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: Cells

Article Title: TRPA1 as a Key Regulator of Keratinocyte Homeostasis and Inflammation in Human Skin

doi: 10.3390/cells15020192

Figure Lengend Snippet: TRPA1 silencing modestly enhances keratinocyte expression of multiple chemokines. Keratinocytes obtained from three different donors were cultured for 18 h in the presence or absence of IFN-γ and TNF-α, in both control cells and silencing-treated cells, and then analyzed by real-time PCR, as described in the Methods section.

Article Snippet: Cytokine stimulations with recombinant human (rh) IFN-γ (200 U/mL), TNF-α (50 ng/mL), and IL-17A (50 ng/mL) (R&D Systems, Minneapolis, MN, USA), as well as treatments with TRPA1 agonists, were conducted on keratinocyte cultures kept in keratinocyte basal medium (KBM-GOLD, Clonetics, Lonza, Basel, Switzerland).

Techniques: Expressing, Cell Culture, Control, Real-time Polymerase Chain Reaction

Journal: Nucleic acids research

Article Title: Internal RNA 2'O-methylation in the HIV-1 genome counteracts ISG20 nuclease-mediated antiviral effect.

doi: 10.1093/nar/gkac996

Figure Lengend Snippet: Figure 7. Restriction profile of ISG20 WT and the R53A/D90A mutant on replication competent HIV-1. (A) HeLa P4C5 cells were treated or not with 1000 IU/ml human IFN-2a for 18h and transfected with a siRNA control (−) or targeting ISG20 (+), as indicated. At 48h post-transfection, ISG20 expression was assessed by western-blot using anti-ISG20 antibodies. (B–D) HeLa P4C5 cells were treated or not with 1000 IU/ml human IFN-I for 18 h and transfected with a siRNA control (−) or targeting ISG20 (+), as indicated. At 48h post-transfection, cells were infected with HIV-1 NL4-3 produced in WT or in FTSJ3-KO cells. At 6 h post-infection, cells were harvested and total RNA and DNA were extracted for specific qPCR analysis. (B) The expression of endogenous ISG20 upon IFN-I induction was evaluated by RT-qPCR. (C) Quantification of ectopic expression of transfected ISG20 WT and double mutant by RT-qPCR. (D) The amount of HIV-1 RT products was estimated by qPCR. Data represent the mean of three independent experiments performed in duplicate ± SD. ****P < 0.0001, as determined by one-way ANOVA with Bonferroni post hoc test. ns, non-significant.

Article Snippet: When indicated, HeLa P4C5 and Jurkat cells were treated with recombinant human IFN- 2a (R&D Systems).

Techniques: Mutagenesis, Transfection, Control, Expressing, Western Blot, Infection, Produced, Quantitative RT-PCR