Journal: Research Square

Article Title: Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity

doi: 10.21203/rs.3.rs-802084/v1

Figure Lengend Snippet: Higher anti-RBD IgM seroconversion rate in respiratory samples compared to paired blood samples of COVID-19 patients. a ELISA titration curves against the SARS-CoV-2 receptor-binding domain (RBD) for IgM, IgG, and IgA in COVID-19 respiratory and paired blood samples and non-COVID-19 respiratory samples. Dotted lines within each graph indicates the cut-off used to determine end-point titres. b Endpoint titres of SARS-CoV-2 RBD antibodies between (i) respiratory samples of COVID-19 and non-COVID-19 patients, and (ii) plasma and respiratory samples of COVID-19 patients. (i) Bars indicate median with interquartile range. Dotted line indicates the detection level. (ii) Dotted lines connect the most closely matched plasma and respiratory samples from each patient. Statistical significance was determined with Mann-Whitney test. c ELISA titration curves against the SARS-CoV-2 RBD for 3 COVID-19 patients with serial respiratory samples. d Heatmap of percentage (%) inhibition tested by surrogate virus neutralization test (sVNT) and anti-RBD ELISA titres. e Correlation between anti-RBD antibody titres and (%) sVNT inhibition. Correlation was determined with Spearman’s correlation. f Number of (i) samples and (ii) patients with seroconverted anti-RBD IgM, IgG, IgA and positive % sVNT inhibition. Pink curved lines surrounding the donut graphs indicate the samples/patients with seroconverted IgM. Earliest samples were used for each patient when determining seroconversion which was defined as average titre +2xSD of non-COVID-19 samples. Positive % sVNT inhibition was defined as % sVNT inhibition ≥ 20%.

Article Snippet: DuoSet ELISA ancillary reagent kit (R&D Systems) was used for respiratory fluids and in-house reagents with the same composition were used for plasma samples.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Binding Assay, Clinical Proteomics, MANN-WHITNEY, Inhibition, Virus, Neutralization

Journal: The Journal of Clinical Investigation

Article Title: CDK12/13 inactivation triggers STING-mediated antitumor immunity in preclinical models

doi: 10.1172/JCI193745

Figure Lengend Snippet: ( A ) Enrichment of the indicated pathways in Myc-CaP cells treated with YJ1206 at 1 μM for 24 hours. Adj., adjusted. ( B ) Immunoblot of the indicated proteins in Myc-CaP cells treated with YJ1206 at increasing concentrations for 4 hours, or siRNAs targeting Cdk12 and/or Cdk13 . Nontargeting siRNA was used as control. GAPDH was used as a loading control. ( C ) Top: Analysis of the indicated gene expression by RT-qPCR in Myc-CaP cells treated with YJ1206 at 1 μM for 15 hours, or siRNAs targeting Cdk12 and/or Cdk13 . Nontargeting siRNA was used as control. Bottom left: IFN-β ELISA results in Myc-CaP cells treated as described above. Bottom right: Flow cytometry assessing surface MHC-I expression in Myc-CaP cells treated as described above. ( D ) Immunoblot of the noted proteins in B16-F10 cells treated with YJ1206 at increasing concentrations for 4 hours. ( E and F ) Flow cytometry median fluorescence intensity (MFI) quantifications of surface MHC-I ( E ) or PD-L1 ( F ) in the indicated cells treated with YJ1206 at 1 μM or 3 μM for 15 hours. ( G ) Quantification of immunofluorescence DNA/RNA hybrid (red) staining in Myc-CaP cells treated with 1 μM YJ1206 for 4 hours or siRNA targeting Cdk12 and/or Cdk13 , with/without RNase H. Representative images are in . Nontargeting siRNA was used as control. Forty (siRNA treatment) or 20 (YJ1206 or DMSO treatment) cells were used per data point. Data in C , E , and F are displayed as mean ± SD of triplicate experiments. Data in G are presented as box-and-whisker plots, with the median (center line), 25th–75th percentiles (box), and minimum to maximum values (whiskers). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by 2-tailed t test. NS, not significant. Bonferroni’s correction was applied for multiple comparisons in C and E – G .

Article Snippet: IFN-β ELISA (R&D Systems, DY8234-05 and DY008B) and cGAMP ELISA (Cayman Chemical, 501700) kits were used per the manufacturer’s protocols.

Techniques: Western Blot, Control, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Fluorescence, Immunofluorescence, Staining, Whisker Assay

Journal: The Journal of Clinical Investigation

Article Title: CDK12/13 inactivation triggers STING-mediated antitumor immunity in preclinical models

doi: 10.1172/JCI193745

Figure Lengend Snippet: ( A and B ) Representative images ( A ) or quantification ( B ) of immunofluorescence assessing PCNA-RNAPII PLA foci in Myc-CaP cells treated with siRNA targeting Cdk12 and/or Cdk13 , or YJ1206 at 3 μM for 4 hours, with or without DRB or triptolide treatment. ( C – E ) Representative images ( C ) or quantification ( D and E ) of dsDNA ( C , left, and D ) and ssDNA ( C , right, and E ) in Myc-CaP cells treated with 3 μM YJ1206 for 4 hours, with or without DRB treatment. Scale bars: 5 μm. ( F ) ELISA measuring cGAMP levels in Myc-CaP cells treated with YJ1206 at the indicated concentrations. ( G ) Immunoblot of the noted proteins in Myc-CaP cells treated with YJ1206 at 1 μM, with or without DRB for 4 hours. Data in D and E are presented as box-and-whisker plots, with the median (center line), 25th–75th percentiles (box), and minimum to maximum values (whiskers). Data are displayed as mean ± SEM in F of triplicate experiments. One hundred cells were used per data point in B , D , and E . **** P < 0.0001 by 2-tailed t test. NS, not significant. Bonferroni’s correction was applied for multiple comparisons.

Article Snippet: IFN-β ELISA (R&D Systems, DY8234-05 and DY008B) and cGAMP ELISA (Cayman Chemical, 501700) kits were used per the manufacturer’s protocols.

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Whisker Assay

Journal: International journal of molecular sciences

Article Title: Surgical Site-Released Tissue Is Potent to Generate Bone onto TCP and PCL-TCP Scaffolds In Vitro.

doi: 10.3390/ijms242115877

Figure Lengend Snippet: Figure 6. (a–c) ELISA boxplots of the osteogenic markers osteocalcin (a), osteopontin (b), and collagen I (c). Boxplots compare the concentration of ocn, opn, and col I in the control group with the PCL-TCP and β-TCP group over the cultivation time of 6 weeks. Groups are further subdivided into the condition’s agarose and glue, to examine if the chosen adhesive, used for the attachment of the scaffolds onto the plastic wells, makes a difference in protein expression. The boxblots indicating the median within the 25–75% percentile. The dots represent single outliners during measurements.

Article Snippet: Enzyme-linked immunosorbent assays (ELISAs) (human Duoset ELISA kit, R&D Systems, Bio Techne, Minneapolis, MN, USA) were then performed using the supernatants at a dilution of 1:10 for osteocalcin (1:50; pure for collagen and pure for osteopontin) according to the manufacturer’s protocol and measured at 450 nm using a Multiscan Ascent reader (Thermo Scientific).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Control, Adhesive, Expressing

Journal: Nature Communications

Article Title: TREM2 promotes lung fibrosis via controlling alveolar macrophage survival and pro-fibrotic activity

doi: 10.1038/s41467-025-57024-0

Figure Lengend Snippet: A TREM2 (+/+) and TREM2 (-/-) Mo-AMs were isolated by flow sorting from mice that were treated BLM for 3 weeks, and apoptosis evaluated. n = 4 mice per group; mean ± SEM; unpaired two-tailed Student’s t -test. B Numbers of Mo-AMs in the BALs. n = 6 and 5 mice, respectively; mean ± SEM; unpaired two-tailed Student’s t -test. C BMDMs were transfected with control (con) and TREM2 siRNAs and then cultured in the presence of 10 ng/ml M-CSF for 2 days. Cell apoptosis was determined 6 h after M-CSF withdrawal. n = 4 independent culture of cells per group; mean ± SD; one-way ANOVA with Bonferroni’s multiple comparisons test. The experiment was repeated three with similar results. D BMDMs were transfected with control and TREM2 siRNAs and then cultured in the presence of 10 ng/ml M-CSF for 2 days. Cells were harvested 6 h after M-CSF withdrawal and the expression of the indicated proteins were determined by Western blotting. The experiment was repeated three times with similar results. E The expression of pro-fibrotic mediators in Mo-AMs from mice treated in “ A ” was determined by real-time PCR. n = 4 mice per group; mean ± SEM; unpaired two-tailed Student’s t- test. F TGF-β1 levels in BALs from mice treated in “ A ” were determined by ELISA. n = 6 and 5 mice, respectively; mean ± SEM; unpaired two-tailed Student’s t -test. G BAL TGF-β1 levels in TREM2 (+/+) and TREM2 (-/-) mice instilled with saline or BLM for 3 weeks. n = 3, 3, 7, and 6 mice, respectively; mean ± SEM; one-way ANOVA with Bonferroni’s multiple comparisons test. H , I Bubble plots for the indicated pro-fibrotic and anti-apoptotic genes in the macrophages from BLM treated mouse lungs ( H ) and human IPF lungs ( I ) were re-produced from scRNA-seq datasets GSE132771 ( n = 2 mouse lungs) and GSE128033 ( n = 5 IPF lungs), respectively. Source data are provided as a Source Data file.

Article Snippet: Levels of TGF-β1, IL-1β and KC in the BALs were determined using the respective DuoSet ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Isolation, Two Tailed Test, Transfection, Control, Cell Culture, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Saline, Produced

Journal: iScience

Article Title: Rearrangement of T Cell genome architecture regulates GVHD

doi: 10.1016/j.isci.2022.104846

Figure Lengend Snippet:

Article Snippet: IFNg DuoSet ELISA Kit , BD Systems , DY485.

Techniques: Recombinant, Staining, Protease Inhibitor, Cell Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Knock-In, Expressing, Software