Journal: Cell Research

Article Title: Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration

doi: 10.1038/s41422-020-00393-6

Figure Lengend Snippet: a Schematics of generating stable C2C12 cell lines that are capable of Tet-induced necroptosis or apoptosis, through overexpressing MLKL or tBid, respectively. b Immunoblotting analysis of MLKL-Flag, tBid-Flag, and Cleaved Caspase-3 expression in cell death-inducible C2C12 cell lines with or without tetracycline treatment. C2C12- Mlkl -TetON cells were treated with 1 μg/mL tetracycline for 12 h to induce necroptosis, and C2C12- tBid -TetON cells were treated with 1 μg/mL tetracycline for 6 h to induce apoptosis. Whole cell lysates were subjected to SDS-PAGE and immunoblotting analysis using antibodies as indicated. GAPDH serves as the loading control. The asterisk (*) denotes the non-specific band. Experiments were repeated independently for more than three times. c Schematic experimental design for the collection and application of cell death conditioned medium. d Representative phase-contrast images (left) and quantification of MuSC cell numbers (right) after 48 h culturing. 20,000 freshly isolated MuSCs were seeded in 35 mm dish and cultured with the indicated medium for 48 h. NCM necroptosis conditioned medium, ACM apoptosis conditioned medium. Scale bars, 100 μm. The data are expressed as the means ± SD of 3 independent experiments. e Proliferation analysis of NCM-cultured MuSCs by BrdU assay. Freshly isolated MuSCs were cultured in F-10 medium or NCM for 48 h, followed by 10 μM BrdU labeling for 2 h and analyzed by FACS. f Quantification of the BrdU + MuSCs as representatively shown in e . The data are expressed as the means ± SD of 4 independent experiments. g Immunofluorescence staining of Pax7 (green) and MyoD (red) in F-10 or NCM-expanded MuSCs. Cells were cultured and expanded for 2 passages in the corresponding medium followed by immunofluorescence staining as described in Materials and Methods. Nuclei were identified by staining with DAPI. Scale bars, 50 μm. h Quantification of the percentages of Pax7 hi MyoD low , Pax7 hi MyoD hi , Pax7 low MyoD hi and Pax7 low MyoD low subpopulations of MuSCs as shown in g . Histogram represents the percentages of each subpopulation out of 300 cells per condition. Fluorescent intensity was measured by Image Pro-Plus as described in Materials and Methods. The data are expressed as the means ± SD of 6 images. i Representative immunofluorescence staining of MyHC (green) in differentiated MuSCs. Nuclei were identified by staining with DAPI. Scale bars, 50 μm. Experiments were repeated independently for more than three times. j Representative immunofluorescence staining of Laminin (green) merged with red fluorescent engrafted transplanted MuSCs. Red fluorescent MuSCs isolated from R26 mT/mG transgenic mice were expanded in F-10 medium or NCM for 2 passages and then transplanted into X-ray irradiated recipient, the injured nonfluorescent Rag2 −/− ;Il2rg −/− TA muscles. Freshly isolated MuSCs were transplanted and used as a positive control. Cross-sections of TA muscles were harvested at 28 days after transplantation and prepared for immunofluorescence staining of Laminin. Nuclei were identified by staining with DAPI. Scale bars, 25 μm. k Quantification of the engrafted Tomato + myofibers as shown in j . The number of Tomato + myofibers from 24 fields (Leica SP8 microscopy with 20x objective magnification) were quantified for each mouse. Each dot represents an individual mouse. The data are expressed as the means ± SD. n = 6 for each group of recipient mice. P values for d and k were determined by unpaired two-tailed t -test; P value for f was determined by unpaired two-tailed t -test with Welch’s correction; P values for h were determined by two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.005.

Article Snippet: For in vivo antibody delivery, individual 50 μL of 15 μg/mL monoclonal antibody (anti-EGF, Santa Cruz Biotechnology, cat# sc-374255; anti-Tenascin-C, Abcam, cat# ab3970; anti-EGFR/Cetuximab, MedChemExpress, cat# HY-P9905) was injected into TA muscles of recipient ( Rag2 −/− ; Il2rg −/− ) mice every other day for 12 days, starting from one day after CTX injection.

Techniques: Western Blot, Expressing, SDS Page, Isolation, Cell Culture, BrdU Staining, Labeling, Immunofluorescence, Staining, Transgenic Assay, Irradiation, Positive Control, Transplantation Assay, Microscopy, Two Tailed Test

Journal: Cell Research

Article Title: Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration

doi: 10.1038/s41422-020-00393-6

Figure Lengend Snippet: a Coomassie blue staining of the purified recombinant mouse TNC (1–700 amino acids). b Immunoblotting analysis of the GST pull-down products with the anti-EGFR antibody. 500 ng/mL GST-tagged TNC (1–700aa) was used as the bait to incubate with MuSCs, and subsequently performed GST pull-down assay as described in Materials and Methods. Experiments were repeated independently for more than three times. c Immunoblotting analysis of EGFR activation in MuSCs. NCM was derived from both the control ( sg-control ) and Tnc knockout ( sg-Tnc ) C2C12- Mlkl -TetON cells. MuSCs were treated with combinations of 50 ng/mL EGF, 500 ng/mL GST-TNC-1-700, and 10 μM EGFR inhibitor in F-10 or NCM as indicated for 4 h. Whole cell lysates were subjected to SDS-PAGE and immunoblotting analysis using antibodies as indicated. HSP70 serves as the loading control. Experiments were repeated independently for more than three times. EGFR inhibitor: Afatinib. d Immunoblotting analysis of EGFR pathway activation in MuSCs. MuSCs were cultured in different conditions for 48 h. Whole cell lysates were subjected to SDS-PAGE and immunoblotting analysis using antibodies as indicated. F-10, F-10 medium; sg-ctl NCM, NCM derived from the control C2C12- Mlkl -TetON cells; sg-Tnc NCM, NCM derived from the Tnc knockout C2C12- Mlkl -TetON cells; NCM + EGFRi, normal NCM with EGFR inhibitor (10 μM Afatinib). HSP70 serves as the loading control. Experiments were repeated independently for more than three times. e Quantification of MuSCs that were cultured in NCM with TNC or EGFR neutralizing antibodies. MuSCs were cultured for 48 h. Cell proliferation was determined by measuring intracellular ATP levels using CellTiter-Glo assay. The data are expressed as the means ± SD of 3 technical repeats. NCM + α-TNC, NCM with antibody against TNC to neutralize TNC; NCM + α-EGFR, NCM with antibody against EGFR to block EGFR activation. The antibodies were titrated to different doses as indicated. f Quantification of Egfr knockout MuSCs that were cultured in NCM. MuSCs were isolated from Cas9-expressing mice and infected by AAV encoding sgRNA against Egfr ( sg-Egfr) or the scramble control ( sg-control) . The Egfr and the control knockout MuSCs were cultured in F10 or NCM for 48 h. Cell proliferation was determined by measuring intracellular ATP levels using CellTiter-Glo assay. The data are expressed as the means ± SD of 3 technical repeats. g Immunoblotting analysis of the knockout efficiency of EGFR in AAV-infected MuSCs. The asterisk (*) denotes the non-specific band. h Representative immunofluorescence staining of p-EGFR (green) and Pax7 (red) in freshly isolated MuSCs (2 days after CTX injection). MuSCs were isolated by FACS and fixed on PDL/Collagen-I pre-coated coverslip, as described in Materials and Methods, followed by immunofluorescence staining using the antibodies as indicated. Nuclei were identified by staining with DAPI. Scale bars, 5 μm. i Quantification of the p-EGFR + /Pax7 + cells as shown in h . MuSCs isolated from 3 mice were pooled together for immunofluorescence staining in each group. The histogram represents the percentage of p-EGFR + /Pax7 + cells out of 200 Pax7 + cells per genotype. The data are expressed as the means ± SD of 6 images. j Representative H&E staining of TA muscle cross sections from injured Rag2 −/− ;Il2rg −/− mice with antibodies neutralizing TNC/EGFR activation. After CTX injection, individual anti-TNC, anti-EGFR, or anti-EGF antibody was injected intramuscularly every other day as described in Materials and Methods. Twelve days later, TA samples were harvest and prepared for H&E staining. Scale bars, 40 μm. k Quantification of myofiber sizes from cross-sectional areas as representative shown in j . Histogram graph represents averaged myofiber size. The sizes of each 900 adjacent regenerating myofibers with central nuclei were measured for every mouse. Each dot represents an individual mouse. The data are expressed as the means ± SD. n = 6 for each group of mice. P values for e , f , and k , were determined by one-way ANOVA with Tukey’s multiple comparisons test; P value for i was determined by unpaired two-tailed t -test with Welch’s correction. ns, non-significant; * P < 0.05; *** P < 0.005.

Article Snippet: For in vivo antibody delivery, individual 50 μL of 15 μg/mL monoclonal antibody (anti-EGF, Santa Cruz Biotechnology, cat# sc-374255; anti-Tenascin-C, Abcam, cat# ab3970; anti-EGFR/Cetuximab, MedChemExpress, cat# HY-P9905) was injected into TA muscles of recipient ( Rag2 −/− ; Il2rg −/− ) mice every other day for 12 days, starting from one day after CTX injection.

Techniques: Staining, Purification, Recombinant, Western Blot, Pull Down Assay, Activation Assay, Derivative Assay, Knock-Out, SDS Page, Cell Culture, Glo Assay, Blocking Assay, Isolation, Expressing, Infection, Immunofluorescence, Injection, Two Tailed Test

Journal: Scientific Reports

Article Title: A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells

doi: 10.1038/s41598-021-90096-8

Figure Lengend Snippet: Bispecific antibodies specific to IL-2Rβ and IL-2Rγ induce phosphorylation of STAT5. ( A ) Summary of IL-2RB and IL-2RG monospecific UniAb binders identified during antibody lead discovery. ( B , C , D ) Heatmaps showing fold-induction of phosphorylated STAT5 (pSTAT5) in CD8 + T-cells from human PBMCs treated with individual monospecific UniAbs or αIL2Rβ and αIL2Rγ monospecific UniAbs in a 1:1 molar ratio mixture ( B ), αIL-2Rβ/γ bispecific UniAbs ( C ), or rhIL-2 positive control ( D ) at 50 nM for 1 h. In the antibody graphic on the right, the green and blue arms represent IL2RG and IL2RB binding domains, respectively. pSTAT5 levels were determined by flow cytometry and reported as geometric mean fluorescent intensity (gMFI) over the gMFI of unstimulated cells.

Article Snippet: Offrate measurements were done for the following: human IL-2Rβ (AcroBiosystems), human IL-2Rγ (Sino Biological), cynomolgus IL-2Rβ (Sino Biological), cynomolgus IL-2Rγ (expressed and purified in house using ExpiCHO expression system followed by Ni–NTA His-tag purification), mouse IL-2Rγ (Sino Biological), mouse IL-2Rβ (Sino Biological), human IL-2Rα (Sino Biological), IL-4R (Sino Biological), IL-7R (Sino Biological), IL-9R (R&D Systems) and IL-21R (Sino Biological).

Techniques: Positive Control, Binding Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells

doi: 10.1038/s41598-021-90096-8

Figure Lengend Snippet: BsAb-1 and BsAb-2 induce T-cell proliferation and accelerated GVHD in a mouse model engrafted with human PBMCs. ( A ) BALB/c mice (n=3 per group per time point) were administered 1 mg/kg BsAb-1 or BsAb-2 by tail-vein injection. Serum was collected at 6 time points over two weeks and tested together by ELISA for human IgG4 using rhIL-2Rγ as a capture antigen. Error bars show SD. Pharmacokinetic parameters were determined using Phoenix pharmacokinetic software. ( B-D ) Irradiated NSG mice (5 per treatment group) were engrafted with 20 million human PBMCs each. Animals were then treated with either vehicle only, rhIL-2 (daily), or BsAb-1 or BsAb-2 (twice weekly) until ≥ 20% body weight loss. ( B ) Relative body weight of treated mice after injection of PBMCs. Horizontal lines indicate mean, and error bars show SEM. p values were calculated using a two-way RM ANOVA test. ( C and D ) Percentage of engrafted CD8 + T-cells ( C ) and CD4 + T-cells ( D ) which showed at least one round of division by CSFE staining. Representative histograms shown in Fig. . p values were calculated using a one-way ANOVA. Horizontal lines indicate mean, and error bars show SEM ( B ) or SD ( C and D ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Offrate measurements were done for the following: human IL-2Rβ (AcroBiosystems), human IL-2Rγ (Sino Biological), cynomolgus IL-2Rβ (Sino Biological), cynomolgus IL-2Rγ (expressed and purified in house using ExpiCHO expression system followed by Ni–NTA His-tag purification), mouse IL-2Rγ (Sino Biological), mouse IL-2Rβ (Sino Biological), human IL-2Rα (Sino Biological), IL-4R (Sino Biological), IL-7R (Sino Biological), IL-9R (R&D Systems) and IL-21R (Sino Biological).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Software, Irradiation, Staining