Journal: BMC Developmental Biology

Article Title: Interleukin-6 increases inner cell mass numbers in bovine embryos

doi: 10.1186/s12861-019-0182-z

Figure Lengend Snippet: Transcript abundances for IL6 , IL6R and IL6ST from zygotes, 2-cell embryos, 8-cell embryos, morulae and blastocysts. Total RNA was isolated from 3 to 5 pools of 10 embryos from each developmental stage before reverse transcription. The relative abundance of each target transcript is expressed as fold change from the embryo stage containing the lowest abundance for the specified transcript by using the 2 [-ddCt] approach. Corresponding means and SEMs are indicated by the bars. Different superscripts within each transcript indicates differences ( P < 0.05)

Article Snippet: For all studies, a concentrated IL6 stock (10 μg/ml, recombinant bovine, Kingfisher Biotech, St. Paul, MN, USA) was prepared in SOF containing 1% [ w / v ] bovine serum albumin (BSA; Sigma-Aldrich) and stored in aliquots at − 80 °C.

Techniques: Isolation, Reverse Transcription

Journal: BMC Developmental Biology

Article Title: Interleukin-6 increases inner cell mass numbers in bovine embryos

doi: 10.1186/s12861-019-0182-z

Figure Lengend Snippet: Cleavage and blastocyst formation across each study

Article Snippet: For all studies, a concentrated IL6 stock (10 μg/ml, recombinant bovine, Kingfisher Biotech, St. Paul, MN, USA) was prepared in SOF containing 1% [ w / v ] bovine serum albumin (BSA; Sigma-Aldrich) and stored in aliquots at − 80 °C.

Techniques:

Journal: BMC Developmental Biology

Article Title: Interleukin-6 increases inner cell mass numbers in bovine embryos

doi: 10.1186/s12861-019-0182-z

Figure Lengend Snippet: Embryonic ICM and TE cell counts in Day 8 blastocysts

Article Snippet: For all studies, a concentrated IL6 stock (10 μg/ml, recombinant bovine, Kingfisher Biotech, St. Paul, MN, USA) was prepared in SOF containing 1% [ w / v ] bovine serum albumin (BSA; Sigma-Aldrich) and stored in aliquots at − 80 °C.

Techniques:

Journal: BMC Developmental Biology

Article Title: Interleukin-6 increases inner cell mass numbers in bovine embryos

doi: 10.1186/s12861-019-0182-z

Figure Lengend Snippet: Representative images of differential cell staining in blastocysts collected at day 8 post-fertilization. Embryos either received 0 or 100 ng/ml IL6 beginning at day 5 post-fertilization. Panel A: Blastocysts were harvested at day 8, fixed, immunostained, and physically flattened between a slide and coverslip. Photographs represent a single plane of focus. Nuclei representing TE are indicated by CDX2 + /DAPI + staining (green) and the ICM nuclei are CDX2 − /DAPI + (blue). Control embryo number 1 had 42 ICM cells and 94 TE cells, while control embryo number 2 had 53 ICM cells and 120 TE cells. IL6-treated embryo number 1 had 86 ICM cells and 99 TE cells, while IL6-treated embryo number 2 had 76 ICM cells and 143 TE cells

Article Snippet: For all studies, a concentrated IL6 stock (10 μg/ml, recombinant bovine, Kingfisher Biotech, St. Paul, MN, USA) was prepared in SOF containing 1% [ w / v ] bovine serum albumin (BSA; Sigma-Aldrich) and stored in aliquots at − 80 °C.

Techniques: Staining, Control

Journal: BMC Developmental Biology

Article Title: Interleukin-6 increases inner cell mass numbers in bovine embryos

doi: 10.1186/s12861-019-0182-z

Figure Lengend Snippet: Pooled ICM cell counts and ICM to TE ratios from all studies. All 100 ng/ml treatments of IL6 and associated controls were utilized, regardless of time point of treatment. No other doses of IL6 (1, 10 or 200 ng/ml) are included in this figure. Data from different studies are indicated by different symbols. Panel A : Individual ICM counts for embryos receiving either no treatment or 100 ng/ml IL6. Panel B : Individual ICM:TE ratios for embryos receiving either no treatment or 100 ng/ml IL6. Corresponding means and SEMs are indicated by the bars. Different superscripts within each panel indicates differences ( P < 0.05)

Article Snippet: For all studies, a concentrated IL6 stock (10 μg/ml, recombinant bovine, Kingfisher Biotech, St. Paul, MN, USA) was prepared in SOF containing 1% [ w / v ] bovine serum albumin (BSA; Sigma-Aldrich) and stored in aliquots at − 80 °C.

Techniques:

Journal: BMC Developmental Biology

Article Title: Interleukin-6 increases inner cell mass numbers in bovine embryos

doi: 10.1186/s12861-019-0182-z

Figure Lengend Snippet: Primers used for quantitative RT-PCR

Article Snippet: For all studies, a concentrated IL6 stock (10 μg/ml, recombinant bovine, Kingfisher Biotech, St. Paul, MN, USA) was prepared in SOF containing 1% [ w / v ] bovine serum albumin (BSA; Sigma-Aldrich) and stored in aliquots at − 80 °C.

Techniques: Sequencing

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: Journal of Thoracic Disease

Article Title: SIX1 mediates pulmonary arterial hypertension endothelial dysfunction through the IL-6/STAT3 axis

doi: 10.21037/jtd-2026-1-0101

Figure Lengend Snippet: SuHx group induces pulmonary arterial hypertension and increased mRNA expression of IL-6. (A,B) RVSP was significantly higher in the pulmonary hypertension group (SuHx) compared to the control group (Control) (n=6; ****, P<0.0001). (C) The right ventricular hypertrophy index [RV/(LV + S), Fulton index] was also significantly increased in the SuHx group relative to the Control group (n=6; ****, P<0.0001). (D) H&E staining revealed marked vessel wall thickening and lumen narrowing in small pulmonary arteries with diameters <50 µm and 50–100 µm in the SuHx group. Black arrows indicate vascular structures. Scale bars: 20 µm, 100 µm. (E,F) Quantitative analysis of vascular thickness in all mice was performed using Image J software. The intimal thickness was calculated using the formula: (outer diameter – inner diameter)/outer diameter. The bar graph shows pulmonary artery thickness (µm) on the y-axis, with the Control and SuHx groups on the x-axis; each dot represents the mean vascular thickness of an individual mouse. Pulmonary artery thickness was significantly greater in the SuHx group than in the Control group (n=6; *, P<0.05; **, P<0.01). (G,H) Collagen deposition in pulmonary vessels was assessed by Masson staining. Black arrows indicate vessels, and blue areas represent collagen fibers. The percentage of collagen deposition was calculated as: (collagen fiber area/total vessel area) × 100%. Quantitative analysis of perivascular collagen deposition in all mice was performed using Image J software. Each dot represents the mean percentage of collagen deposition in an individual mouse. The results showed that the percentage of perivascular collagen deposition was significantly higher in the SuHx group than in the Control group (n=6; **, P<0.01). Scale bars: 20 µm, 100 µm. (I) RT-qPCR analysis of lung tissue showed that IL-6 mRNA expression levels were significantly higher in the SuHx group than in the Control group (n=6; **, P<0.01). Statistical analysis: all quantitative data were obtained from 6 independent mice. Data were analyzed using GraphPad Prism 10.1.2 software. Data are presented as mean ± standard deviation. Normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was evaluated using the F-test. The results showed that the data conformed to a normal distribution (P>0.05) and exhibited homogeneity of variance (P>0.05). Comparisons between two groups were performed using an independent samples t -test (two-tailed), and P<0.05 was considered statistically significant. H&E, hematoxylin-eosin; IL-6, interleukin-6; LV, left ventricle; mRNA, messenger RNA; RT-qPCR, reverse transcription quantitative polymerase chain reaction; RV, right ventricular; RVSP, right ventricular systolic pressure; S, septum.

Article Snippet: A working stock of recombinant human IL-6 (C610007, Sangon Biotech, Shanghai, China) was prepared in sterile phosphate-buffered saline (PBS).

Techniques: Expressing, Control, Staining, Software, Quantitative RT-PCR, Standard Deviation, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Thoracic Disease

Article Title: SIX1 mediates pulmonary arterial hypertension endothelial dysfunction through the IL-6/STAT3 axis

doi: 10.21037/jtd-2026-1-0101

Figure Lengend Snippet: SIX1 mediates IL-6-induced STAT3 activation and interaction in HUVECs. (A) CellTiter-Glo assay was used to assess the proliferation rate of HUVECs stimulated with different concentrations of IL-6 (0–200 ng/mL) for 24 h. The results showed that, compared to the NC group, IL-6 at 100 ng/mL exhibited the most significant pro-proliferative effect (****, P<0.0001). (B) RT-qPCR results indicated that, compared to the NC group and other IL-6 concentrations, the mRNA expression level of SIX1 was highest in the IL-6 (100 ng/mL) group (**, P<0.01). (C) Western blot analysis of SIX1 protein expression in HUVECs treated with NC or IL-6 (100 ng/mL, 24 h). Compared with the NC group, IL-6 stimulation significantly upregulated SIX1 protein expression (*, P<0.05). Subsequent experiments were performed using IL-6 at 100 ng/mL. (D,E) The knockdown efficiency of SIX1 was assessed by Western blot and RT-qPCR. Compared with IL-6 combined with siRNA negative control (IL-6 + siNC), the IL-6 + SIX1 siSIX1-3 (IL-6 + siSIX1-3) group exhibited the highest knockdown efficiency (*, P<0.05; ****, P<0.0001). (F,G) Western blot analysis of SIX1 , total STAT3, p-STAT3, and β-actin protein expression in HUVECs under different treatment conditions: NC, vehicle control, IL-6 (100 ng/mL, 24 h), IL-6 combined with siNC (IL-6 + siNC), IL-6 combined with SIX1 siRNA (IL-6 + siSIX1), LIF stimulation (30 ng/mL, 24 h) as an alternative activator of the JAK-STAT pathway, and IL-6 neutralizing antibody pretreatment followed by IL-6 stimulation (anti-IL-6 + IL-6). Quantitative results showed that compared to the NC group, IL-6 significantly increased the expression of SIX1 and p-STAT3 (**, P<0.01); compared to the IL-6 + siNC group, the IL-6 + siSIX1 group exhibited decreased p-STAT3 and SIX1 protein expression (*, P<0.05; ***, P<0.001); compared to the IL-6 group, the anti-IL-6 + IL-6 group also showed significantly reduced SIX1 and p-STAT3 expression (**, P<0.01). (H) IF analysis of SIX1 (green) and total STAT3 (red) colocalization in HUVECs under different treatment conditions. Nuclei were stained with DAPI (blue). Merged images show colocalization signals in white. Scale bar: 10 μm. (I) Quantitative analysis of colocalization signals using Image J software. Pearson’s R and Overlap R were used to evaluate the degree of colocalization, following the same method as in (F). The results showed that compared to the NC group, the IL-6 group exhibited significantly increased colocalization of SIX1 and STAT3 (**, P<0.01; ***, P<0.001); compared to the IL-6 + siNC group, the IL-6 + siSIX1 group showed significantly reduced colocalization (**, P<0.01; ***, P<0.001); compared to the IL-6 group, the anti-IL-6 + IL-6 group also exhibited significantly reduced colocalization (**, P<0.01; ***, P<0.001). Statistical analysis: all quantitative data were obtained from three independent experiments, each performed in triplicate. Each data point in the figures represents the mean of one independent experiment (n=3). Data analysis was performed using GraphPad Prism 10.1.2 software. All data are presented as mean ± standard deviation. Normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was evaluated using the F-test. The results confirmed that the data followed a normal distribution (P>0.05) and exhibited homogeneous variances (P>0.05). Comparisons between two groups were conducted using an independent samples t -test (two-tailed). Multiple group comparisons were performed using one-way ANOVA, followed by Tukey’s multiple comparison test for pairwise comparisons when overall significance was detected. A P value <0.05 was considered statistically significant. ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; HUVEC, human umbilical vein endothelial cell; IF, immunofluorescence; IL-6, interleukin-6; LIF, leukemia inhibitory factor; mRNA, messenger RNA; NC, negative control; p-STAT3, phosphorylated STAT3; RT-qPCR, reverse transcription quantitative polymerase chain reaction; siNC, negative control siRNA; siRNA, small interfering RNA; siSIX1-3, the third of three independent SIX1-targeting siRNAs.

Article Snippet: A working stock of recombinant human IL-6 (C610007, Sangon Biotech, Shanghai, China) was prepared in sterile phosphate-buffered saline (PBS).

Techniques: Activation Assay, Glo Assay, Quantitative RT-PCR, Expressing, Western Blot, Knockdown, Negative Control, Control, Staining, Software, Standard Deviation, Two Tailed Test, Comparison, Immunofluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Journal of Thoracic Disease

Article Title: SIX1 mediates pulmonary arterial hypertension endothelial dysfunction through the IL-6/STAT3 axis

doi: 10.21037/jtd-2026-1-0101

Figure Lengend Snippet: SIX1 promotes IL-6-induced excessive proliferation and migration of HUVECs. (A) EdU assay was performed to assess the proliferation of HUVECs. Proliferating cells were labeled with EdU (green), and nuclei were stained with DAPI (blue). Scale bar: 100 μm. Quantitative analysis of (A) shown in (D) revealed that compared to the NC group, IL-6 stimulation significantly promoted cell proliferation (***, P<0.001), whereas knockdown of SIX1 (IL-6 + siSIX1) significantly suppressed the IL-6-induced proliferative effect (compared to the IL-6 + siNC group, **, P<0.01). (B) Wound healing assay was performed to evaluate the migration ability of HUVECs. Yellow lines indicate the wound edges. Scale bar: 200 μm. Quantitative analysis of (B) shown in (E) demonstrated that compared to the NC group, IL-6 stimulation significantly promoted cell migration (**, P<0.01), whereas knockdown of SIX1 significantly suppressed the IL-6-induced migratory effect (compared to the IL-6 + siNC group; **, P<0.01). (C) CellTiter-Glo assay was used to measure cell proliferation rate. The results showed that compared to the NC group, HUVECs in the IL-6 group exhibited a higher proliferation rate (****, P<0.0001), whereas knockdown of SIX1 significantly attenuated this effect (compared to the IL-6 + siNC group, ****, P<0.0001), further confirming that SIX1 knockdown inhibits HUVEC proliferation. Statistical analysis: all quantitative data were obtained from three independent experiments, each performed in triplicate. Each data point in the figures represents the mean of one independent experiment (n=3). Data analysis was performed using GraphPad Prism 10.1.2 software. All data are presented as mean ± standard deviation. Normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was evaluated using the F-test. The results confirmed that the data followed a normal distribution (P>0.05) and exhibited homogeneous variances (P>0.05). Multiple group comparisons were performed using one-way ANOVA, followed by Tukey’s multiple comparison test for pairwise comparisons when overall significance was detected. A P value <0.05 was considered statistically significant. ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin-6; NC, negative control; siNC, negative control siRNA; siSIX1, the most efficient SIX1‑targeting siRNA, corresponding to siSIX1‑3.

Article Snippet: A working stock of recombinant human IL-6 (C610007, Sangon Biotech, Shanghai, China) was prepared in sterile phosphate-buffered saline (PBS).

Techniques: Migration, EdU Assay, Labeling, Staining, Knockdown, Wound Healing Assay, Glo Assay, Software, Standard Deviation, Comparison, Negative Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Human Recombinant Interleukin-6 and Leukemia Inhibitory Factor Improve Inner Cell Mass Cell Number but Lack Cryoprotective Activities on In Vitro-Produced Bovine Blastocysts

doi: 10.3390/ani15050668

Figure Lengend Snippet: Effects of human recombinant IL6, IL11, and LIF supplementation on blastocyst development or blastocyst stage. Embryos were treated with either 0 (control) or 100 ng/mL IL6, IL11, or LIF from d 5–8 post-fertilization ( n = 344–367 embryos/treatment over 6 replicates). Blastocyst stage was assessed on d 7 and 8. Shown are d 7 blastocyst development (based on numbers of cleaved embryos ( A ), proportion of blastocyst stages on d 7 ( B ), d 8 blastocyst development (based on numbers of cleaved embryos) ( C ), and proportion of blastocyst stages on d 8 ( D )). ( A , C ) show means and standard error of the means (SEM). Different superscripts indicate statistical differences within each graph ( p ≤ 0.05).

Article Snippet: Concentrated stocks of recombinant human IL6, IL11, and LIF (10 μg/mL; R&D Systems, Minneapolis, MN, USA) were prepared in SOF base stock solution with 1% [ w / v ] bovine serum albumin (BSA).

Techniques: Recombinant, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Human Recombinant Interleukin-6 and Leukemia Inhibitory Factor Improve Inner Cell Mass Cell Number but Lack Cryoprotective Activities on In Vitro-Produced Bovine Blastocysts

doi: 10.3390/ani15050668

Figure Lengend Snippet: Human recombinant IL6 and LIF influence the cellular composition of IVP bovine blastocysts. Embryos were treated with either 0 (control) or 100 ng/mL IL6, IL11, or LIF from d 5–8 post fertilization. A subset of blastocysts was processed for immunofluorescence for quantification of ICM and TE cell numbers on d 8 ( n = 35–38 blastocysts/treatment over 6 replicates). Shown are total cell numbers ( A ), number of trophectoderm (TE) ( B ) and inner cell mass (ICM) cells ( C ), and the ICM/TE ratio ( D ). ( E – H ) contain representative images of blastocysts from the control, IL6, IL11, and LIF treatments, respectively. Scale bar = 180 μm. Different superscripts indicate statistical differences within each graph ( p ≤ 0.05). ( A – C ) show means and standard error of the means (SEM). ( D ) shows the median ICM/TE ratio with first quartile boxes from all datelines.

Article Snippet: Concentrated stocks of recombinant human IL6, IL11, and LIF (10 μg/mL; R&D Systems, Minneapolis, MN, USA) were prepared in SOF base stock solution with 1% [ w / v ] bovine serum albumin (BSA).

Techniques: Recombinant, Control, Immunofluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Human Recombinant Interleukin-6 and Leukemia Inhibitory Factor Improve Inner Cell Mass Cell Number but Lack Cryoprotective Activities on In Vitro-Produced Bovine Blastocysts

doi: 10.3390/ani15050668

Figure Lengend Snippet: Treatment with IL6 and LIF influence cell composition in regular and advanced blastocysts. Embryos were treated with 100 ng/mL of either IL6, IL11, or LIF from d 5–8 post fertilization. On d 8, regular ( n = 15–17 blastocysts/treatment over 6 replicates) and advanced (expanded and hatching) ( n = 17–23 blastocysts/treatment over 6 replicates) blastocysts were fixed and processed for immunofluorescence and quantification of inner cell mass (ICM) and trophectoderm (TE) cells. Shown are total number of cells in regular blastocysts ( A ), total number of TE cells in regular blastocysts ( B ), total number of ICM cells in regular blastocysts ( C ), ICM/TE ratio in regular blastocysts ( D ), total number of cells in advanced blastocysts ( E ), total number of TE cells in advanced blastocysts ( F ), total number of ICM cells in advanced blastocysts ( G ), and ICM/TE ratio in advanced blastocysts ( H ). Different letters indicate statistical differences ( p ≤ 0.05). ( A – C , E – G ) show means and standard error of the means (SEM). ( D , H ) show the median ICM/TE ratio with first quartile boxes from all datelines.

Article Snippet: Concentrated stocks of recombinant human IL6, IL11, and LIF (10 μg/mL; R&D Systems, Minneapolis, MN, USA) were prepared in SOF base stock solution with 1% [ w / v ] bovine serum albumin (BSA).

Techniques: Immunofluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Human Recombinant Interleukin-6 and Leukemia Inhibitory Factor Improve Inner Cell Mass Cell Number but Lack Cryoprotective Activities on In Vitro-Produced Bovine Blastocysts

doi: 10.3390/ani15050668

Figure Lengend Snippet: Human recombinant IL6, IL11, and LIF supplementation did not improve IVP embryo cryotolerance. Embryos were treated with either 0 (control), or 100 ng/mL IL6, IL11, or LIF from d 5–8 post fertilization. On d 8, a subset of blastocysts was slow frozen and thawed for 24 h (n = 74–97 blastocysts/treatment over 6 replicates). Re-expanded and hatched blastocysts were processed for immunofluorescence and quantification of the inner cell mass (ICM) and trophectoderm (TE) cells ( n = 25–39 embryos/treatment over 6 replicates). Shown are the blastocyst re-expansion after cryopreservation and thawing ( A ), blastocyst hatching after cryopreservation and thawing ( B ), total cell number of sampled frozen and thawed blastocysts ( C ), trophectoderm (TE) cells of sampled frozen and thawed blastocysts ( D ), ICM cells of sampled frozen and thawed blastocysts ( E ), and ICM/TE ratio of sampled frozen and thawed blastocysts ( F ). Different letters indicate statistical differences ( p ≤ 0.05). ( A – E ) show means and standard error of the means (SEM). ( F ) shows the median ICM/TE ratio with first quartile boxes from all datelines.

Article Snippet: Concentrated stocks of recombinant human IL6, IL11, and LIF (10 μg/mL; R&D Systems, Minneapolis, MN, USA) were prepared in SOF base stock solution with 1% [ w / v ] bovine serum albumin (BSA).

Techniques: Recombinant, Control, Immunofluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Human Recombinant Interleukin-6 and Leukemia Inhibitory Factor Improve Inner Cell Mass Cell Number but Lack Cryoprotective Activities on In Vitro-Produced Bovine Blastocysts

doi: 10.3390/ani15050668

Figure Lengend Snippet: Percentage of TUNEL positive cells after thawing in IL6, IL11, and LIF treated embryos. Embryos were treated 100 ng/mL of either IL6, IL11, or LIF from d 5–8 post fertilization. A subset of blastocysts was slow frozen and thawed for 24 h. Re-expanded and hatched blastocysts were processed for immunofluorescence and quantification of apoptosis (TUNEL) in the inner cell mass (ICM) and trophectoderm (TE) cells ( n = 25–39 blastocysts/treatment over 6 replicates). Shown are the percentage of total TUNEL positive cells in frozen and thawed embryos ( A ), percentage of TUNEL positive TE cells ( B ), and percentage of TUNEL positive ICM cells ( C ). ( D – H ) contain representative images of blastocysts from DNase-treated positive controls, non-treated controls, and the IL6, IL11, and LIF treatments, respectively. Scale bar = 180 μm. ( A – C ) show means and standard error of the means (SEM). Different letters indicate statistical differences ( p ≤ 0.05).

Article Snippet: Concentrated stocks of recombinant human IL6, IL11, and LIF (10 μg/mL; R&D Systems, Minneapolis, MN, USA) were prepared in SOF base stock solution with 1% [ w / v ] bovine serum albumin (BSA).

Techniques: TUNEL Assay, Immunofluorescence