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