Journal: Nature Neuroscience

Article Title: Aging and injury drive neuronal senescence in the dorsal root ganglia

doi: 10.1038/s41593-025-01954-x

Figure Lengend Snippet: a , Representative images of SA-β-gal activity staining (blue) in the lumbar DRG of young (11–16 weeks) and aged (20–24 months) mice. Percent SA-β-gal-positive pixels per DRG area (right) ( n = 6 young, 5 aged mice; two-tailed unpaired t -test, P = 0.0153). Scale bar, 100 µm. b , Representative RNAscope images for senescence markers p21 and p16 with SASP factor IL6 in whole DRG section. Scale bar, 100 µm. c , d , Quantification of neuronal expression of each marker ( c ) or in combination ( d ) expressed as a percent of total DRG neurons ( n = 5 young, 4 aged mice, two-tailed unpaired t -test, p21 + , P = 0.0014; p16 + , P = 0.006; IL6 + , P = 0.0031; p21 + IL6 + , P = 0.0005; p16 + IL6 + , P = 0.3076; p21 + p16 + IL6 + , P = 0.1560). e , Analysis of IL6 -expressing DRG neuron population to show co-expression with senescence markers p21 and/or p16 in young and aged mice ( n = 5 young, 4 aged mice). f , Quantification of IL6 protein levels by ELISA assay in young or aged plasma ( n = 6 young, 5 aged mice, two-tailed unpaired t -test, P = 0.0474). All data are expressed as the mean ± s.e.m. NS, not significant.

Article Snippet: For in vitro (culture) preparations, coverslips were pre-incubated with either IL6 (50 ng ml −1 with carrier, prepared from 100 μg ml −1 stock solution in 0.1% BSA; R&D Systems, catalogue number 406-ML) or control media (with 0.00005% BSA) for 1 hour before recording, and aCSF containing the same concentration of either IL6 or control BSA was applied (29−32 °C).

Techniques: Activity Assay, Staining, Two Tailed Test, RNAscope, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Nature Neuroscience

Article Title: Aging and injury drive neuronal senescence in the dorsal root ganglia

doi: 10.1038/s41593-025-01954-x

Figure Lengend Snippet: a , Schematic of SNI and DRG tissue analysis timepoints (BioRender). b , qPCR from lumbar DRG in young (11–16 weeks) mice ( n = 4 control; n = 4 SNI young mice; two-tailed unpaired t -test; Supplementary Table ). c , Left, RNAscope image of DRG slice in young mice. Scale bar, 100 µm. Right, number of DRG neurons expressing p21 (upper) or p16 (lower) in young mice ( n = 5 uninjured mice, n = 4, 7-day and 3-week post-SNI mice, n = 3, 7-week post-SNI mice; one-way ANOVA, p21 : uninj versus 7 days or 3 weeks, P < 0.0001; uninj versus 7 weeks, P = 0.0002. p16 : uninj versus 3 weeks, P = 0.0117; uninj versus 7 weeks, P = 0.0002; 7 days versus 7 weeks, P = 0.0085). d , e , Re-analysis of Renthal et al. . RNA-seq dataset using young adult mouse DRG. d , Percentage of p16 ( Cdkn2a + )-expressing senescent cells relative to all DRG cells after ScNT. Cells are negative for Lmnb1 and Top2a to filter out any nonsenescent cells. Glia, satellite glia and Schwann cells; Immune & other cells, neutrophils, macrophages, B cells, fibroblasts, endothelial cells and pericytes; Neuron, all DRG neurons. Dot plot ( e ) of senescence marker gene expression by DRG neurons after ScNT. SenMayo genes are significant, at least one timepoint (Supplementary Table ). f , RNAscope image of DRG slice in aged (20–24 months) mice. Scale bar, 100 µm. Number of DRG neurons expressing either p21 (right) or p16 (right) in aged mice ( n = 4 uninjured mice, n = 3 post-SNI mice/timepoint; one-way ANOVA, p21 : uninj versus 7 days, P = 0.0335; uninj versus 3 weeks, P = 0.0298. p16 : uninj versus 7 days, P = 0.0182; uninj versus 3 weeks, P = 0.0009; uninj versus 7 weeks, P = 0.0008). g , p21 + p16 + co-expressing DRG neurons ( n = 5 young uninjured mice, n = 4 young 3-week post-SNI mice; n = 4 aged uninjured mice, n = 3 aged 3-week post-SNI mice; one-way ANOVA, young uninj versus SNI, P = 0.0002; aged uninj versus SNI, P < 0.0001; young SNI versus aged SNI, P = 0.0003). h , p21 + p16 + IL6 + (asterisk) neuron by RNAscope. Scale bar, 10 µm. i , IL6 + DRG neurons co-expressing p21 and/or p16 ( n = 5 young uninjured mice, n = 4 aged uninjured mice, n = 4 young 3-week post-SNI mice, n = 3 aged 3-week post-SNI mice; one-way ANOVA; Supplementary Table ). j , IL6 + DRG neurons co-express p21 and/or p16 at 3 weeks after SNI ( n = 3 mice per group). All data are mean values ± s.e.m. Ag, aged; d, days; h, hours; NS, not significant; wk, weeks; Yg, young.

Article Snippet: For in vitro (culture) preparations, coverslips were pre-incubated with either IL6 (50 ng ml −1 with carrier, prepared from 100 μg ml −1 stock solution in 0.1% BSA; R&D Systems, catalogue number 406-ML) or control media (with 0.00005% BSA) for 1 hour before recording, and aCSF containing the same concentration of either IL6 or control BSA was applied (29−32 °C).

Techniques: Control, Two Tailed Test, RNAscope, Expressing, RNA Sequencing, Marker, Gene Expression

Journal: Nature Neuroscience

Article Title: Aging and injury drive neuronal senescence in the dorsal root ganglia

doi: 10.1038/s41593-025-01954-x

Figure Lengend Snippet: a , Analysis of cell diameter (µm) of p21 + IL6 + , p16 + IL6 + or p21 + p16 + IL6 + co-positive neurons in the DRG at 3 weeks after nerve injury in young (11–16 weeks) and aged (20–24 months) mice (young: n = 215 p21 + IL6 + neurons; n = 51 p16 + IL6 + neurons; n = 102 p21 + p16 + IL6 + neurons; aged: n = 155 p21 + IL6 + neurons; n = 21 p16 + IL6 + neurons; n = 46 p21 + p16 + IL6 + neurons). b , Representative RNAscope images of young or aged DRG co-labeled for the ion channel Trpv1, senescence marker p21 and SASP factor/cytokine IL6. Merged images also have DAPI overlay (gray). For IL6 signal, intense puncta signal with white center are positive neurons, and fainter/dull blue is background. Arrows: Trpv1 + senescent neurons; asterisks: Trpv1 − senescent neurons. Scale bars, 100 µm and 20 µm (insets). c , d , Quantification of Trpv1 neuron population and its co-expression with p21 and/or IL6 in young ( c ) and aged ( d ) L3/4 DRG of uninjured (controls) and 3 weeks after SNI ( n = 3 uninjured young mice, n = 972 Trpv1 + neurons; n = 3 SNI young mice, n = 1,548 Trpv1 + neurons; n = 4 uninjured aged mice, n = 1,056 Trpv1 + neurons; n = 3 SNI aged mice, n = 1,292 Trpv1 + neurons). wk, weeks.

Article Snippet: For in vitro (culture) preparations, coverslips were pre-incubated with either IL6 (50 ng ml −1 with carrier, prepared from 100 μg ml −1 stock solution in 0.1% BSA; R&D Systems, catalogue number 406-ML) or control media (with 0.00005% BSA) for 1 hour before recording, and aCSF containing the same concentration of either IL6 or control BSA was applied (29−32 °C).

Techniques: RNAscope, Labeling, Marker, Expressing

Journal: Nature Neuroscience

Article Title: Aging and injury drive neuronal senescence in the dorsal root ganglia

doi: 10.1038/s41593-025-01954-x

Figure Lengend Snippet: a , Representative traces from p16 -expressing neurons demonstrating repetitive firing (left), hyperpolarization-activated current (Ih) presence (middle) and the firing parameters rheobase and AP latency (right). b , Clusters identified with the hierarchical density-based algorithm HDBSCAN after UMAP alignment of individual neurons constructed with diameter (range, 14–41 µm), firing properties and intrinsic currents. Discrete clusters – are identified by color ( n = 82 recorded DRG neurons from young (11–16 weeks) and aged (20–24 months) mice). UMAP highlighting senescence marker p16 (orange) ( c ), p21 (pink) ( d ) and the SASP factor IL6 (blue) ( e ). f , Heatmap depicting parameters from left to right as follows: clusters (cool gradient), gene expression (black, no expression; light teal, expression), diameter and physiology parameters (warm gradient; higher normalized values are lighter and lower values are darker). Senescence marker p16 and SASP factor IL6 groups contain neurons with high-firing phenotypes (>100 total APs fired during current steps), which is outlined over increasing depolarizing current steps (lower left panel). Ih current amplitude was also measured at decreasing hyperpolarizing steps (lower right panel). g , Senescent neurons (p21 in magenta, p16 in orange and IL6 in blue) display the DRG nociceptor-associated property of wide APs (half-width above 0.5 ms, gray dotted line). h , IL6 application increases evoked firing in senescence marker-expressing neurons in monolayer culture (155.5 ± 18 APs, n = 26 cells from 11 mice for control; 209.7 ± 19.06 APs , n = 27 cells from 13 mice for IL6 application; U = 234.5, P = 0.038, two-sided, Mann–Whitney test; neuronal expression of p21 in magenta, p16 in orange, p21 and p16 in green and p21 and IL6 in blue). All data are mean values ± s.e.m.

Article Snippet: For in vitro (culture) preparations, coverslips were pre-incubated with either IL6 (50 ng ml −1 with carrier, prepared from 100 μg ml −1 stock solution in 0.1% BSA; R&D Systems, catalogue number 406-ML) or control media (with 0.00005% BSA) for 1 hour before recording, and aCSF containing the same concentration of either IL6 or control BSA was applied (29−32 °C).

Techniques: Expressing, Construct, Marker, Gene Expression, Control, MANN-WHITNEY

Journal: Nature Neuroscience

Article Title: Aging and injury drive neuronal senescence in the dorsal root ganglia

doi: 10.1038/s41593-025-01954-x

Figure Lengend Snippet: a , b , Representative RNAscope images from young or aged human L4 DRG showing expression of p21 and p16 senescence markers (enlarged left images with DAPI; scale bar, 100 µm). The large globular signal present in both channels is autofluorescent lipofuscin and not RNAscope signal (small puncta). c , Quantification of p21 + and p16 + neurons in the young and aged human DRG as a percent of total DRG neurons ( n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). d , Quantification of IL6 -expressing neurons as a percent of total DRG neurons ( n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). e , Analysis of IL6 + neuron population and quantification of the co-expression of senescence markers p21 and/or p16 . f , Quantification of neurons co-expressing senescence markers p21 and/or p16 with IL6 as a percent of total DRG neurons ( n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). g , Percent of DRG neurons that are ATF3 + in young and aged human DRG ( n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). h , Example image depicting a single human neuron positive for ATF3 (nuclear-localized, immunohistochemistry) and p21 (RNAscope, puncta). Scale bars, 20 µm. Analysis of ATF3 + neuron population and quantification of the co-expression with p21 in young and aged human DRG (right, donuts) ( n = 64 young ATF3 + DRG neurons, n = 54 aged ATF3 + DRG neurons). i , Total percentage of TRPV1 + neurons as a percent of total DRG neurons in young and aged human DRG. Quantification of the subsets of TRPV1 + neurons that co-express either p21 or p16 by RNAscope (boxed right) ( n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). j , Single representative human neurons (quantified in k ) showing co-expression of TRPV1 with p21 and/or p16 . DAPI in gray. Scale bars, 20 µm. k , Venn diagram of human DRG neurons that express TRPV1 , p16 and p21 . Aged DRG display a greater overlapping fraction of TRPV1 + neurons expressing either or both senescence markers p21 and p16 compared to young neurons. n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG. All data are mean values ± s.e.m.

Article Snippet: For in vitro (culture) preparations, coverslips were pre-incubated with either IL6 (50 ng ml −1 with carrier, prepared from 100 μg ml −1 stock solution in 0.1% BSA; R&D Systems, catalogue number 406-ML) or control media (with 0.00005% BSA) for 1 hour before recording, and aCSF containing the same concentration of either IL6 or control BSA was applied (29−32 °C).

Techniques: RNAscope, Expressing, Immunohistochemistry

Journal: Nature Neuroscience

Article Title: Aging and injury drive neuronal senescence in the dorsal root ganglia

doi: 10.1038/s41593-025-01954-x

Figure Lengend Snippet: Cell diameters (µm) of human DRG neurons co-expressing either p21 + IL6 + , p16 + IL6 + , or p21 + p16 + IL6 + , as a percent of total neurons counted in each population in young ( a ) and aged ( b ) DRG (n = 2 young female (33yo and 32yo) DRG: n = 19 p21 + IL6 + DRG neurons, n = 96 p16 + IL6 + DRG neurons, n = 71 p16 + p21 + IL6 + DRG neurons; n = 2 aged male and female (65yo) DRG: n = 126 p21 + IL6 + DRG neurons, n = 84 p16 + IL6 + DRG neurons, n = 271 p16 + p21 + IL6 + DRG neurons).

Article Snippet: For in vitro (culture) preparations, coverslips were pre-incubated with either IL6 (50 ng ml −1 with carrier, prepared from 100 μg ml −1 stock solution in 0.1% BSA; R&D Systems, catalogue number 406-ML) or control media (with 0.00005% BSA) for 1 hour before recording, and aCSF containing the same concentration of either IL6 or control BSA was applied (29−32 °C).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Expression of CD74 in bladder cancer and its suppression in association with cancer proliferation, invasion and angiogenesis in HT-1376 cells

doi: 10.3892/ol.2018.8309

Figure Lengend Snippet: Association between MIF and CD74 expression with clinical and pathological characteristics of patient samples.

Article Snippet: The CD74 shRNA lentiviral particles (cat. no., sc-35023-V; 1.0×10 6 /200 μl; Santa Cruz Biotechnology, Inc.) targeting the human CD74 transcript and control shRNA lentiviral particles (cat. no., sc-108080; 1.0×10 6 /200 μl; Santa Cruz Biotechnology, Inc.) were used to knock down CD74 in HT-1376 cells.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Expression of CD74 in bladder cancer and its suppression in association with cancer proliferation, invasion and angiogenesis in HT-1376 cells

doi: 10.3892/ol.2018.8309

Figure Lengend Snippet: Expression of MIF and CD74 in tissue samples and cells, and protein expression levels in HT-1376 cells following knockdown of CD74. (A) Little or no expression of immunoreactive CD74 was identified in the urothelial layers of normal bladder and NMIBC samples, but sections of MIBC samples exhibited strong immunoreactive signals of CD74. In contrast, MIF demonstrated strong immunoreactions in the normal and UCB samples. (B) Western blotting assay results for MIF and CD74 in the cultured urothelial SV-HUV-1, SW780, 5637, T24, J82 and HT-1376 cell lines. HT-1376 was the only one identified to express CD74, but all cells expressed MIF to a certain extent. (C) Immunofluorescence microscopy (×100, magnification) indicated positive CD74 staining in HT-1376 cells. (D) CD74 shRNA lentiviral particles abrogated the RNA expression of CD74 in the HT-1376 cells compared with untreated and scramble cells. (E) CD74 short hairpin RNA lentiviral particles abrogated the protein expression of CD74 in the HT-1376 cells when compared with the untreated and scramble cells. (F) Knockdown of CD74 modulated the expression levels of not only Cyclin D1, Cyclin E, but also intranuclear NF-κB p65, pAkt, pErk1/2. (G and H) However, no significant changes in Bcl-2, Bad, cleaved PARP and Erk1/2 were observed among all groups. *intranuclear NF-κB. NMIBC, non-muscle-invasive bladder cancer; MIBC, muscle-invasive bladder cancer; UCB, urothelial cell carcinoma of the bladder; MIF, macrophage migration inhibitory factor; CD74, cluster of differentiation; p65, transcription factor p65; Akt, RAC-alpha serine/threonine protein kinase; p, phosphorylated; Erk1/2, Extracellular regulated protein kinase 1/2; Bcl-2, B-cell lymphoma 2; Bcl-xL, Bcl-extra large; Bax, Bcl-2-associated X protein; PARP, poly(adenosine 5′-diphosphate-ribose) polymerase.

Article Snippet: The CD74 shRNA lentiviral particles (cat. no., sc-35023-V; 1.0×10 6 /200 μl; Santa Cruz Biotechnology, Inc.) targeting the human CD74 transcript and control shRNA lentiviral particles (cat. no., sc-108080; 1.0×10 6 /200 μl; Santa Cruz Biotechnology, Inc.) were used to knock down CD74 in HT-1376 cells.

Techniques: Expressing, Knockdown, Western Blot, Cell Culture, Immunofluorescence, Microscopy, Staining, shRNA, RNA Expression, Migration

Journal: Oncology Letters

Article Title: Expression of CD74 in bladder cancer and its suppression in association with cancer proliferation, invasion and angiogenesis in HT-1376 cells

doi: 10.3892/ol.2018.8309

Figure Lengend Snippet: In vitro analysis of the effects of CD74-knockdown cells. (A) Knockdown of CD74 attenuated cell proliferation in HT-1376 cells compared with the scramble group. *P<0.05 and **P<0.01 vs. Control shRNA. (B) Flow cytometry indicated that knockdown of CD74 significantly increased the proportion of G1 stage cells, decrease G2 stage and S stage ones, compared with scramble shRNA cells. (C) The cell invasion assay demonstrated that knockdown of CD74 significantly attenuated the invasion ability of HT-1376 cells, (D) and the cells of two groups were counted (mean ± SD) and the data of CD74 shRNA group were presented by a percentage of control shRNA group. (E and F) ELISA test indicated that the secretion of (E) VEGF and MMP-9 (F-a) was significantly reduced in CD74-knockdown-HT-1376 cells compared to the shRNA control, while not significantly reduced in MMP-2 (F-b). CD74, cluster of differentiation; VEGF, vascular endothelial growth factor; MMP, matrix metalloproteinase; sh, short hairpin.

Article Snippet: The CD74 shRNA lentiviral particles (cat. no., sc-35023-V; 1.0×10 6 /200 μl; Santa Cruz Biotechnology, Inc.) targeting the human CD74 transcript and control shRNA lentiviral particles (cat. no., sc-108080; 1.0×10 6 /200 μl; Santa Cruz Biotechnology, Inc.) were used to knock down CD74 in HT-1376 cells.

Techniques: In Vitro, Knockdown, Control, shRNA, Flow Cytometry, Invasion Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncology Letters

Article Title: Expression of CD74 in bladder cancer and its suppression in association with cancer proliferation, invasion and angiogenesis in HT-1376 cells

doi: 10.3892/ol.2018.8309

Figure Lengend Snippet: In vivo analysis of the effects of CD74-knockdown cells. (A) Volume of tumors derived from CD74-knockdown cells was significantly decreased compared with those derived from control cells in vivo . (B) The average weight of the tumors was significantly decreased in in the CD74 shRNA group. (C) Untreated HT-1376 xenograft tumors exhibited strong CD74 signals by immunochemistry. (D) CD74 in the wild-type J82 tumor was indicated using immunostaining. *P<0.05 and **P<0.01 vs. Control shRNA. Magnification, ×100.

Article Snippet: The CD74 shRNA lentiviral particles (cat. no., sc-35023-V; 1.0×10 6 /200 μl; Santa Cruz Biotechnology, Inc.) targeting the human CD74 transcript and control shRNA lentiviral particles (cat. no., sc-108080; 1.0×10 6 /200 μl; Santa Cruz Biotechnology, Inc.) were used to knock down CD74 in HT-1376 cells.

Techniques: In Vivo, Knockdown, Derivative Assay, Control, shRNA, Immunostaining

Journal: Nature Communications

Article Title: Inhibition of glycine transporter-1 in the dorsal vagal complex improves metabolic homeostasis in diabetes and obesity

doi: 10.1038/ncomms13501

Figure Lengend Snippet: ( a ) Schematic representation of working hypothesis: glycine transporter-1 (GlyT1) facilitates the cellular uptake of glycine in the dorsal vagal complex (DVC). Chemical (via DVC ALX infusion) or genetic (via DVC lentiviral injection of GlyT1 shRNA) inhibition of GlyT1 increases extracellular glycine levels in the DVC, which potentiates the activation of DVC N -methyl- D -aspartate receptors (NMDAr) to regulate glucose production and glucose tolerance, and food intake and body weight gain. MK-801, NMDAr ion channel blocker; 7-chlorokynurenic acid, 7CKNA-antagonist to the GluN1 subunit of NMDAr. ( b ) Plasma glucose levels (inset: integrated area under the curve (AUC)) and ( c ) plasma insulin levels during ivGTT with DVC infusion of ALX ( n =8, black squares) or saline ( n =7, white squares). † P <0.04, †† P <0.0008 determined by Sidak's multiple comparisons test following repeated-measures ANOVA. * P <0.05 determined by t -test. ( d ) Glucose infusion rates and ( e ) glucose production during clamps with DVC infusion of saline ( n =11), ALX ( n =9), MK801 ( n =9), ALX+MK801 ( n =5), ALX+7CKNA ( n =5), Ad-MM+ALX ( n =5) or Ad-GluN1 shRNA+ALX ( n =5). ( d : * P <0.002 versus saline, MK801, ALX+MK801, and ALX+7CKNA determined by ANOVA and Dunnett's post hoc test; † P <0.002 versus Ad-GluN1 shRNA+ALX determined by t -test; ( e ) * P <0.02 versus saline, MK801, ALX+MK801 and ALX+7CKNA determined by ANOVA and Dunnett's post hoc test; † P <0.0008 versus Ad-GluN1 shRNA+ALX determined by t -test.) ( f ) Glucose infusion rates and ( g ) glucose production during clamps with DVC ALX infusion in vagotomized ( n =7) or sham-operated ( n =5) rats or DVC saline infusion in vagotomized ( n =7) or sham-operated rats ( n =5) rats. (for f and g * P <0.01 compared with all the other groups determined by ANOVA and Dunnet's post hoc test). ( h ) Extracellular glycine levels within the DVC following DVC infusion of ALX ( n =7) or saline ( n =7) in microdialysis studies. * P <0.03 versus saline determined by t- test. Data are shown as the mean+s.e.m.

Article Snippet: In separate sets of experiments, a lentivirus expressing shRNA to GlyT1 (LV-GlyT1 shRNA, 1.0 × 10 6 infectious units; sc-270432-V, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mismatch sequence as a control (LV-MM, 1.0 × 10 6 infectious units; sc-108080, Santa Cruz Biotechnology) was injected in the DVC.

Techniques: Injection, shRNA, Inhibition, Activation Assay, Clinical Proteomics, Saline

Journal: Nature Communications

Article Title: Inhibition of glycine transporter-1 in the dorsal vagal complex improves metabolic homeostasis in diabetes and obesity

doi: 10.1038/ncomms13501

Figure Lengend Snippet: ( a ) Representative western blots and protein levels of plasma membrane GlyT1 (55, 70 and 90 kDa isoforms) normalized to insulin receptor (IR) in DVC wedges of rats 13-day post DVC lentiviral (LV) injection of GlyT1 shRNA (black bars, n =14) or a mismatch sequence (MM; white bars, n =11) as a control. * P <0.01, ** P <0.001 determined by t -test. ( b – d ) Representative western blots and protein levels of plasma membrane GlyT1 (70 and/or 90 kDa isoforms) normalized to IR in sp5, Sp5C, Sp5I (L), sp5, Sp5C, Sp5I (R) and py wedges of rats 13-day post DVC LV injection of GlyT1 shRNA (black bars, n =5) or MM (white bars, n =5). ( e ) Glucose infusion rates and ( f ) glucose production during clamps in rats injected with LV-MM ( n =7), LV-GlyT1 shRNA ( n =7) or LV-GlyT1 shRNA with DVC MK801 infusion ( n =6). ( e : * P <0.006; f : * P <0.003 versus LV-MM control and LV-GlyT1 shRNA+MK801 determined by ANOVA and Dunnett's post hoc test). Data are shown as the mean+s.e.m.

Article Snippet: In separate sets of experiments, a lentivirus expressing shRNA to GlyT1 (LV-GlyT1 shRNA, 1.0 × 10 6 infectious units; sc-270432-V, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mismatch sequence as a control (LV-MM, 1.0 × 10 6 infectious units; sc-108080, Santa Cruz Biotechnology) was injected in the DVC.

Techniques: Western Blot, Clinical Proteomics, Membrane, Injection, shRNA, Sequencing, Control

Journal: Nature Communications

Article Title: Inhibition of glycine transporter-1 in the dorsal vagal complex improves metabolic homeostasis in diabetes and obesity

doi: 10.1038/ncomms13501

Figure Lengend Snippet: ( a ) Experimental protocol for b – e . ( b ) Glucose infusion rates and ( c ) glucose production during clamps in 28-d HFD-fed rats with DVC lentivirus (LV) injection of GlyT1 shRNA ( n =10) or a mismatch sequence (MM, n =9) as a control. ( b , c : * P <0.001 versus 28d-HFD+MM determined by t -test.) ( d ) Body weights on the morning of clamp experiments in 28-d HFD-fed rats with DVC LV MM or GlyT1 shRNA. * P <0.04 determined by t -test. ( e ) Percentage body weight change on days 4 and 5 following DVC injection of LV-MM (white circles, n =9) or GlyT1 shRNA (black circles, n =9) in 28-d HFD-fed rats and of MM fed with regular chow (white squares, n =5). *P <0.05 compared with all other groups determined by ANOVA and Dunnett's post hoc test. Data are shown as the mean+s.e.m.

Article Snippet: In separate sets of experiments, a lentivirus expressing shRNA to GlyT1 (LV-GlyT1 shRNA, 1.0 × 10 6 infectious units; sc-270432-V, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mismatch sequence as a control (LV-MM, 1.0 × 10 6 infectious units; sc-108080, Santa Cruz Biotechnology) was injected in the DVC.

Techniques: Injection, shRNA, Sequencing, Control

Journal: Nature Communications

Article Title: Inhibition of glycine transporter-1 in the dorsal vagal complex improves metabolic homeostasis in diabetes and obesity

doi: 10.1038/ncomms13501

Figure Lengend Snippet: ( a ) Experimental protocol for feeding experiments in rats that received DVC injection of glycine (black squares, n =11) or saline (white squares, n =11). ( b ) Cumulative food intake during the feeding experiment. ( c ) Daily food intake on day 1 and day 2 after food was returned during the feeding experiment. ( d ) Percentage body weight gain on day 1 or day 2 after the feeding experiment. ( e ) Experimental protocol for feeding experiments in the rats that received DVC injection of ALX (black squares, n =8) or saline (white squares, n =8). ( f ) Cumulative food intake during the feeding experiment. ( g ) Daily food intake on day 1 and day 2 after food was returned during the feeding experiment. ( h ) Percentage body weight gain on day 1, day 2 or day 3 after the feeding experiment. ( i ) Percentage body weight change on day 4 following DVC injection of LV-MM ( n =8) or GlyT1 shRNA ( n =10) in regular chow-fed rats. ( j ) Cumulative food intake during the feeding experiment in rats injected with LV-MM ( n =6) or GlyT1 shRNA ( n =7). ( k ) Daily food intake on day 1 and day 2 after food was returned during the feeding experiment. ( l ) Percentage body weight gain on day 1, day 2 or day 3 after the feeding experiment. † P <0.05, †† P <0.01, ††† P <0.001 determined by t- test at each time point, ‡ P <0.05 determined by t -test at each time. Data are shown as the mean+s.e.m.

Article Snippet: In separate sets of experiments, a lentivirus expressing shRNA to GlyT1 (LV-GlyT1 shRNA, 1.0 × 10 6 infectious units; sc-270432-V, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mismatch sequence as a control (LV-MM, 1.0 × 10 6 infectious units; sc-108080, Santa Cruz Biotechnology) was injected in the DVC.

Techniques: Injection, Saline, shRNA

Journal: Central-European Journal of Immunology

Article Title: The effect of interleukin 17 and Toll-like receptor 2 on CD11b expression and apoptosis of neutrophils in zymosaninduced arthritis and paw oedema

doi: 10.5114/ceji.2014.43712

Figure Lengend Snippet: Ly6G + neutrophils in blood of mice with zymosan-induced arthritis. A) Representative dot-plot histogram (from n = 5 mice per group in 5 experiments) showing the frequencies of Ly6G + CD11b + cells among granulocytes in whole blood of PBS and ZIA mice. B) Flow cytometry analysis of whole blood showing the percentages of early apoptotic Annexin V + Ly6G + cells. Data represent the mean ± SEM ( n = 5 mice per group in 5 experiments), ** p < 0.01, Student's t test. C) Representative histograms (from n = 5 mice per group in 5 experiments) showing the expression of CD11b on Annexin V + and Annexin V − neutrophils. Both populations were gated within total Ly6G + cell pool in whole blood. D) Amounts of survival factor GM-CSF in serum of ZIA and PBS mice at day 7. Data represent the mean ± SEM ( n = 5 mice per group in 5 experiments), * p < 0.05, Student's t test

Article Snippet: Neutrophil apoptosis was determined after 0, 6, 12 and 24 hours by Annexin V Apoptosis Detection Kit (Abcam).

Techniques: Flow Cytometry, Expressing

Journal: Central-European Journal of Immunology

Article Title: The effect of interleukin 17 and Toll-like receptor 2 on CD11b expression and apoptosis of neutrophils in zymosaninduced arthritis and paw oedema

doi: 10.5114/ceji.2014.43712

Figure Lengend Snippet: Effect of IL-17 and TLR2 stimulation on the dynamic of Ly6G + cell apoptosis. Apoptosis of purified blood Ly6G + CD11b + cells from A) PBS group and B) ZIA group during culture period of 12 hours, 37°C, 5% CO 2 measured by commercial Annexin V kit. Neutrophils were stimulated with zymosan (20 µg/ml) in the presence or the absence of IL-17 (40 ng/ml). Apoptosis was evaluated at each time point in 6 samples per group and the values represent the mean ± SEM of Annexin V + neutrophils. ** p < 0.01, *** p < 0.001, Student's t test. C) CD11b expression on Annexin V − neutrophils in cultures from the ZIA and PBS groups. Neutrophils from 6-hour cultures were collected, washed and stained with Abs against Ly6G and CD11b and then labelled with Annexin V. During flow cytometry analysis, Annexin V + and Annexin V − cells were gated within the Ly6G + population. Mean fluorescent intensity (MFI) of CD11b on Annexin V − neutrophils was recorded. In order to compare different experiments, the values of MFI were normalised to the control of unstimulated neutrophils isolated from PBS group and expressed as a % versus the control (dashed line). The values represent the mean ± SEM ( n = 5 animals per group in 3 experiments), ** p < 0.01, *** p < 0.001, Student's t -test

Article Snippet: Neutrophil apoptosis was determined after 0, 6, 12 and 24 hours by Annexin V Apoptosis Detection Kit (Abcam).

Techniques: Purification, Expressing, Staining, Flow Cytometry, Isolation

Journal: Central-European Journal of Immunology

Article Title: The effect of interleukin 17 and Toll-like receptor 2 on CD11b expression and apoptosis of neutrophils in zymosaninduced arthritis and paw oedema

doi: 10.5114/ceji.2014.43712

Figure Lengend Snippet: Apoptosis of blood Ly6G + CD11b + cells to IL-17 and TLR2 ligand stimulation in the presence of GM-CSF. Representative density-plot histograms showing apoptosis of neutrophils from A) PBS and B) ZIA group. Blood neutrophils incubated in medium containing GM-CSF (50 ng/ml) were stimulated with zymosan (20 µg/ml) in the absence or presence of IL-17 (40 ng/ml) and cultured at 37°C, 5% CO 2 for 24 hours. Apoptosis was measured by Annexin V-PI staining kit and analysed by flow cytometry. C) Effect of IL-17 and TLR2 on the frequencies of Annexin V + Ly6G + cells in the GM-CSF loaded environment. Data represent the mean ± SEM of Annexin V + cells ( n = 5 animals per group in 3 experiments). * p < 0.05, *** p < 0.001, Student's t test.

Article Snippet: Neutrophil apoptosis was determined after 0, 6, 12 and 24 hours by Annexin V Apoptosis Detection Kit (Abcam).

Techniques: Incubation, Cell Culture, Staining, Flow Cytometry

Journal: Central-European Journal of Immunology

Article Title: The effect of interleukin 17 and Toll-like receptor 2 on CD11b expression and apoptosis of neutrophils in zymosaninduced arthritis and paw oedema

doi: 10.5114/ceji.2014.43712

Figure Lengend Snippet: Administration of IL-17 in zymosan-induced paw oedema. A) Schedule of IL-17 treatment and the induction of paw inflammation. BALB/c mice were i.p. injected with IL-17 or PBS. After 30 minutes they received a sub-plantar injection of 30 µl zymosan suspension (20 mg/ml) at the hind paw and PBS at the contralateral one. B) Paw thickness measured in the medial-lateral direction at different time points post-zymosan injection using a calliper. The results are expressed as the difference (D) in mm between baseline and measurements after zymosan/PBS injection. Each time point represents the mean ± SEM ( n = 7 animals per group in 3 experiments), ANOVA. C) Ly6G + CD11b + cells in whole blood and D) expression of CD11b on Annexin V − neutrophils detected by flow cytometry. E) The frequencies of Annexin V + Ly6G + cells in blood and the amounts of GM-CSF in serum 4 hours after the induction of ZIO. Values in C, D, E and F are the mean ± SEM ( n = 7 animals per group in 3 experiments). * p < 0.05, ** p < 0.01, *** p < 0.001, Student's t test

Article Snippet: Neutrophil apoptosis was determined after 0, 6, 12 and 24 hours by Annexin V Apoptosis Detection Kit (Abcam).

Techniques: Injection, Expressing, Flow Cytometry

Journal: Nature Genetics

Article Title: Single-nucleus multi-omic profiling of human placental syncytiotrophoblasts identifies cellular trajectories during pregnancy

doi: 10.1038/s41588-023-01647-w

Figure Lengend Snippet: a , UMAP shows CTB and STB nuclei profiled with integration of snRNA-seq and snATAC-seq in early pregnancy. b , Heatmap shows pseudotime ordering of the 23,746 DARs in early pregnancy (left). Zoomed-in genomic tracks show the cis -element accessibility of representative genes. The normalized accessibility of DARs is presented with color intensities. c , TF-mining heatmap shows candidate master TFs of each nucleus type. The NES and expression z score are presented with dot color and dot size, respectively. Dots with bold edges show selected TFs used for network construction. d , Regulatory networks with three layers (TF, cis -element and target gene) represent the cis -regulatory architecture covering the complete STB differentiation process. The gene percentage, peak accessibility and gene expression score are presented with circle sizes, edge width and color intensities, respectively. e , smFISH staining (left) and fluorescence intensities (right) show the colocalization of TFs ( STAT5A and FOSL2 ) and representative genes ( PAPPA and FLT1 ) of eSTB mature 1 and eSTB mature 2. hCG, STB marker. The white dotted lines show the outline of CTB. The white lines represent the plotted tracks of fluorescence intensity. f , UMAP shows CTB and STB nuclei profiled with integration of snRNA-seq of STB-CT30, STB-BL and placenta villi in early pregnancy. g , Heatmaps show marker gene expression in STB-BL, STB-CT30 and placental villi in early pregnancy. The expression levels are presented with color intensities. Binarized gene expression levels (positive or negative) are calculated and visualized beside heatmaps for the two marker genes, PAPPA and FLT1 . h , Pseudotime ordering shows three differential trajectories of CTB and STB nuclei (upper left). The differentiation time is presented with color intensities. Quantitative classifications for each cluster (upper right) and specific pseudotime of cluster 9 and cluster 7 on the integrated UMAP (bottom) are shown. i , Heatmap shows the different expression patterns of marker genes, master TFs and representative hormones among in vitro trophoblast models and in vivo placental villi in early pregnancy. The z scores are presented with color intensities. Examples of genes with similar expression patterns are highlighted in bold black, and those with different patterns are colored in red.

Article Snippet: For the induction of STB, hTSCs were seeded in a six-well plate precoated with 2.5 μg ml −1 Collagen I at a density of 1 × 10 5 cells per well and cultured in STB medium (DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% Penicillin–Streptomycin, 0.3% BSA, 1% ITS-X supplement, 2.5 μM Y27632, 2 μM forskolin (MedChemExpress) and 4% Knockout Serum Replacement (KSR, Gibco).

Techniques: Expressing, Gene Expression, Staining, Fluorescence, Marker, In Vitro, In Vivo

Journal: Nature Genetics

Article Title: Single-nucleus multi-omic profiling of human placental syncytiotrophoblasts identifies cellular trajectories during pregnancy

doi: 10.1038/s41588-023-01647-w

Figure Lengend Snippet: a , The schematic illustrates the lentiviral vector used for STAT5A and MITF overexpression. b , Bright-field images show hTSCs-BL-STAT5A OE and STB-BL-STAT5A OE . Scale bar: 100 μm. c , RT–qPCR analysis of STAT5A and target gene expression in STB-BL with DOX-inducible overexpression of STAT5A . Data are shown as mean ± s.d. P values by multiple unpaired two-tailed t test. n = 3 independent experiments. The dotted lines in c and e represent the baseline 1. d , Bright-field images show hTSCs-BL-MITF OE and STB-BL-MITF OE . Scale bar: 100 μm. e , RT–qPCR analysis of MITF and target gene expression in STB-BL with DOX-inducible overexpression of MITF . Data are shown as mean ± s.d. P values by multiple unpaired two-tailed t test. n = 3 independent experiments. f , Verification and visualization of eGRN enhancer-gene regulatory events near two target genes of two STB mature 2 specific TF regulators, CEBPB and FOSL2. Genomic tracks are calculated and compared with CEBPB and FOSL2 CUT&Tag, CEBPB ChIP–seq datasets, which are prepared with STB-BL and hTSC-BL cell lines. IgG mock is used for negative control. Regulatory events are highlighted with the following criteria: eGRN target gene regions (enhancers) should overlap STB-BL CUT&Tag peak(s) and/or ChIP–seq peak(s) but are depleted in hTSC-BL. g , Schematic representation of trophoblast organoids derivation. hTSC-BL are transferred to matrigel droplets on day 0 (D0) and maintained in trophoblast organoid medium to analyze on day 6 (D6). h , Bright fields and immunofluorescence analysis of trophoblast markers ( CDH1 and hCG) in trophoblast organoids derived from hTSCs-BL with DOX-inducible overexpression of MITF . Scale bars: 250 μm for bright-field images, 20 μm for immunofluorescence images. i , ELISA analysis of CSH2 expression in trophoblast organoids derived from hTSCs-BL with DOX-inducible overexpression of MITF . Data are shown as mean ± s.d. P values by unpaired two-tailed t test. n = 3 independent experiments. CDS, coding sequence; hPGK, human phosphoglycerate kinase 1 promoter; P2A: porcine teschovirus-1 2A peptide; Puro, puromycin; TRE3GS, TRE3GS inducible promoter; Tet-on 3G, Tet-on 3G transactivator protein; WPRE, Woodchuck hepatitis virus’s post-transcriptional regulatory element; OE, overexpression.

Article Snippet: For the induction of STB, hTSCs were seeded in a six-well plate precoated with 2.5 μg ml −1 Collagen I at a density of 1 × 10 5 cells per well and cultured in STB medium (DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% Penicillin–Streptomycin, 0.3% BSA, 1% ITS-X supplement, 2.5 μM Y27632, 2 μM forskolin (MedChemExpress) and 4% Knockout Serum Replacement (KSR, Gibco).

Techniques: Plasmid Preparation, Over Expression, Quantitative RT-PCR, Targeted Gene Expression, Two Tailed Test, ChIP-sequencing, Negative Control, Immunofluorescence, Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Leptin enhances hypothalamic lactate dehydrogenase A (LDHA)–dependent glucose sensing to lower glucose production in high-fat–fed rats

doi: 10.1074/jbc.RA117.000838

Figure Lengend Snippet: High-fat feeding impairs hypothalamic glucose sensing. A, experimental protocol. B, cumulative food intake. *, p < 0.05 compared with regular chow (RC)–fed. Body weight (C), fat and lean mass (D), and free water and total water mass (E) of HFD (n = 7)- versus RC (n = 5)-fed rats are shown. F, glucose infusion rate. G, glucose production in basal (white bars) and clamp (black bars) conditions. H, glucose uptake during the clamps. n = 7 for RC-MBH saline, n = 6 for RC-MBH glucose, n = 7 for HFD-MBH saline, and n = 7 for HFD-MBH glucose. Error bars represent S.E. *, p < 0.05 compared with RC-saline, HFD-saline, and HFD-glucose. LV, lentivirus.

Article Snippet: Immediately following brain surgery, a group of rats received 3 μl of lentivirus expressing shRNA to LDHA (1.0 × 10 6 infectious units; sc-270631-V, Santa Cruz Biotechnology, Inc., Dallas, TX) or a mismatch sequence as a control (1.0 × 10 6 infectious units; sc-108080, Santa Cruz Biotechnology, Inc.) through each side of the MBH catheters that target the arcuate nucleus ( 29 , 30 ).

Techniques: Saline

Journal: The Journal of Biological Chemistry

Article Title: Leptin enhances hypothalamic lactate dehydrogenase A (LDHA)–dependent glucose sensing to lower glucose production in high-fat–fed rats

doi: 10.1074/jbc.RA117.000838

Figure Lengend Snippet: Molecular knockdown of hypothalamic LDH negates leptin's impact on glucose sensing. A, representative Western blot of LDHA and LDHB in the MBH of rats that received MBH injections of either MM (n = 4) or LDHA shRNA lentivirus (n = 5). Quantification of LDHA or LDHB levels normalized to β-tubulin expressed as -fold change over MM is shown below. Error bars represent S.E. *, p < 0.05 compared with MM. Glucose infusion rate (B), glucose production in basal (white bars) and clamp (black bars) conditions (C), and glucose uptake during the clamps (D) (n = 7 per group) are shown. Error bars represent S.E. *, p < 0.05 compared with RC-MBH saline and RC-MBH glucose + LDHA shRNA; †, p < 0.05 compared with HFD-MBH saline + MM, HFD-MBH glucose + MM, HFD-MBH glucose + LDHA shRNA, and HFD-MBH glucose + leptin + LDHA shRNA.

Article Snippet: Immediately following brain surgery, a group of rats received 3 μl of lentivirus expressing shRNA to LDHA (1.0 × 10 6 infectious units; sc-270631-V, Santa Cruz Biotechnology, Inc., Dallas, TX) or a mismatch sequence as a control (1.0 × 10 6 infectious units; sc-108080, Santa Cruz Biotechnology, Inc.) through each side of the MBH catheters that target the arcuate nucleus ( 29 , 30 ).

Techniques: Knockdown, Western Blot, shRNA, Saline

Journal: The Journal of Biological Chemistry

Article Title: Leptin enhances hypothalamic lactate dehydrogenase A (LDHA)–dependent glucose sensing to lower glucose production in high-fat–fed rats

doi: 10.1074/jbc.RA117.000838

Figure Lengend Snippet: Plasma insulin and glucose concentrations during the basal and clamp periods relating to Errors represent S.E. (basal, 60–90 min; clamp, 180–210 min).

Article Snippet: Immediately following brain surgery, a group of rats received 3 μl of lentivirus expressing shRNA to LDHA (1.0 × 10 6 infectious units; sc-270631-V, Santa Cruz Biotechnology, Inc., Dallas, TX) or a mismatch sequence as a control (1.0 × 10 6 infectious units; sc-108080, Santa Cruz Biotechnology, Inc.) through each side of the MBH catheters that target the arcuate nucleus ( 29 , 30 ).

Techniques: Clinical Proteomics, shRNA, Saline

Journal: bioRxiv

Article Title: Molecular Imaging of the TGF-β Activating Integrin αvβ6 Detects Chronic Lung Allograft Dysfunction

doi: 10.64898/2026.01.07.698265

Figure Lengend Snippet: (A) Flow cytometric analysis of POD 16-tolerant and CLAD allografts with (A) representative contour plots gated on live CD45-allograft cells (N=5/group) and (B) Intracellular Sftpc expression analysis on CLAD allograft CD326+ αvβ6+ cells. The histogram shown is representative of three independent experiments. (C) Plot of POD 16 allograft numbers of live CD45-CD326+ αvβ6+ cells shown with a mean ± standard deviation from the mean for N=5 per group. Representative immunohistochemical stains with αvβ6 specific antibodies with indicated magnification and scales (N≥4/group).

Article Snippet: Sections were then stained with 1:200 αvβ6 antibodies (bs-5791R, Bioss for mouse tissue; AB00891-23.0, Absolute Antibody for human tissue) overnight at 4°C.

Techniques: Expressing, Standard Deviation, Immunohistochemical staining

Journal: bioRxiv

Article Title: Molecular Imaging of the TGF-β Activating Integrin αvβ6 Detects Chronic Lung Allograft Dysfunction

doi: 10.64898/2026.01.07.698265

Figure Lengend Snippet: αvβ6 immunohistological staining of allografts from recipients that underwent club cell injury and received (A) CCL2 neutralizing Abs, (B) CD8α depleting Abs, respectively, and (A, B) Control Ig injections followed by POD 16 [ 64 Cu]Cu-DOTA-A20-K16R PET/CT imaging. Images shown are representative of N≥4 per group. Scale bars: 400 μm for 10x, 100 μm for 40x.

Article Snippet: Sections were then stained with 1:200 αvβ6 antibodies (bs-5791R, Bioss for mouse tissue; AB00891-23.0, Absolute Antibody for human tissue) overnight at 4°C.

Techniques: Staining, Control, Positron Emission Tomography-Computed Tomography, Imaging

Journal: bioRxiv

Article Title: Molecular Imaging of the TGF-β Activating Integrin αvβ6 Detects Chronic Lung Allograft Dysfunction

doi: 10.64898/2026.01.07.698265

Figure Lengend Snippet: (A, Left Panel) Representative autoradiographic images following incubation of [ 64 Cu]Cu-DOTA-A20-K16R with human explanted lung tissue sections from a patient with CLAD and Control lung tissue from a donor lung obtained prior to transplantation. Preincubation with an excess of a cold probe sharply reduces autoradiographic activity (Cold Probe/CLAD). (A, Right Panel) corresponding trichrome-stained lung sections that were incubated with radiotracer. Images are representative of independently conducted experiments, with (B) violin plots showing combined data from 3 explanted CLAD transplants with or without cold-probe blockade and 3 control lungs per group. Two-sided Mann-Whitney U t-test *p<0.05, ***p<0.001. Representative αvβ6 (C) immunohistochemical and (D) immunofluorescent staining of human explanted lungs with CLAD and control lungs (N=3/group). Arrows denote clusters of cells expressing αvβ6. Scale bars: 400 μm for 10x, 100 μm for 40x.

Article Snippet: Sections were then stained with 1:200 αvβ6 antibodies (bs-5791R, Bioss for mouse tissue; AB00891-23.0, Absolute Antibody for human tissue) overnight at 4°C.

Techniques: Incubation, Control, Transplantation Assay, Activity Assay, Staining, MANN-WHITNEY, Immunohistochemical staining, Expressing