p426 snr52p grna  (Addgene inc)

Journal: bioRxiv

Article Title: DNA Damage Response Proteins Are Involved in the Formation of Defective HIV-1 Proviruses

doi: 10.64898/2026.03.31.715508

Figure Lengend Snippet: A) Flow cytometry gating strategy for K562 VPR cells transduced with CRISPRa-gRNA and LTatC[M]L lentiviral vectors. Example shown for CRISPRa-HLTF technical replicates. B) Single mCherry+/double-positive Cerulean+/mCherry+ (mCherry/Dp) cell ratio for candidate genes in K562-VPR cells transduced with corresponding CRISPRa-gRNA and LTatC[M]L. Data shown relative to non-targeting control gRNA, marked by the dotted line. Each dot represents one technical replicate. C) mCherry/Dp ratio for candidate genes in K562 cells transfected with corresponding siRNA and transduced with LTatC[M]L. From the left: transfection with siRNAs targeting single genes; transfection with combination of siRNAs targeting two genes. Data shown relative to scrambled control siRNA, marked by the dotted line. Each dot represents one technical replicate. All data was statistically tested using mixed-effect logistic regression model, p value <0.1 was considered significant. **** = p <0.001, *** p <0.01, ** p <0.05.

Article Snippet: The Golden Gate reaction contained 25 ng backbone plasmid, 1 μl of 1:10 diluted annealed gRNA oligonucleotides, 0.5 μl BsmBI-v2, 0.5 μl T4 DNA ligase (Thermo Scientific, EL0011), 1 μl 10 mM ATP (New England Biolabs, P0756S), 1 μl NEB Buffer r3.1 (New England Biolabs), and nuclease-free water to the final reaction volume.

Techniques: Flow Cytometry, Transduction, Control, Transfection

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs dampens 2′3′-cGAMP-mediated STING signaling. (A) Graph represents mean (±SEM, n = 3 independent experiment) CXCL10 and CCL5 levels as measured in supernatant of T98G cells treated or not with 2 µM NU7441 for 1 h prior to transfection or not of 10 µg/ml 2′3′-cGAMP for 6 h. Statistical significance was calculated by two-tailed Student's t test. (B) T98G cells were pretreated with the NU7441 (2 µM), NU7026 (10 µM), and AZD7648 (5 µM) DNA-PKcs inhibitors for 1 h prior to transfection of 2′3′-cGAMP (10 µg/ml) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (C) T98G cells were pretreated with the NU7441 (2 µM) for 1 h prior to transfection with dsDNA (2 µg) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (D) T98G cells were transfected for DNA-PKcs– or KU70-targeting siRNAs or a control nontargeting siRNA for 48 h prior to analysis of knockdown efficiency by WB using the indicated antibodies. Representative WB; n = 3 independent experiments. (E) T98G cells were engineered to express control nontargeting or PRKDC-targeting gRNA. Representative WB; n = 3 independent experiments. (F) THP-1 CTRL, THP1 cGAS−/− , and THP1 STING−/− were pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection or not of 2′3′-cGAMP (10 µg/ml) for 6 h. WB analysis was performed using the indicated antibodies. Representative WB of three to five independent experiments. (G) Densitometric quantification of band intensities of the p-IRF3/IRF3 ratio from the WB in F. Results shown as % of induction of 2′3′-cGAMP response ( n = 3–5 independent experiments). (H) Densitometric quantification of band intensities of the pSTING/STING ratio from the WB in F. Results shown as % of induction of 2′3′-cGAMP response ( n = 3–5 independent experiments). (I) Graph represents mean (±SEM, n = 3 independent experiment) IFNβ levels as measured in supernatant of THP1 CTRL , THP1 cGAS−/− , and THP1 STING−/− pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection or not of 2′3′-cGAMP (10 µg/ml) for 6 h. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were engineered to express control nontargeting or PRKDC-targeting gRNA. Representative WB of three independent experiments. (K) T98G cells engineered to express control nontargeting or STING-targeting gRNAs were treated or not with 2 µM of NU7441 prior to transfection or not of 10 µg/ml 2′3′-cGAMP and gene expression analysis. Graphs present the mean (±SEM, n = 5 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (L) As in K, except that WB analysis was performed using the indicated antibodies. Representative WB of three independent experiments. (M) As in L, except that T98G cells expressing an IFNAR-targeting gRNA were used. Representative WB of three independent experiments. (N) THP-1 cells were pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection with dsDNA (2 µg) for up to 24 h. Gene expression analysis was conducted at 3, 6, 16, and 24 h. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (O) Cell culture supernatants were collected at 24 h in experiment performed as in N, and cytokine/chemokine levels were analyzed using a proteome profiler array. Heatmap representation of relative spot intensities is shown (mean of three independent experiments). (P) STING-deficient THP-1 cells engineered to express human STING haplotypes (STING-H232, STING-AQ, and STING-HAQ) were pretreated or not with the NU7441 (2 µM) for 1 h prior to transfection of 2′3′-cGAMP (10 µg/ml) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (Q) As in P, except that IFNβ and CXCL10 levels were quantified by ELISA in supernatants. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Related to . Source data are available for this figure: .

Article Snippet: To generate the T98G IFNAR−/− knockout and control cell lines, lentiviral particles were produced by co-transfection of 2 × 10 6 293T cells with 5 μg of LentiCRISPRv2GFP plasmid (#82416; Addgene) expressing the gRNA targeting the gene of interest or nontargeting control gRNA, 5 μg of psPAX2, and 1 μg of pMD2.G, using the standard calcium phosphate transfection protocol.

Techniques: Transfection, Two Tailed Test, Gene Expression, Control, Knockdown, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs decreases the ability of STING agonists to trigger an antiviral response. (A) T98G cells were treated with the 10 µM diABZI, 1 µM E7766, or 10 mg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with VSV-GFP for 16 h prior to DAPI nuclear staining and image acquisition. Images are representative of three independent experiments. (B) As in A, except that cells were infected with the MPXV clade 2b strain S2626 for 48 h. Images are representative of three independent experiments. (C) T98G cells engineered to express control nontargeting or DNA-PKcs–targeting gRNA were treated with 10 µg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with VSV-GFP at MOI 0.3 for 16 h, prior to DAPI nuclear staining and image acquisition. Graph shows the mean (±SEM, n = 3 independent experiments) percentage of infected (GFP + ) cells as measured by fluorescent microscopy. Statistical significance was assessed using two-tailed Student's t test. (D) Gating strategy for macrophages used in . (E) Histograms show the percentage of infected (GFP + ) cells as measured by flow cytometry, following treatment with STING agonists, in the presence or absence of NU7441, at two different MOIs. (F) Primary macrophages from healthy donors 1, 2, and 3 were pretreated with NU7441 prior to STING agonist treatment. Cells treated with a STING agonist, and they were set as 100% infection, and the effect of adding a NU7441 was assessed relative to this condition. Scale bars, 500 μm. **: P < 0.01; *: P < 0.05. Data are from at least three independent experiments. Related to .

Article Snippet: To generate the T98G IFNAR−/− knockout and control cell lines, lentiviral particles were produced by co-transfection of 2 × 10 6 293T cells with 5 μg of LentiCRISPRv2GFP plasmid (#82416; Addgene) expressing the gRNA targeting the gene of interest or nontargeting control gRNA, 5 μg of psPAX2, and 1 μg of pMD2.G, using the standard calcium phosphate transfection protocol.

Techniques: Infection, Staining, Control, Microscopy, Two Tailed Test, Flow Cytometry