Journal: Antimicrobial Agents and Chemotherapy

Article Title: Activation of Stimulator of Interferon Genes in Hepatocytes Suppresses the Replication of Hepatitis B Virus

doi: 10.1128/aac.00771-17

Figure Lengend Snippet: Figure 1. Stable expression of cGAS and STING in HepAD38 cells successfully 574

Article Snippet: Human cGAS cDNA clone was 122 purchased from OriGene and the coding region was subcloned into puromycin resistant gene 123 expressing retroviral vector pQCXIP (Clontech) between Not I and BamH I sites.

Techniques: Expressing

Journal: Life Science Alliance

Article Title: Attenuation of cGAS/STING activity during mitosis

doi: 10.26508/lsa.201900636

Figure Lengend Snippet: (A) Mitotic phospho-IRF3 and phospho-STING responses to pGL3. Asynchronous and arrested cells were stimulated by transfection of 500 ng pGL3 or water for 90 min before Western blots. (B) Mitotic cGAMP responses to pGL3. Asynchronous and arrested cells were transfected with 500 ng pGL3. Non-internalized transfection complexes were removed by media change 2 h after the initial transfection. 5 h post-transfection, cGAMP levels were measured by ELISA. * P < 0.05, n = 2 biological replicates, with n = 2 technical replicates each. (C, D) cGAS subcellular localization and Golgi morphology in asynchronous (C) and arrested (D) HaCaT cells. Scale bars = 10 μm. (E) Mitotic phospho-IRF3 and phospho-STING responses to exogenous cGAMP. Asynchronous and arrested cells were treated with 25 μg/ml 2′-3′cGAMP for 2 h before Western blots. Source data are available for this figure.

Article Snippet: Pooled scramble (sc-37007), cGAS (sc-95512), STING (sc-92042), and IRF3 (sc-35710) siRNA duplexes were obtained from Santa Cruz Biotechnologies.

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo

doi: 10.3390/cancers13123058

Figure Lengend Snippet: Correlation of the oncolytic activity of T-VEC with the expression of stimulator of interferon genes (STING) and cyclic GMP-AMP synthase (cGAS) in a panel of melanoma cell lines. ( A ) Western Blot analysis of STING and cGAS with respect to housekeeping protein ß-actin using lysates of 20 melanoma cell lines. ( B ) Densitometric quantification of STING and cGAS, normalized for ß-actin and shown as the mean and standard error of three independent Western blot experiments. ( C ) Spearman correlation coefficient, ( D ) box plot, and ( E ) ROC curve analyses of all responder and non-responder cell lines with respect to STING (upper part) and cGAS expression (lower part) and corresponding susceptibility to T-VEC induced cell death. p values for box plots were calculated using the Mann–Whitney test; ROC curves analyzed the area under the curve (AUC).

Article Snippet: The following primary antibodies were used: Rabbit monoclonal antibody against STING (D2P2F, Cell Signaling; dilution for patient samples 1:50, dilution for cell lines 1:200), murine monoclonal antibody against Nectin-1 (R1.302.12, Santa Cruz, Heidelberg, Germany; dilution for patient samples 1:50), rabbit polyclonal antibody against Nectin-1 (AB_2736197, Invitrogen/Thermo Fisher; dilution for cell lines 1:100), murine monoclonal antibody against HVEM (CW10, Santa Cruz; dilution 1:500 for patient samples and cell lines), rabbit polyclonal antibody against cGAS (NBP1-86761, Novus Biologicals/Bio-Techne, Wiesbaden Nordenstadt, Germany; dilution for patient samples 1:200), and rabbit monoclonal antibody against cGAS (D1D3G) (Cell Signaling; dilution for cell lines 1:200).

Techniques: Activity Assay, Expressing, Western Blot, MANN-WHITNEY

Journal: Cancers

Article Title: Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo

doi: 10.3390/cancers13123058

Figure Lengend Snippet: Correlation of biomarkers evaluated by flow cytometry, Western blot, and immunohistochemistry with the oncolytic activity of T-VEC in melanoma cell lines. Spearman correlation coefficient analysis for ( A ) Nectin-1 and HVEM expression, measured by flow cytometry (FACS) and immunohistochemistry (IHC), ( B ) STING and cGAS expression, evaluated by Western blot (WB) and immunohistochemistry, and ( C ) expression of all four biomarkers in immunohistochemistry with the oncolytic activity of T-VEC in 20 melanoma cell lines. Data show the mean of three independent experiments for each biomarker and MTT assay.

Article Snippet: The following primary antibodies were used: Rabbit monoclonal antibody against STING (D2P2F, Cell Signaling; dilution for patient samples 1:50, dilution for cell lines 1:200), murine monoclonal antibody against Nectin-1 (R1.302.12, Santa Cruz, Heidelberg, Germany; dilution for patient samples 1:50), rabbit polyclonal antibody against Nectin-1 (AB_2736197, Invitrogen/Thermo Fisher; dilution for cell lines 1:100), murine monoclonal antibody against HVEM (CW10, Santa Cruz; dilution 1:500 for patient samples and cell lines), rabbit polyclonal antibody against cGAS (NBP1-86761, Novus Biologicals/Bio-Techne, Wiesbaden Nordenstadt, Germany; dilution for patient samples 1:200), and rabbit monoclonal antibody against cGAS (D1D3G) (Cell Signaling; dilution for cell lines 1:200).

Techniques: Flow Cytometry, Western Blot, Immunohistochemistry, Activity Assay, Expressing, Biomarker Discovery, MTT Assay

Journal: Cancers

Article Title: Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo

doi: 10.3390/cancers13123058

Figure Lengend Snippet: Oncolytic effect of T-VEC upon injection into 35 malignant melanoma lesions. ( A ) Waterfall plot showing the response rate of each individual lesion as increase or decrease of the tumor volume, calculated as (length × width × thickness)/2 when the maximum anti-tumor effect had been achieved. ( B ) Based on this criterion, 14 and 21 lesions were respectively categorized as non-responders (+129.89% to −29%) and responders (−30% to −100%). ( C ) Representative example of Nectin-1, HVEM, STING, and cGAS immunostaining in a melanoma lesion responding (upper panel) or not responding (lower panel) to intratumoral T-VEC injection. Images provide an overview and details at higher magnification (inserts); corresponding size bars are included. ( D ) Spearman correlation coefficient analysis of Nectin-1, HVEM, STING, and cGAS immunostaining with the oncolytic activity of T-VEC inoculated into the respective lesion. ( E ) Box plots (with median, interquartile ranges, minimum, and maximum values) and ( F ) ROC curve analysis of responder and non-responder lesions with respect to Nectin-1, HVEM, STING, and cGAS immunostaining. p values for box plots were calculated using the Mann–Whitney test; p values for ROC curves analyzed the area under the curve.

Article Snippet: The following primary antibodies were used: Rabbit monoclonal antibody against STING (D2P2F, Cell Signaling; dilution for patient samples 1:50, dilution for cell lines 1:200), murine monoclonal antibody against Nectin-1 (R1.302.12, Santa Cruz, Heidelberg, Germany; dilution for patient samples 1:50), rabbit polyclonal antibody against Nectin-1 (AB_2736197, Invitrogen/Thermo Fisher; dilution for cell lines 1:100), murine monoclonal antibody against HVEM (CW10, Santa Cruz; dilution 1:500 for patient samples and cell lines), rabbit polyclonal antibody against cGAS (NBP1-86761, Novus Biologicals/Bio-Techne, Wiesbaden Nordenstadt, Germany; dilution for patient samples 1:200), and rabbit monoclonal antibody against cGAS (D1D3G) (Cell Signaling; dilution for cell lines 1:200).

Techniques: Injection, Immunostaining, Activity Assay, MANN-WHITNEY

Journal: Nature metabolism

Article Title: CRAT links cholesterol metabolism to innate immune responses in the heart

doi: 10.1038/s42255-023-00844-5

Figure Lengend Snippet: a,b, Combined deletion of caspase-1 In CRAT-mKO male and female mice inhibited the increases in IL-1β level in serum (a) and hearts (b). c,d, Representative images and quantitative analysis showed that the infiltration of F4/80+ macrophages in CRAT-mKO male and female hearts was significantly inhibited by combined deletion of caspase-1. Scale bar, 25 μm. DAPI, 4,6-diamidino-2-phenylindole. e–g, HW:TL ratios (e) and echocardiographic analyses (f,g) at 2 months after tamoxifen injection indicated that combined deletion of caspase-1 reversed the compromised cardiac function induced by CRAT deficiency. EF, ejection fraction; FS, fractional shortening. h–j, CRAT-WT and CRAT-mKO male and female mice were injected with AAV9-sh-control, AAV9-sh-cGas or AAV9-sh-Aim2 virus (5 × 1011 GC per mouse) at 1 week before tamoxifen administration. After 8 weeks, echocardiographic analyses were performed to evaluate cardiac contractile function (h). CMs were then isolated for western blots to examine the roles of cGAS and AIM2 in inflammasome activation induced by CRAT depletion in vivo (i,j). n = 6 (a,b,d), n = 8 (e), n = 7–8 (f,g, WT), n = 6 (f,g, Casp1 KO), n = 6 (h, WT, Sh-cGas), n = 7 (h, Sh-Aim2) and n = 6 (j) mice per group. Data are shown as mean ± s.e.m. Analysis was carried out by two-way ANOVA followed by Fisher’s LSD multiple comparison test (a,b,d–g,h,j).

Article Snippet: Generation of lentiviral particles Lentiviral vectors encoding Crat , cGas , Pparα , Aim2 shRNAs or control vectors were obtained from Origene.

Techniques: Injection, Control, Virus, Isolation, Western Blot, Activation Assay, In Vivo, Comparison

Journal: Nature metabolism

Article Title: CRAT links cholesterol metabolism to innate immune responses in the heart

doi: 10.1038/s42255-023-00844-5

Figure Lengend Snippet: a,b, Heat map and GSEA of ISGs in NRVMs transduced with lentivirus expressing control shRNA (WT) or Crat shRNA (KD). c, RT–PCR confirmed the induction of ISGs in CRAT-deficient NRVMs. d–h, RT–PCRs indicated that increased expression of ISGs in CRAT-deficient NRVMs was dependent on cGAS. i, Western blots confirmed cGAS-dependent induction of some ISGs in CRAT-deficient NRVMs.j, Quantification of cytosolic mtDNA in control or CRAT-deficient NRVMs. k, Quantification of cytosolic mtDNA in adult CMs isolated from CRAT-WT or CRAT-mKO male and female hearts. l, Treatment with cyclosporin A (CsA, 10 μM) significantly inhibited ISG expression in CRAT-deficient NRVMs. n = 6 (c), n = 6 (d–h, Sh-control), n = 7 (d–f,h, Sh-cGas), n = 5 (g, Sh-cGas and Sh-Crat), n = 5 (j), n = 6 (k) and n = 4 (l) biologically independent samples per group. Data are presented as mean ± s.e.m. Analysis was carried out by two-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test (d–h) and unpaired two-tailed Student’s t-test (c,j–l).

Article Snippet: Generation of lentiviral particles Lentiviral vectors encoding Crat , cGas , Pparα , Aim2 shRNAs or control vectors were obtained from Origene.

Techniques: Transduction, Expressing, Control, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Comparison, Two Tailed Test

Journal: Nature metabolism

Article Title: CRAT links cholesterol metabolism to innate immune responses in the heart

doi: 10.1038/s42255-023-00844-5

Figure Lengend Snippet: Loss of CRAT promotes cholesterol catabolism through the bile acid synthesis pathway, leading to intracellular accumulation of intermediates of bile acid synthesis such as 7-HOCA, which is sufficient to promote mtDNA release into cytosol and trigger cGAS–STING-dependent type I interferon responses. Further, type I interferon responses elicited by CRAT deficiency lead to a substantial increase in AIM2 expression and subsequent activation of the DNA-sensing AIM2 inflammasome, which in turn promotes proteolytic maturation of IL-1β and CM pyroptosis. Eventually, genetic deletion of CRAT in CMs leads to myocardial inflammation and results in DCM. Collectively, we have identified a mechanism by which cardiac energy metabolism, cholesterol homeostasis and CM-intrinsic innate immune responses are interconnected via a CRAT-mediated bile acid synthesis pathway, which contributes to chronic myocardial inflammation and HF progression.

Article Snippet: Generation of lentiviral particles Lentiviral vectors encoding Crat , cGas , Pparα , Aim2 shRNAs or control vectors were obtained from Origene.

Techniques: Expressing, Activation Assay