Journal: Open Medicine

Article Title: HLA-DRB5 promotes immune thrombocytopenia via activating CD8 + T cells

doi: 10.1515/med-2024-0955

Figure Lengend Snippet: Expressions of HLA-DRB5 and MHC-II in peripheral blood of ITP mice. (a) and (b) ITP murine models were constructed for 15 days. PLT count in peripheral blood was measured every 2 days from the first day, and HLA-DRB5 mRNA expression in peripheral blood was measured by qRT-PCR from the first day (Days 1, 2, 3, 5, 7, 9, 11, 13, 15). (c) and (d) After the ITP modeling for 15 days, analysis of HLA-DRB5 mRNA expression in peripheral blood was performed by qRT-PCR. (e)–(g) Western blot was used to measure HLA-DRB5 protein expression in peripheral blood of ITP murine models. Relative expression was normalized to GAPDH. Data from all triplicate experiments were expressed as mean ± standard deviation. Comparison between two groups was carried out using independent-samples t -test. ** p < 0.01, *** p < 0.001, vs Control. Abbreviation: ITP, immune thrombocytopenia; HLA-DRB5, human leukocyte antigen class II heterodimer β5; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.

Article Snippet: Then, primary antibodies against HLA-DRB5 (21702-1-AP, 30 kDa, Proteintech, Chicago, IL, China), MHC-II (ab139365, 29 kDa; Abcam, Cambridge, UK) and GAPDH (ab9485, 36 kDam; Abcam, UK) were used to incubate the membranes at 4°C overnight.

Techniques: Construct, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Comparison, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Open Medicine

Article Title: HLA-DRB5 promotes immune thrombocytopenia via activating CD8 + T cells

doi: 10.1515/med-2024-0955

Figure Lengend Snippet: Effects of HLA-DRB5 knockdown on PLT count and expressions of MHC-II, CD80 and CD86 in peripheral blood of ITP mice. (a) ITP murine models were constructed for 15 days, during which sh-HLA-DRB5 adenovirus or negative control was injected into mice by tail vein once a week for 2 weeks. After the ITP modeling for 15 days, analysis of HLA-DRB5 mRNA expression in peripheral blood was performed by qRT-PCR. (b) and (c) Western blot was used to measure HLA-DRB5 protein expression in peripheral blood of ITP murine models. Relative expression was normalized to GAPDH. (d) PLT count in peripheral blood was measured every 2 days from the first day. (e)–(g) QRT-PCR was utilized to detect MHC-II, CD80 and CD86 mRNA expressions in peripheral blood of ITP murine models. Relative expression was normalized to GAPDH. Data from all triplicate experiments were expressed as mean ± standard deviation. Comparison among multiple groups was carried out using one-way analysis of variance. * p < 0.05, *** p < 0.001, vs Control; +++ p < 0.001, vs ITP + vector. Abbreviation: sh-HLA-DRB5, short hairpin RNA targeting HLA-DRB5.

Article Snippet: Then, primary antibodies against HLA-DRB5 (21702-1-AP, 30 kDa, Proteintech, Chicago, IL, China), MHC-II (ab139365, 29 kDa; Abcam, Cambridge, UK) and GAPDH (ab9485, 36 kDam; Abcam, UK) were used to incubate the membranes at 4°C overnight.

Techniques: Knockdown, Construct, Negative Control, Injection, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Comparison, Control, Plasmid Preparation, shRNA

Journal: Open Medicine

Article Title: HLA-DRB5 promotes immune thrombocytopenia via activating CD8 + T cells

doi: 10.1515/med-2024-0955

Figure Lengend Snippet: Effects of HLA-DRB5 knockdown on the population of PBMCs with MHC-II + , CD80 + , CD86 + and CD8 + in ITP mice. (a)–(d) ITP murine models were constructed for 15 days, during which sh-HLA-DRB5 adenovirus or negative control was injected into mice by tail vein once a week for 2 weeks. Fluorescent analysis of MHC-II, CD80 and CD86 in PBMCs of ITP mice was performed using fluorescence-labeled antibodies in combination with fluorescent microscopy (magnification: ×200, scale bar: 100 μm). (e)–(g) Flow cytometry combined with fluorescence-labeled CD8 and CD4 antibodies was performed to detect the percentage of CD8 + T cells and CD4 + T cells in PBMCs of ITP mice. Data from all triplicate experiments were expressed as mean ± standard deviation. Comparison among multiple groups was carried out using one-way analysis of variance. * p < 0.05, *** p < 0.001, vs Control; + p < 0.05, +++ p < 0.001, vs ITP + vector. Abbreviation: MHC-II, major histocompatibility complex II; PBMCs, peripheral blood mononuclear cells.

Article Snippet: Then, primary antibodies against HLA-DRB5 (21702-1-AP, 30 kDa, Proteintech, Chicago, IL, China), MHC-II (ab139365, 29 kDa; Abcam, Cambridge, UK) and GAPDH (ab9485, 36 kDam; Abcam, UK) were used to incubate the membranes at 4°C overnight.

Techniques: Knockdown, Construct, Negative Control, Injection, Fluorescence, Labeling, Microscopy, Flow Cytometry, Standard Deviation, Comparison, Control, Plasmid Preparation, Immunopeptidomics

Journal: Molecular Therapy. Nucleic Acids

Article Title: Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

doi: 10.1016/j.omtn.2020.01.012

Figure Lengend Snippet: Time-Course Study of SNP-Selective Knockdown (A) mRNA knockdown in hiPSC-derived cardiomyocytes was evaluated at six time points over a 2-week period using allele-specific droplet digital PCR. At day 0, 250 nM rs715-T targeting ASO A259 was added via gymnosis. SNP-matched knockdown is seen for up to 2 weeks, whereas the SNP-mismatched allele does not show knockdown. Data were normalized to the no ASO day 2 time point for each allele. Mean ± SD from three independent experiments is shown. Significance between T alleles (no ASO versus ASO) was determined by two-way ANOVA followed by Sidak’s multiple comparisons test (*p < 0.05, ***p < 0.001). (B) Quantification of β-MHC in iCell 2 human iPSC-derived cardiomyocyte (hiPSC-CM) with and without addition of ASO A259. β-MHC is not reduced at any time point, suggesting protein compensation by the SNP-mismatched allele. Bar graph represents three independent experiments (mean ± SD). Protein levels were normalized to the no ASO group at each time point.

Article Snippet: A custom antibody specific for β-MHC was generated to allow for discrimination between myosin isoforms (iCell 2 cells also contain α-MHC).

Techniques: Knockdown, Derivative Assay, Digital PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

doi: 10.1016/j.omtn.2020.01.012

Figure Lengend Snippet: Study of SNP-Selective Knockdown in Mice (A) Allele-specific mRNA quantitation from mouse left ventricle (LV; 8-week-old males) 1 week following ASO dosing (*p < 0.05, **p < 0.01, ***p < 0.001 comparing C and T allele abundance within a group as determined by t test). All five compounds significantly reduce the C allele compared with the T allele. Two compounds give significant knockdown of the C allele compared with the C allele in the saline group ( ### p < 0.001 comparing with saline C allele as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test). n = 7–12 per group. (B) Total α-MHC protein quantitation from mouse LV 1 week following ASO dosing. No significant difference was seen relative to the saline group, as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. An α-MHC-specific antibody was used that showed no reactivity to recombinant β-MHC. (C) ASO concentrations in heart (LV), liver, and kidney. On average, ASO concentration is 37× higher in kidney and 16× higher in liver compared with heart. Mean ± SD shown.

Article Snippet: A custom antibody specific for β-MHC was generated to allow for discrimination between myosin isoforms (iCell 2 cells also contain α-MHC).

Techniques: Knockdown, Quantitation Assay, Saline, Protein Quantitation, Recombinant, Concentration Assay

Journal: Cancer & Metabolism

Article Title: Ets-1 global gene expression profile reveals associations with metabolism and oxidative stress in ovarian and breast cancers

doi: 10.1186/2049-3002-1-17

Figure Lengend Snippet: Functional annotation clustering of genes associated with Ets-1 overexpression

Article Snippet: 8 , INTERPRO , IPR010579:MHC class I, alpha chain, C-terminal , 0.01 , 14.77.

Techniques: Functional Assay, Activity Assay, Binding Assay

Journal: PLoS ONE

Article Title: Hydrogen Sulfide Suppresses Outward Rectifier Potassium Currents in Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.1371/journal.pone.0050641

Figure Lengend Snippet: A: Light microscopy and immunocytochemistry image of H9 hESC-CMs dissociated from contracting EBs. Scale bar: 100 µm. Fluorescent images of H9 hESC-CMs stained with antibodies against α-actinin, β-MHC and Titin showed typical cardiac sarcomeres. Scale bar: 30 µm. B1∼2: Action potential trace of three subtypes of cardiomyocytes derived from H9 hESCs and corresponding specific action potential properties. APA: action potential amplitude. dV/dt max : maximal rate of depolarization or maximal upstroke velocity. APD50: action potential duration at 50%. APD90: action potential duration at 90%.

Article Snippet: Sigma), β-myosin heavy chain or β-MHC (Alexis Biochemicals, FL, USA) followed by Alexa Fluo® 488 goat anti-rabbit IgG (Invitrogen, CA, USA); and cardiac Titin (1∶10) (Sigma-Alrich, MO, USA) followed by Alexa Fluo® 555 donkey anti-rabbit IgG.

Techniques: Light Microscopy, Immunocytochemistry, Staining, Derivative Assay