Journal: Frontiers in microbiology

Article Title: Paracrine IFN Response Limits ZIKV Infection in Human Sertoli Cells.

doi: 10.3389/fmicb.2021.667146

Figure Lengend Snippet: FIGURE 2 | IFN response limits ZIKV infection in Sertoli cells. SC were exposed to ZIKV for 1h (MOI 3) and ZIKV infection and MX1 protein levels were evaluated by (A) immunofluorescence assay (IFA) using mouse monoclonal against the ZIKV E protein (green) and rabbit polyclonal against MX1 (red), respectively. Nuclei were stained with DAPI (blue). Images were taken at 100× magnification. (B) ZIKV titers in SC supernatant were (black line curve) determined by plaque assay (n = 3 for each time point) and the percentage of ZIKV-positive cells (green line curve) was quantified via IFA (n = 2 field per coverslip for each time point). (C) Changes in MX1 protein levels were quantified by mean fluorescence intensity (MFI) of IFA coverslips (n = 4) from each time point using ImageJ software, reported as MFI fold-change compared to mock. (D) Western blot analysis of MX1 protein levels at 24 and 48 h post-infection. b-actin and IRF3 were used as loading controls and each lane represents an independent experiment. (E) Secreted IFN-β in SC supernatant (n = 4 for each time point), determined by human IFN-β ELISA (R&D Systems); dotted line denotes detection limit of assay. (F) Zoomed images of IFA to depict co-localization of ZIKV and MX1 at 120h post-infection. (G,H) SC were treated with 5 pg/mL (1 IU/mL) of recombinant human IFN-β (rhIFN-β; R&D systems) 24 h prior to and upon ZIKV infection (MOI 1) and were replenished with the rhIFN-β (5 pg/mL) at 24 h post-infection. (G) ZIKV genome copies were measured in infected SC with and without IFN-β treatment at 48 h post-infection by RT-qPCR. (H) Gene expression of MX1 and IFIT1 was evaluated at 48 h post-infection by RT-qPCR in both mock and infected SC with and without IFN-β treatment and reported as fold-change compared to mock untreated. The housekeeping gene GAPDH was used to normalize fold-change for all gene expression assays. Significance determined by Student’s t-test for all assays, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Images were taken at 100× magnification. (B) ZIKV titers in SC supernatant were (black line curve) determined by plaque assay (n = 3 for each time point) and the percentage of ZIKV-positive cells (green line curve) was quantified via IFA (n = 2 field per coverslip for each time point). (C) Changes in MX1 protein levels were quantified by mean fluorescence intensity (MFI) of IFA coverslips (n = 4) from each time point using ImageJ software, reported as MFI fold-change compared to mock. (D) Western blot analysis of MX1 protein levels at 24 and 48 h post-infection. b-actin and IRF3 were used as loading controls and each lane represents an independent experiment. (E) Secreted IFN-β in SC supernatant (n = 4 for each time point), determined by human IFN-β ELISA (R&D Systems); dotted line denotes detection limit of assay. (F) Zoomed images of IFA to depict co-localization of ZIKV and MX1 at 120h post-infection. (G,H) SC were treated with 5 pg/mL (1 IU/mL) of recombinant human IFN-β (rhIFN-β; R&D systems) 24 h prior to and upon ZIKV infection (MOI 1) and were replenished with the rhIFN-β (5 pg/mL) at 24 h post-infection. (G) ZIKV genome copies were measured in infected SC with and without IFN-β treatment at 48 h post-infection by RT-qPCR. (H) Gene expression of MX1 and IFIT1 was evaluated at 48 h post-infection by RT-qPCR in both mock and infected SC with and without IFN-β treatment and reported as fold-change compared to mock untreated.

Techniques: Infection, Staining, Plaque Assay, Software, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Quantitative RT-PCR, Gene Expression

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , Schematic showing the region of the 3’ UTR of STING that is recognized by the miR-576-3p seed sequence. b , HBEC were transfected with control or miR-576-3p (M-576-3p) mimic for 72 h. Cells were then mock infected or infected with VSV-GFP at an MOI 3 for 3 h. Total RNA was harvested from cells and STING mRNA levels were determined by qPCR and normalized to levels of β-actin. c , HBEC were transfected with miRNA mimic (M-576-3p) or inhibitor (I-576-3p) as in b and mock-infected or infected with VSV-GFP at MOI 0.1 for 18 h. Cell lysates were harvested and subjected to western blot analysis with anti-STING antibodies. β-actin serves as loading control. d,e , HBEC were transfected with 1 µg/ml of poly (I:C) for 6 h ( d ) or treated with 100 U/ml of IFNβ for 18 h ( e ) and levels of pri-mir-576 and SEC24B mRNA were analyzed by qPCR. f,g , HBEC were transfected with poly (I:C) as in d after siRNA knockdown of NFκB (P65) or IRF3. Relative pri-mir-576 ( f ) or SEC24B mRNA ( g ) levels were measured by qPCR. h , HBEC expressing control or miR-576-3p mimic (M-576-3p) or an siRNA targeting STING were transfected with poly (I:C) and levels of IFNβ mRNA were measured by qPCR. i , HBEC were transfected with control or miR-576-3p mimic (M-576-3p) and pre-treated with 100 U/ml of IFNβ prior to VSV infection. Cell viability was determined by measuring ATP levels of mock or infected cells. j , HBEC expressing control or miR-576-3p inhibitor (I-576-3p) were transfected with poly (I:C) and levels of IFNβ were measured by qPCR. Data are representative of three independent experiments. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Sequencing, Transfection, Control, Infection, Western Blot, Knockdown, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , HBEC were transfected with 1 µg/ml of poly (I:C) for 6 h or treated with 100 U/ml of IFNβ for 18 h and levels of mature miR-576-3p were determined by qPCR. b , HBEC were mock infected or infected with HSV-1 at MOI 10 for 3 h and levels of miR-576-3p were measured by qPCR. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01. ns, non significant with respect to control.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Transfection, Infection, Two Tailed Test, Control

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , Model illustrates regulation of IFN expression by miR-576-3p as a feedback mechanism. Targets of miR-576-3p are indicated by inhibitory red lines. b , Public available microRNA array datasets (GSE37425 and GSE37426) of synovial or renal tissues from RA or SLE patients, respectively, along with controls were obtained from the GEO database . Normalized expression values of miR-576-3p from each sample were used to calculate the relative levels and p-values of miR-576-3p for SLE or RA patients as compared to controls. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Expressing, Two Tailed Test

Journal: Chemical science

Article Title: Dual-targeting biomimetic delivery for anti-glioma activity via remodeling the tumor microenvironment and directing macrophage-mediated immunotherapy.

doi: 10.1039/c7sc04853j

Figure Lengend Snippet: Fig. 4 (A) The cytotoxicity test (IC50) in the U87 and GL261 cells. (B) The cytotoxicity test in the U87 cells cultured with M1-CM or M2-CM. (C) The expression of MRs in TAM1 and TAM2 and the polarization modulation of the drugs. (D) The re-education of TAM2 treated with drugs and the expression of MRs and B7-H4. The ELISA analysis of the expression of TNF-a (E) and TGF-b1 (F) in TAM1 and TAM2 after drug treatment. The ELISA analysis of the expression of TNF-a (G) and TGF-b1 (H) in TAM1 and TAM2 cocultured with U87 cells after treatment.

Article Snippet: The mouse IFN-g Elisa Kit was purchased from R&D Systems (USA).

Techniques: Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Clinical Epigenetics

Article Title: The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry

doi: 10.1186/s13148-017-0312-z

Figure Lengend Snippet: RRx-001 induced IFN response through upregulation of type I and III IFN expression and JAK/STAT pathway. Cells were transiently (24 h) treated with 0.5 μM RRx-001 or 0.5 μM 5-AZA and subsequently maintained in drug-free medium for an additional 7 days. RRx-001 induced a significant increase in type I IFN (IFN-β) ( a ) and type III IFN (IL-29/IL-28B) ( b ) secretion into culture medium by HCT 116 cells as measured by ELISA. Transcript levels of IL29 / IL28A were also increased as determined by qPCR ( c ). ISGs ( IFI27 , IFi44 , IFI44L , and IFI6 ) were upregulated by 5-AZA and RRx-001 but blocked by the JAK/STAT inhibitor ruxolitinib (rux) at 2 μM concentration ( d ). Expression of ISGs ( IRF7 , ISG15 , DDX58 , and OASL ) was also upregulated in HCT 116 cells cultured in conditioned medium containing secreted IFNs induced by 5-AZA and RRx-001 as determined by qPCR ( e )

Article Snippet: Levels of type I IFN (IFN-β) and type III IFN (IL-29/IL-28B) were determined using a VeriKine Human IFN-beta ELISA Kit (PBL Assay Science, Piscataway Township, NJ, USA) and a Human IL-29/IL-28B (IFN-lambda 1/3) DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to manufactures’ instructions, respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture