mhc  (Developmental Studies Hybridoma Bank)

Journal: iScience

Article Title: Histone methyltransferase SET-18/SMYD2-mediated activation of NADase TIR-1d/SARM1 increases mtROS to promote aging

doi: 10.1016/j.isci.2026.114649

Figure Lengend Snippet: SMYD2-mediated activation of SARM1 reduces NAD + level to promote C2C12 myotube atrophy via increase of mtROS (A and B) The effect of smyd2 RNAi in reducing C2C12 myotubes atrophy was abolished by overexpression of SARM1. In A–7F, smyd2 siRNA (siSMYD2) was co-transfected with/without the overexpression plasmid of sarm1 (OE SARM1) into the differentiated C2C12 myotubes. #1 and #2 were two of siRNAs targeting the distinct coding sequences of smyd2 . The non-targeting siRNA (siNT) was used as control. The transfected myotubes were then treated with or without H 2 O 2 , respectively. (A) The representative images of C2C12 myotubes with MHC staining. Red, MHC protein; Blue, the nuclei stained by DAPI. (B) The analysis of myotube diameter and fusion index. The fusion index was indicated as the value of the number of nuclei present in myotubes divided by the number of total nuclei in MHC + cells. Error bars represent SEM. Myotube diameter: without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) = 0.0003, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) < 0.0001, ∗∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) < 0.0001; with H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) < 0.0001, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0365; Fusion index: without H 2 O 2 : ∗p (siSMYD2 #1 vs. siNT) = 0.0162, ∗p (siSMYD2 #2 vs. siNT) = 0.0171, ∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0191, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0101; with H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0017, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0449; one-way ANOVA. (C and D) Overexpression of SARM1 attenuated the influences of smyd2 RNAi in up-regulating MHC mRNA level and downregulating the mRNA levels of atrophy-related genes in C2C12 myotubes with/without H 2 O 2 treatments. The mRNA levels of MHC (C), MuRF1 and Atrogin-1 (D) in myotubes were measured by RT-qPCR and normalized to those of control groups without H 2 O 2 treatment. β-actin was used as an internal reference. Error bars represent SEM. n = 3–4 biological replicates. MHC mRNA level: without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗p (siSMYD2 #2 vs. siNT) = 0.0025, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0003, ∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0025; with H 2 O 2 : ∗∗p (siSMYD2 #1 vs. siNT) = 0.0065, ∗∗p (siSMYD2 #2 vs. siNT) = 0.0069, ∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0246, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0306; MuRF1 mRNA level: without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) = 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0172, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0111; with H 2 O 2 : ∗p (siSMYD2 #1 vs. siNT) = 0.0140, ∗p (siSMYD2 #2 vs. siNT) = 0.0196, ∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0189, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0407; Atrogin-1 mRNA level: without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) = 0.0006, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0018, ∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0030; with H 2 O 2 : ∗∗p (siSMYD2 #1 vs. siNT) = 0.0031, ∗∗p (siSMYD2 #2 vs. siNT) = 0.0073, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0002; one-way ANOVA. (E) Overexpression of SARM1 attenuated the increased NAD + level of C2C12 myotubes with smyd2 RNAi treatment. The NAD + levels of myotubes were detected with microplate reader and normalized to those of control without H 2 O 2 treatment. Error bars represent SEM. n = 3 biological replicates. ∗p (siSMYD2 #1 vs. siNT) = 0.0100, ∗p (siSMYD2 #2 vs. siNT) = 0.0451, ∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0041, ∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0069; one-way ANOVA. (F) Overexpression of SARM1 alleviated the effect of smyd2 RNAi in decreasing mtROS level in C2C12 myotubes with/without H 2 O 2 treatments. The mtROS and nuclei (Blue) in myotubes were stained with MitoSOX and DAPI, respectively. The stained myotubes were detected by confocal microscopy (the representative images showed on the left), followed by quantitative analysis of fluorescence intensity (on the right). Error bars represent SEM. Without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) < 0.0001, ∗∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) < 0.0001; with H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) < 0.0001, ∗∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) < 0.0001; one-way ANOVA.

Article Snippet: The C2C12 myotubes were incubated with anti-myosin heavy chain (MHC) antibody (MF20, DSHB) followed by fluorescent secondary antibody (C2181, Sigma Chemical) to identify MHC expression.

Techniques: Activation Assay, Over Expression, Transfection, Plasmid Preparation, Control, Staining, Quantitative RT-PCR, Confocal Microscopy, Fluorescence

Journal: Cancer Immunology Research

Article Title: Phenotypic Characterization and Prognostic Impact of CD103 + Tissue-Resident Memory T Cells in Diffuse Large B-cell Lymphoma

doi: 10.1158/2326-6066.CIR-25-0445

Figure Lengend Snippet: Characterization of the T RM population in DLBCL by spectral flow cytometry. A, Representative flow plots of CD103 + CD69 + T cells, highlighted in red. B, % CD103 + CD69 + of T cells per COO ( n = 2–4). C, UMAP reduction of T cells collected from patients with DLBCL ( n = 8) and rLN ( n = 2). Colors represent clusters determined by PhenoGraph clustering ( k = 45). D, Overlay of COO classification of DLBCL samples ( n = 2 for each). E, Overlay of markers of interest is highlighted in red. F, Heatmap showing the percent positivity of all T-cell phenotype markers in spectral panel. G, Boxplots showing the percent of CD103 + (red) or CD103 − (gray) T cells (CD45 + CD3 + CD5 + CD2 + cells) expressing each marker for samples from patients with DLBCL ( n = 10). P values displayed on plot were calculated using paired t tests for each marker. H, Intracellular IFNγ and TNFα expression by CD103 + or CD103 − T cells measured by flow cytometry after bulk single-cell lymphoma biopsy samples ( n = 3) or healthy donor peripheral blood mononuclear cells ( n = 1) were cultured alone, with PMA/ionomycin, or with HLA class I and class II CEF peptide pools for 24 hours. I, Enumeration of remaining live B cells by flow cytometry after 24-hour coculture of sorted CD103 + or CD103 − T cells (sorted as CD8 + or CD4 + cells) with autologous sorted B cells from single-cell biopsy samples of patients with lymphoma ( n = 4). Samples containing CD103 + and CD103 − cells from the same donor are connected with a line. P value generated using a ratio paired t test. The bar graph represents the mean, with error bars representing SEM. For box plots, the center line represents the median, the box represents the IQR, and the whiskers represent 1.5× the IQR. ICS, intracellular staining; UNCLASS, unclassified.

Article Snippet: Bulk-cell suspensions (1 × 10 6 ) were cultured alone, with MHC class I and class II cytomegalovirus, Epstein–Barr virus, and influenza virus (CEF) peptides (Miltenyi Biotech, cat. #130-098-426 and Cedarlane, cat. #PT-PA-CEFT-001-1), or with 1× cell activation cocktail [phorbol 12-myristate 13-acetate (PMA) and ionomycin; BioLegend, cat. #423301] for 24 hours in RPMI-1640 supplemented with 1% (vol/vol) GlutaMAX (Gibco, cat. #35050061), 1% (vol/vol) penicillin–streptomycin (Thermo Fisher Scientific, cat. #15140122), 50 μmol/L β-mercaptoethanol (Sigma-Aldrich, cat. #M3148), 5% (vol/vol) male AB-plasma human serum (Sigma-Aldrich, cat. #H4522), and 5 U/mL IL2 (PeproTech, cat. #200-02) in 96-well flat-bottom plates.

Techniques: Flow Cytometry, Expressing, Marker, Cell Culture, Generated, Staining