Journal: Nature Aging

Article Title: Epigenetic dysregulation in aged muscle stem cells drives mesenchymal progenitor expansion via IL-6 and Spp1 signaling

doi: 10.1038/s43587-025-01002-0

Figure Lengend Snippet: a , Representative images of co-cultures of control or i Ezh2 −/− MuSCs and FAPs. The number of Pax7 + cells is reduced and FAPs are increased in i Ezh2 −/− MuSCs and FAPs co-cultures. The experiment was independently repeated three times with similar results. b , Bulk-RNA-seq tracks of IL-6 and Spp1 in FAPs isolated from either control or i Ezh2 −/− mice. c , EdU incorporation in co-cultures of MuSCs and PDGFRα EGFP + FAPs untreated or treated with either IL-6R or Spp1 neutralizing antibodies. MuSCs were isolated from either control PDGFRα EGFP or i Ezh2 −/− ; PDGFRα EGFP mice; FAPs were isolated from control PDGFRα EGFP mice. Arrows indicate GFP + EdU + cells. Right panel shows quantification of PDGFRα EGFP + EdU + cells. n= number of regions counted (n = 10), with cells pooled from three independent experiments. ** P = 0.0069, *** P = 0.0004 (untreated Ctrl MuSCs vs. untreated i Ezh2 −/− MuSCs), *** P = 0.0005 (untreated i Ezh2 −/− MuSCs vs. IL6R-treated i Ezh2 −/− MuSCs), ns= non-significant. d , αSMA immunostaining of co-cultured MuSCs and PDGFRα EGFP + FAPs untreated or treated with either IL-6R antibody or Spp1 neutralizing antibodies. MuSCs were isolated as described above. Right panel shows quantification of PDGFRα EGFP + αSMA + cells. n= number of regions counted (n = 10), with cells pooled from three independent experiments. * P = 0.0223 (untreated Ctrl MuSCs vs. untreated i Ezh2 −/− MuSCs), * P = 0.0169 (untreated i Ezh2 −/− MuSCs vs. IL6R-treated i Ezh2 −/− MuSCs), ** P = 0.0038, ns= non-significant. Two-way ANOVA with Sidak’s multiple comparisons ( c, d ).

Article Snippet: A solution of the anti-IL-6R monoclonal antibody (1 mg ml −1 ) (Bio X Cell, BE0047) and the mouse Spp1 polyclonal antibody (200 μg ml −1 ) (R&D Systems, AF808) were prepared in PBS shortly before the injections.

Techniques: Control, RNA Sequencing, Isolation, Immunostaining, Cell Culture

Journal: Nature Aging

Article Title: Epigenetic dysregulation in aged muscle stem cells drives mesenchymal progenitor expansion via IL-6 and Spp1 signaling

doi: 10.1038/s43587-025-01002-0

Figure Lengend Snippet: a , Representative images of Pax7 immunostaining and GFP reactivity of co-cultures with MuSCs isolated from either adult (top panel) or aged (bottom panel) (24-month-old) mice at 3 dpi and FAPs isolated from Pax7 creER ; PDGFRα EGFP adult mice at 3 dpi ( n = 3 independent experiments). b , EdU incorporation, GFP reactivity and αSMA immunostaining of co-cultured MuSCs and GFP + FAPs. Arrows indicate PDGFRα EGFP + EdU + cells. Right panel shows quantification of PDGFRα EGFP + , PDGFRα EGFP + EdU + and PDGFRα EGFP + αSMA + cells. n = number of regions counted, with cells pooled from three independent experiments ( n = 45 for PDGFRα EGFP + and PDGFRα EGFP + EdU + and n = 17 for PDGFRα EGFP + αSMA + ), **** P < 0.0001, two-tailed unpaired t -test. c , Bar plots showing IL-6 and Spp1 concentrations in the supernatants from adult, i Ezh2 −/− or aged MuSCs. Data are presented as mean ± s.d. ( n = 4 independent experiments) * P = 0.0461 (IL-6 panel) * P = 0.0321 (Spp1 panel), ** P = 0.0069 (IL-6 panel), *** P = 0.0001 (Spp1 panel). One-way ANOVA with Dunnett’s multiple comparison. d , Spp1 immunostaining of TA muscle sections from adult, i Ezh2 −/− and aged mice at 7 dpi. DAPI marks nuclei. Three muscle sections per mouse were analyzed ( n = 3 mice). e , EdU incorporation in co-cultures of MuSCs and PDGFRα EGFP + FAPs untreated or treated with either IL-6R or Spp1 neutralizing antibodies. MuSCs were isolated from either adult or aged mice; FAPs were isolated from Pax7 creER ; PDGFRα EGFP mice. Arrows indicate GFP + EdU + cells. Bottom panel shows quantification of PDGFRα EGFP + EdU + cells. n = number of regions counted, with cells pooled from three independent experiments ( n = 22) **** P = < 0.0001. f , αSMA immunostaining of co-cultured MuSCs and PDGFRα EGFP + FAPs untreated or treated with either IL-6R antibody or Spp1 neutralizing antibodies. MuSCs were isolated as described above. Right panel shows quantification of PDGFRα EGFP + αSMA + cells. n = number of regions counted, with cells pooled from three independent experiments ( n = 22) **** P = < 0.0001, NS, non-significant. Two-way ANOVA with Sidak’s multiple comparisons ( e , f ). αIL6R, against-IL6R antibody; αSppl1, against-Spp1 antibody.

Article Snippet: A solution of the anti-IL-6R monoclonal antibody (1 mg ml −1 ) (Bio X Cell, BE0047) and the mouse Spp1 polyclonal antibody (200 μg ml −1 ) (R&D Systems, AF808) were prepared in PBS shortly before the injections.

Techniques: Immunostaining, Isolation, Cell Culture, Two Tailed Test, Comparison

Journal: Nature Aging

Article Title: Epigenetic dysregulation in aged muscle stem cells drives mesenchymal progenitor expansion via IL-6 and Spp1 signaling

doi: 10.1038/s43587-025-01002-0

Figure Lengend Snippet: a , Schematic of IL-6R and Spp1 antibodies injection in aged (24-month-old) mice. b , Pdgfrα immunostaining of TA muscle sections from adult or aged mice injected with either PBS or Spp1 and IL-6R antibodies at 14 dpi. DAPI identifies cell nuclei. On the right is the quantification of Pdgfrα + cells. Two muscle sections per mouse were analyzed. Data are presented as mean ± s.d., two-tailed unpaired t -test, n = 4 mice, * P = 0.0425. c , Representative images of Spp1 immunostaining of TA muscle sections from aged mice injected with either PBS or Spp1 and IL-6R antibodies at 14 dpi. DAPI identifies cell nuclei. The experiment was independently repeated three times with similar results. d , Laminin immunostaining of TA muscle sections from aged mice treated with PBS (untreated) or with Spp1 and IL-6R antibodies at 14 dpi. DAPI identifies cell nuclei. Right panel shows quantification of myofiber CSA analyzed. Two muscle sections per mouse were analyzed. Data are presented as mean ± s.d., two-tailed unpaired t -test. n = 8 TAs from four PBS-treated or against-Spp1 antibody-treated and against-IL6R antibody-treated mice, respectively, * P = 0.0197 (0−200-μm 2 fibers). e , Picrosirius Red staining and quantification of fibrotic area in TA muscle sections from aged mice injected with PBS or with Spp1 and IL-6R antibodies at 14 dpi. Two muscle sections per mouse were analyzed. Data are presented as mean ± s.d., two-tailed unpaired t -test. n = 6 TA from three PBS-treated or against-Spp1 antibody-treated and against-IL-6R antibody-treated mice-treated mice, respectively, * P = 0.0338. αIL6R, against-IL6R antibody; αSppl1, against-Spp1 antibody.

Article Snippet: A solution of the anti-IL-6R monoclonal antibody (1 mg ml −1 ) (Bio X Cell, BE0047) and the mouse Spp1 polyclonal antibody (200 μg ml −1 ) (R&D Systems, AF808) were prepared in PBS shortly before the injections.

Techniques: Injection, Immunostaining, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Enhancement of activation-induced T cell proliferation by SIRPG in a CD47-independent manner

doi: 10.1101/2025.05.01.651731

Figure Lengend Snippet: A . PBMC from healthy donors were stimulated with anti-CD3/anti-CD28 and restimulated with anti-CD3 on day 6. The surface level of SIRPG in CD4+ T cells was analyzed with FACS and shown in the overlay histograms (the left panel). The MFI of SIRPG from three independent donors over the time course is shown in the right panel. Adalimumab (ada), certolizumab (cer), etanercept (eta), tocilizumab (toc), or control IgG1 was added on day 0 during the stimulation. The MFI of SIRPG from non-blasting ( B ) and blasting ( C ) T cells is shown in the overlay histograms. The cumulative results from 3 independent experiments are shown in the corresponding bar graphs. D . PBMC were stimulated in the presence or absence of ada and the surface level of SIRPG was monitored with FACS at indicated time points. Representative data from one donor is shown in the overlay histograms and cumulated results from five donors are shown in the right panel. The data points from the same donors are connected with lines. E . PBMC were stimulated and ada or control IgG was added on day 3. The level of SIRPG was analyzed on day 6. Representative overlay histograms are shown in the left panel and the cumulative results from three donors are shown in the right panel. F . PBMC were treated with ada or control IgG without anti-CD3 stimulation. The surface level of SIRPG in T and non-T cells was analyzed on day 3. Representative overlay histograms are shown in the left panel and cumulative results from three donors are shown in the right panel.

Article Snippet: PBMCs were plated in 24-well plates (2-2.5 millions/1ml/well) pre-coated with anti-CD3 (2,5ug/ml, OKT3, Supplemental Table 3) and soluble anti-CD28 (2,0ug/ml, CD28.2, Supplemental Table 3) and stimulated with adalimumab (125ug/ml), etanercept (50ug/ml), certolizumab pegol (50ug/ml), tocilizumab (100ug/ml), or an isotype control anti-human IgG1 (125ug/ml, #BE0297, BioXCell, Lebanon, NH, USA) for indicated times before analyzing by flow cytometry.

Techniques: Control

Journal: PloS one

Article Title: Association of interleukin-6 signalling with the muscle stem cell response following muscle-lengthening contractions in humans.

doi: 10.1371/journal.pone.0006027

Figure Lengend Snippet: Figure 1. Circulating IL-6 and muscle IL-6 mRNA and IL-6 receptor response muscle lengthening contractions (MLC). (1a) Average serum interleukin-6 (IL-6) concentration. Time 0 hr corresponds to pre-intervention values; all other time-points correspond to post-intervention time (hr). (1b) Relative IL-6 mRNA expression, expressed as fold-change from 0 hr. (1c) Pearson correlation of serum IL-6 concentration versus muscle IL-6 mRNA (fold-change), correlation is representative of the individual data points and presented as mean values ($)6SD (error bars). (1d) Relative IL-6 receptor (IL-6Ra) mRNA expression, expressed as fold-change from 0 hr. (1e) Immunofluorescent image (406) of a muscle cross-section triple-stained for Pax7 (red), IL-6Ra (green) and nuclei (DAPI = blue); IL-6Ra staining is apparent on the sarcolemma and satellite cell membrane. White arrow denotes one of the Pax7+ nuclei which co-localized with IL-6Ra (scale bar = 100 mm). Values are reported as mean6S.E.M. Mean values represent the mean for all 8 subjects per time-point (8 samples per time-point, 40 samples total). *p,0.05 vs. 0 hr. doi:10.1371/journal.pone.0006027.g001

Article Snippet: Immunofluorescence 7 mm sections were cryosectioned and stained with antibodies against Pax7 (neat; DSHB, USA); IL-6 (500 ng/mL, MAB 2061, R&D Systems, USA); p-STAT3 (p-STAT3 Y705 1:100, Cell Signaling Technologies Inc., USA); IL-6Ra (1:50, MCA822, Serotec, UK); PCNA (1:200, ab15497, Abcam Inc., USA); and Laminin (1:1000, L8271, Sigma-Aldrich, Canada).

Techniques: Concentration Assay, Expressing, Staining, Membrane

Journal: BioMed Research International

Article Title: The Role of IL-6RA in UHMWPE Promotes Proliferation in Fibro-Like Synovial Cells

doi: 10.1155/2018/3928915

Figure Lengend Snippet: UHMWPE increased the expression of mIL-6R protein. FLS cells were incubated with UHMWPE (0, 0.01, 0.1, and 1g/L) for 7 d. The expression of IL-6R protein was detected by western blotting assay.

Article Snippet: The primary antibodies included caspase-3(Abcam, USA; ab4051), cleaved-caspase-3 (Cell Signaling Technology, USA; #9661), Bax (Abcam, USA; ab182733), Bcl-2 (Abcam, USA; ab692), IL-6R (Boster, China; A01425-1), β -actin (Bioss, China; bs-0061R), and GAPDH (Abcam, USA; ab8245) at 1:500 dilutions.

Techniques: Expressing, Incubation, Western Blot