotid artery plaque formation. A , Representative magnetic resonance images in cross‐sectional views through the carotid arteries. The red arrow indicates the cross section of the left carotid artery, and the blue arrow indicates the cross section of the right carotid artery. B , Quantitative analysis of left carotid artery diameter in the Ad. HKS and Ad.Null groups. L‐ NAME and NAM blocked the effects of kallistatin (n=10, * P <0.05 vs the Ad.Null‐, L‐ NAME ‐, or NAM ‐treated groups). C , Quantitative analysis of left carotid artery plaque volume in the Ad. HKS and Ad.Null groups. L‐ NAME and NAM blocked the effect of kallistatin; n=10 (* P <0.05 vs the Ad.Null‐, L‐ NAME ‐ or NAM ‐treated groups). D , Plasma MDA levels in apoE −/− mice. E , Plasma TNF ‐α levels in apoE −/− mice. F , Distribution of mouse kallistatin expression in atherosclerotic lesions and normal vascular tissue. Data are presented as the mean± SEM ; n=10, * P <0.05 vs the Ad.Null‐, L‐ NAME ‐, or NAM ‐treated groups. Ad.HKS indicates adenovirus containing kallistatin cDNA; Ad.Null, adenovirus containing null cDNA; L‐NAME, N ω ‐nitro‐L‐arginine methyl ester; MDA, malondialdehyde; NAM, nicotinamide; SEM, standard error of the mean; TNF‐α, tumor necrosis factor‐α.

Article Snippet: Plasma samples were used for the analysis of TNF‐α using a Mouse TNF‐α ELISA (Proteintech, Rosemount, IL) according to the manufacturer's protocol.

Techniques: Shear, Clinical Proteomics, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reduced Plasma Kallistatin Is Associated With the Severity of Coronary Artery Disease, and Kallistatin Treatment Attenuates Atherosclerotic Plaque Formation in Mice

doi: 10.1161/JAHA.118.009562

Figure Lengend Snippet: Kallistatin led to morphological changes in the atherosclerotic plaques of apoE −/− mice. A , Representative images of HE staining. Original magnification is ×10 (n=5 in each group). B , Oil red O staining of carotid specimens of mice in the Ad.Null group, the Ad. HKS group, and L‐ NAME ‐ or NAM ‐treated groups (n=5 in each group). C , Representative images of superoxide formation labeled by the red fluorescence dye dihydroethidium in the carotid artery of Ad.Null mice, Ad. HKS mice, Ad. HKS +L‐ NAME mice, and Ad. HKS + NAM mice (n=5 in each group). D , Representative images of CD 68 immunostaining in lesion areas of the left carotid artery (n=5 in each group). E , Immunohistochemical assessments of the protein level of TNF ‐α in carotid plaque (n=5 in each group). F, Quantitative analyses of CD 68‐positive cells in the 4 groups (n=5 in each group, * P <0.05 vs the Ad.Null‐, L‐ NAME ‐, or NAM ‐treated groups). G , Fluorescence intensity was measured by a fluorescence microscope and quantified with Image software (n=5 in each group, * P <0.05 vs the Ad.Null‐, L‐ NAME ‐, or NAM ‐treated groups). H , Colocalization of PE red fluorescence ( SIRT 1) and FITC green fluorescence ( CD 31) within plaques (×40 magnification) in the carotid arteries of the Ad.Null, Ad. HKS , Ad. HKS +L‐ NAME , and Ad. HKS + NAM groups with labeled cell nuclei (blue) (n=5 in each group). Regions shown at a higher magnification (far right) are indicated by yellow boxes. Endothelial cell‐specific SIRT 1 fluorescence was measured on the luminal side of the internal elastic lamina only and expressed in mean pixel intensity per area (n=5 in each group, # P <0.05 vs the Ad.Null‐ and NAM ‐treated groups). I , Colocalization of PE red fluorescence ( eNOS ) and FITC green fluorescence ( CD 31) within carotid artery plaques (×40 magnification) of the 4 groups with labeled cell nuclei (blue). Regions shown at a higher magnification (far right) are indicated by yellow boxes. Endothelial cell‐specific eNOS fluorescence was measured on the luminal side of the internal elastic lamina only and expressed in mean pixel intensity per area (* P <0.05 vs the Ad.Null‐, L‐ NAME ‐, or NAM ‐treated groups). We observed that red fluorescence ( SIRT 1, eNOS ) and green fluorescence ( CD 31) signals were colocalized in the Ad. HKS group. Scale bar=50 μm. Ad.HKS indicates adenovirus containing kallistatin cDNA; Ad.Null, adenovirus containing null cDNA; DAPI, 4′,6‐diamidino‐2‐phenylindole; DHE, dihydroethidium; eNOS, endothelial nitric oxide synthase; FITC, fluoroisothiocyanate; HE, hematoxylin and eosin; L‐NAME, N ω ‐nitro‐L‐arginine methyl ester; NAM, nicotinamide; PE, phycoerythrin; SIRT1, sirtuin 1; TNF‐α, tumor necrosis factor‐α.

Article Snippet: Plasma samples were used for the analysis of TNF‐α using a Mouse TNF‐α ELISA (Proteintech, Rosemount, IL) according to the manufacturer's protocol.

Techniques: Staining, Labeling, Fluorescence, Immunostaining, Immunohistochemical staining, Microscopy, Software

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reduced Plasma Kallistatin Is Associated With the Severity of Coronary Artery Disease, and Kallistatin Treatment Attenuates Atherosclerotic Plaque Formation in Mice

doi: 10.1161/JAHA.118.009562

Figure Lengend Snippet: Effect of kallistatin protein ( KS ) on TNF ‐α–induced HUVEC s. NADPH oxidase activity ( A ), intracellular ROS accumulation ( B ), relative SIRT 1 mRNA expression levels ( C ), relative eNOS mRNA expression levels ( D ), and SIRT 1 and eNOS protein expression ( E ). Data are presented as the mean± SEM (n=5, * P <0.05 vs the TNF ‐α, TNF ‐α+ KS + NAM , and TNF ‐α+ KS + L‐ NAME groups. # P <0.05 vs the TNF ‐α and TNF ‐α+ KS + NAM groups). eNOS indicates endothelial nitric oxide synthase; HUVEC, human umbilical vein endothelial cells; KS, kallistatin; L‐NAME, N ω ‐nitro‐L‐arginine methyl ester; NAM, nicotinamide; ROS, reactive oxygen species; SIRT1, sirtuin 1; TNF‐α, tumor necrosis factor‐α.

Article Snippet: Plasma samples were used for the analysis of TNF‐α using a Mouse TNF‐α ELISA (Proteintech, Rosemount, IL) according to the manufacturer's protocol.

Techniques: Activity Assay, Expressing