Journal: Skeletal Muscle

Article Title: Aminoguanidine hemisulfate improves mitochondrial autophagy, oxidative stress, and muscle force in Duchenne muscular dystrophy via the AKT/FOXO1 pathway in mdx mice

doi: 10.1186/s13395-024-00371-1

Figure Lengend Snippet: AGH altered fiber type composition via the AKT/FOXO1 signaling pathway. TA muscles from WT, mdx , and AGH-treatment mdx mice were isolated at 8 weeks of age. Gene expression of ( A ) Pax7 , ( B ) Myf5 , ( C ) MyoD , ( D ) Myog , and ( E ) Foxo1 were measured using real-time PCR and values were normalized to 36B4 levels. ( F – J ) Representative blots and quantifications for p-Akt, Akt, p-Foxo1, Foxo1, MyoD, and Myog. Values were normalized to β-tubulin. Values are presented as the mean ± standard error of the mean for each experiment ( n = 5). * p < 0.05 compared with WT control, # p < 0.05 compared with mdx

Article Snippet: Primary antibodies against LC3 (14600-1-AP, 1:1,000), p62 (18420-1-AP, 1:1,000), Pink1 (23274-1-AP, 1:1,000), Prdx3 (10664-1-AP, 1:1,000), Foxo1 (18592-1-AP, 1:1,000), MyH7 (22280-1-AP, 1:1,000), myosin-embryonic (22287-1-AP, 1:1,000), MnSOD (66474-1-Ig, 1:1,000), goat anti-rabbit IgG-HRP (SA00001-2, 1:5,000), and goat anti-mouse IgG-HRP (SA00001-1, 1:5,000) were purchased from Proteintech (Wuhan, China).

Techniques: Muscles, Isolation, Gene Expression, Real-time Polymerase Chain Reaction, Control

Journal: Skeletal Muscle

Article Title: Aminoguanidine hemisulfate improves mitochondrial autophagy, oxidative stress, and muscle force in Duchenne muscular dystrophy via the AKT/FOXO1 pathway in mdx mice

doi: 10.1186/s13395-024-00371-1

Figure Lengend Snippet: Summary diagram of AGH treatment in mdx mice. AGH treatment reduced ROS and apoptosis by restoring mitochondrial autophagy, restored the composition of muscle fiber types, and improved muscle force through the AKT/FOXO1 signaling pathway

Article Snippet: Primary antibodies against LC3 (14600-1-AP, 1:1,000), p62 (18420-1-AP, 1:1,000), Pink1 (23274-1-AP, 1:1,000), Prdx3 (10664-1-AP, 1:1,000), Foxo1 (18592-1-AP, 1:1,000), MyH7 (22280-1-AP, 1:1,000), myosin-embryonic (22287-1-AP, 1:1,000), MnSOD (66474-1-Ig, 1:1,000), goat anti-rabbit IgG-HRP (SA00001-2, 1:5,000), and goat anti-mouse IgG-HRP (SA00001-1, 1:5,000) were purchased from Proteintech (Wuhan, China).

Techniques:

Journal: Communications Biology

Article Title: PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment

doi: 10.1038/s42003-021-02111-3

Figure Lengend Snippet: a CD4 + T cells were purified from AND-Tg Pdcd1 −/− Rag2 −/− mice, stimulated with irradiated B10.BR whole splenocytes with 5 μM MCC 88–103 peptides, and retrovirally transduced with mouse (m) PD-1–EGFP. The cells were plated onto an MCC 88–103 -prepulsed SLB containing I-E k –GPI (200/μm2) and mICAM-1–GPI (100/μm 2 ) without (top) or with mouse mPD-L1–GPI (middle, 150/μm 2 ) or mPD-L2–GPI (bottom, 150/μm 2 ) and real-time imaged by total internal reflection fluorescence microscopy (times are above images; Supplementary Movie ). b Clustering and centripetal movement of PD-1 on the diagonal yellow line in a is presented as horizontal elements in kymographs. c Primary CD4 + T cells expressing mPD-1–EGFP (green) in a were prestained with DyLight 650-labeled anti-TCRβ (H57) Fab (red), plated onto an SLB as in a and real-time imaged by confocal microscopy at 2 (left) or 10 (right) min after contact. Histograms show fold fluorescent intensities of TCRβ (red) and mPD-1 (green) on the diagonal yellow lines in the DIC images. d CD8 + T cells were purified from OT-I-Tg Rag2 −/− mice, stimulated with irradiated C57BL/6 whole splenocytes with 100 nM OVA 257–264 peptide and retrovirally transduced with PD-1–EGFP. The cells were imaged as in a on an SLB containing OVA 257–264 -prepulsed H2-K b –GPI (200/μm 2 ). Histograms are depicted as in c . e The graph shows the percentage of TCR microclusters colocalized with mPD-1 at 2 min after contact in CD4 + T cells in c (left) and CD8 + T cells in d (right) ( n = 5). f AND-TCR T cell hybridomas (2D12) expressing mPD-1–EGFP were prestained with DyLight 650-labeled H57 Fab (red), conjugated with an MCC 88–103 prepulsed (5 μM) I-Ek-expressing APC line, DC-1 cell, not expressing (top) or expressing mPD-L1 (middle) or mPD-L2 (bottom) and real-time imaged by confocal microscopy at 2 min after contact. Histograms show fold fluorescent intensities of TCRβ (red) and mPD-1 (green) on the diagonal yellow lines in the DIC images. All data are representatives of three independent experiments. Bars, 5 μm. Error bars, SD. Statistical analysis was by one-way analysis of variance (ANOVA). **** p < 0.0001.

Article Snippet: Renilla luciferase (RLuc) 8 and HaloTag (Promega)-tagged mPD-1, mPD-L1, or mPD-L2 were generated by PCR and subcloned into the pMXs retroviral vector. pMXs-RLuc8 was constructed by PCR using Yellow Nano-lanterns (kindly provided by Dr. Y. Okada, Riken, Japan ) as a template.

Techniques: Purification, Irradiation, Transduction, Fluorescence, Microscopy, Expressing, Labeling, Confocal Microscopy

Journal: Communications Biology

Article Title: PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment

doi: 10.1038/s42003-021-02111-3

Figure Lengend Snippet: a 2D12 expressing mPD-1–EGFP (green) cells were prestained with DyLight 650–labeled H57 Fab (red) and plated on an SLB with mPD-L1–GPI (left two columns) or mPD-L2–GPI (right two columns) as in Fig. . The cells were real-time imaged by confocal microscopy at 2 min after contact in the absence (top) or presence of anti-PD-1 (29F.1A12, row 2), anti-PD-L1 (MIH5, row 3), anti-PD-L2 (MIH37, row 4) or both anti-PD-L1 and anti-PD-L2 (bottom). b The graph shows the percentage of T cells forming PD-1 microclusters in a ( n = 30). c 2D12 expressing mPD-1–EGFP were imaged as in a in the absence (top) or presence of three different anti-PD-1 mAbs, J43 (row 2 and 3), RMP1-14 (row 4 and 5) or 29 F.1A12 (bottom) at a concentration of 10 or 50 μg/ml. All data are representatives of three independent experiments. Bars, 5 μm. Error bars, SD. Statistical analysis was by one-way analysis of variance (ANOVA). **** p < 0.0001.

Article Snippet: Renilla luciferase (RLuc) 8 and HaloTag (Promega)-tagged mPD-1, mPD-L1, or mPD-L2 were generated by PCR and subcloned into the pMXs retroviral vector. pMXs-RLuc8 was constructed by PCR using Yellow Nano-lanterns (kindly provided by Dr. Y. Okada, Riken, Japan ) as a template.

Techniques: Expressing, Labeling, Confocal Microscopy, Concentration Assay

Journal: Communications Biology

Article Title: PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment

doi: 10.1038/s42003-021-02111-3

Figure Lengend Snippet: a 2D12 transduced with both mPD-1–HaloTag and EGFP–SHP1 (left) or EGFP–SHP2 (right) were preincubated with the HaloTag ligand–Stella650 (red), plated onto the SLB as in Fig. without (top) or with mPD-L1–GPI (150/μm 2 , middle) or mPD-L2–GPI (150/μm 2 , bottom) and real-time imaged by confocal microscopy at 20 s after contact. Histograms show fold fluorescent intensities of mPD-1 (red) and SHP1 (green) or SHP2 (green) on the diagonal yellow lines in the DIC images. b The cells in a (SHP1, left panel; SHP2, right panel) were conjugated with DC-1 cells not expressing (top) or expressing mPD-L1 (middle) or mPD-L2 (bottom) and real-time imaged by confocal microscopy at 20 s after contact. Histograms show fold fluorescent intensities of PD-1(red) and SHP1 (green, left panel) or SHP2 (green, right panel) on the diagonal yellow lines in the DIC images. c 2D12 expressing mPD-1–EGFP were conjugated by MCC 88–103 -prepulsed (5 μM) DC-1 cells (left) or DC-1 expressing mPD-L1 (middle) or mPD-L2 (right) for the indicated times. The cells were lysed, immunoprecipitated for PD-1 by anti-GFP and blotted for SHP1, SHP2, or PD-1. The whole cell lysates (WCLs) were blotted for SHP1, SHP2, or PD-1. The number below each line represents the intensity of the band. All data are representatives of three independent experiments. Bars, 5 μm.

Article Snippet: Renilla luciferase (RLuc) 8 and HaloTag (Promega)-tagged mPD-1, mPD-L1, or mPD-L2 were generated by PCR and subcloned into the pMXs retroviral vector. pMXs-RLuc8 was constructed by PCR using Yellow Nano-lanterns (kindly provided by Dr. Y. Okada, Riken, Japan ) as a template.

Techniques: Transduction, Confocal Microscopy, Expressing, Immunoprecipitation

Journal: Communications Biology

Article Title: PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment

doi: 10.1038/s42003-021-02111-3

Figure Lengend Snippet: a 2D12 transduced with mPD-1–HaloTag were preincubated with the HaloTag ligand–TMR (cyan) and DyLight 488-labeled H57 Fab (green), plated onto the SLB as in Fig. without (top) or with mPD-L1–GPI (150/μm 2 , middle) or mPD-L2–GPI (150/μm 2 , bottom), fixed at 2 min after contact, stained with Alexa Fluor 647-labeled anti-phospho (p) CD3ζ (red) and imaged by confocal microscopy. Histograms show fold fluorescent intensities of TCRβ (green), PD-1 (cyan) and pCD3ζ (red) on the diagonal yellow lines in the DIC images. b The graph shows pCD3ζ/TCRβ fluorescent intensity ratio at the T cell-bilayer interface in a in the absence (left) or presence of mPD-L1–GPI (middle) or mPD-L2–GPI (right) ( n = 20). Horizontal bars, average. c Primary CD4 + T cells in Fig. transduced with mPD-1–GFP (green) were prestained with the DyLight 549-labeled H57 Fab (cyan), plated onto the SLB, fixed, stained for pCD3ζ (red, left panel) or pSLP-76 (red, right panel) and imaged as in a . Histograms show fold fluorescent intensities of TCRβ (cyan), PD-1 (green) and pCD3ζ or pSLP-76 (red) on the diagonal yellow lines in the middle column. d The graphs show fluorescent intensity ratio of pCD3ζ/TCRβ (top) or pSLP-76/TCRβ (bottom) in c in the absence or presence of mPD-L1–GPI or mPD-L2–GPI ( n = 20). Horizontal bars, average. e 2D12 expressing mPD-1 were stimulated with MCC 88–103 -prepulsed DC-1 cells not expressing (left) or expressing mPD-L1 (middle) or mPD-L2 (right) for the indicated times. The WCLs were blotted for pPLCγ1, PLCγ1, pErk1/2, or Erk1/2. The number below each line represents the intensity ratio, pPLCγ/PLCγ or pErk/Erk. f The cells in e were stimulated by 16h-aggregation culture with DC-1 cells (black) or those expressing mPD-L1 (red) or mPD-L2 (green) under the different concentrations of MCC 88–103 and the concentration of IL-2 in each supernatant was measured by ELISA. g The cells in e were stimulated by 16 h-aggregation culture with MCC 88–103 and DC-1 cells expressing mPD-L1 or mPD-L2 in the absence or presence of each antibody as in Fig. and the concentration of IL-2 in each supernatant was measured by ELISA. h The target cell, EL-4 cell, is introduced by RLuc8 and further by mPD-L1 (red) or mPD-L2 (green). Effector primary CD8 + T cells expressing mPD-1 were cocultured with 1 nM OVA 257–264 -pulsed these target EL-4 cells at the indicated E:T ratios for 16 h with or without 10 μg/ml anti-PD-1 (29F.1A12). After treatment with RLuc8 substrate, the intensity of live cells was measured and the percent specific lysis was calculated. i The concentration of IFNγ in culture supernatants in h was measured by ELISA. j The cells in e were stimulated by 16 h-aggregation culture with 10 μM MCC 88–103 and DC-1 cells expressing mPD-L1 and mPD-L2 in the absence or presence of anti-PD-1 (J43: brown, RMP1-14: blue, 29F.1A12: green) as in Fig. at the indicated concentrations and the concentration of IL-2 in each supernatant was measured by ELISA. The right graph shows the recovery rate of IL-2 production by each anti-PD-1 at the different concentrations. All data are representatives of three independent experiments. Bars, 5 μm. Error bars, SD. Statistical analysis was by one-way analysis of variance (ANOVA). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Renilla luciferase (RLuc) 8 and HaloTag (Promega)-tagged mPD-1, mPD-L1, or mPD-L2 were generated by PCR and subcloned into the pMXs retroviral vector. pMXs-RLuc8 was constructed by PCR using Yellow Nano-lanterns (kindly provided by Dr. Y. Okada, Riken, Japan ) as a template.

Techniques: Transduction, Labeling, Staining, Confocal Microscopy, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lysis

Journal: Communications Biology

Article Title: PD-L2 suppresses T cell signaling via coinhibitory microcluster formation and SHP2 phosphatase recruitment

doi: 10.1038/s42003-021-02111-3

Figure Lengend Snippet: a The mPD-L1–GPI (red, middle) and mPD-L2–GPI (green, bottom) were labeled with AlexaFluor 647 and AlexaFluor 488, respectively, and reconstituted into SLBs as in Fig. in the different densities indicated above the images. 2D12 expressing mPD-1–HaloTag (top, cyan) were preincubated with HaloTag ligand–TMR and real-time imaged by confocal microscopy at 2 min after the T cell-bilayer contact. b Images of cells in a at mPD-L1–GPI (75 molecules/μm 2 ) + mPD-L2–GPI (300) (left) or mPD-L1–GPI (300) + mPD-L2–GPI (75) (right). The yellow squares in the left panels are magnified in the right three panels. Yellow enclosures show PD-L1 or PD-L2 clusters colocalized with PD-1 microclusters. Green enclosures show blank where PD-1 microclusters formed. c The graphs show the percentage of T cells forming PD-1 microclusters composed of either PD-L1, PD-L2, or PD-L1 + L2 at the different densities of mPD-L1–GPI and mPD-L2–GPI in a . d Silica beads were coated with the same SLBs as in a with different densities of mPD-L1–GPI and mPD-L2–GPI and prepulsed with MCC 88–103 to use as engineered antigen presenting cells. 2D12 expressing mPD-1 were stimulated by these silica beads for 16 h and the concentration of IL-2 in each supernatant was measured by ELISA. e 2D12 expressing mPD-1–EGFP (green) were prestained with DyLight 650-labeled H57 Fab (red) and real-time imaged on an SLB with both mPD-L1–GPI and mPD-L2–GPI in the absence or presence of each antibody as in Fig. . The graph shows the percentage of T cells forming PD-1 microclusters ( n = 30). f The cells in e were stimulated by 16h-aggregation culture with 10 μM of MCC 88–103 and the concentration of IL-2 in each supernatant was measured by ELISA. All data are representatives of three independent experiments. Bars, 5 μm. Error bars, SD. Statistical analysis was by one-way analysis of variance (ANOVA). * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Renilla luciferase (RLuc) 8 and HaloTag (Promega)-tagged mPD-1, mPD-L1, or mPD-L2 were generated by PCR and subcloned into the pMXs retroviral vector. pMXs-RLuc8 was constructed by PCR using Yellow Nano-lanterns (kindly provided by Dr. Y. Okada, Riken, Japan ) as a template.

Techniques: Labeling, Expressing, Confocal Microscopy, Concentration Assay, Enzyme-linked Immunosorbent Assay