Journal: Cells

Article Title: Cytosolic Immunostimulatory DNA Ligands and DNA Damage Activate the Integrated Stress Response, Stress Granule Formation, and Cytokine Production

doi: 10.3390/cells15020139

Figure Lengend Snippet: The cGAS-STING signaling pathway is required for SG induction by DNA ligands. ( A ) HT1080 cells were treated with siRNA for 36 h to knockdown DDX41, IFI16, or cGAS, followed by transfection with pdA:dT (5 μg/mL) or HT-DNA (4 μg/mL) for 6 h, and cell lysates were analyzed for phosphorylation of STING and TBK1 compared to total STING and total TBK1 normalized to β-actin levels. The ratios of p-STING/total STING and p-TBK1/total TBK1 were quantified by Image J. The efficiency of knockdown by siRNA was compared to control cells and determined by immunoblot analysis. ( B ) WT, cells expressing HA-STING, or STING KO HT1080 cells. ( C ) WT, RIG-I KO, and STING KO HT1080 cells were transfected with the indicated STING ligands for 8 h or indicated times and cell lysates were probed with indicated antibodies on immunoblots; two exposures of STING (L, low and H, high) of the same blot are shown. The ratios of the indicated phospho-proteins/total proteins for the antibodies probed were quantified by Image J. ( D ) WT and STING KO HT1080 cells transfected with pdA:dT (5 μg/mL) or HT-DNA (4 μg/mL) and SG were immunostained with anti-G3BP1, and SG positive cells were quantitated. Percentage of cells showing G3BP1-positive SG puncta was determined from at least 100 cells from three replicates. Results are mean ± SD. ( E ) WT and G3BP1KO HT1080 cells were transfected with pdA:dT (5 μg/mL) or mock-transfected and after 8 h were immunostained for SG markers G3BP1 (red) and TIA1 (green) and nuclei (DAPI, blue), and SG positive cells were analyzed by confocal microscopy. The magnified images correspond to boxed regions. Expression of STING or G3BP1 in KO cells was compared to WT cells by immunoblot analysis. Scale bars, 20 µm. The results presented here are representative of at least three biological repeats. *** p < 0.001.

Article Snippet: Hanks’ balanced salt solution (HBSS) with calcium and magnesium, without phenol red (21-023-CV), was from Corning (Manassas, VA, USA). p-STING (50907), STING (13647), p-TBK1 (5482), TBK1 (3013), p-IRF3 (4947), IRF3 (4302), p-STAT1 (9167), STAT1 (9172), p-eIF2α (3398), eIF2α (5324), p-H2AX (9718), β-actin (3700), IFI16 (14970), DDX41 (15076), HA-Tag (3724), cleaved PARP (5625), cleaved Caspase 3 (9664), anti-mouse IgG, and anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Knockdown, Transfection, Phospho-proteomics, Control, Western Blot, Expressing, Confocal Microscopy

Journal: Cell reports

Article Title: Cell-autonomous inflammation of BRCA1 -deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING

doi: 10.1016/j.celrep.2021.109412

Figure Lengend Snippet: (A) (Upper) UWB1.289 cell lines: UWB1.289 BRCA1 mut with concomitant loss of wild-type (WT) allele through LOH and WT BRCA1 -reconstituted isogenic cell line. (Lower) Western blot (WB) analysis of BRCA1 in nuclear (N) extracts of BRCA1 mut and BRCA1 WT cells. (B) Heatmap of hallmark signatures with significantly different enrichment score between BRCA1 mut and BRCA1 WT isogenic cell lines (adjusted p value of <0.05 after linear regression) at the proteomics level. (C) Volcano plot of differentially expressed proteins in the BRCA1 mut and BRCA1 WT cell lines by MS. Immune-related proteins are highlighted in blue; selected genes of interest are highlighted in red. The position on the right side of the plot indicates higher expression in the BRCA1 mut cell line. Black curves represent significance cutoff (t test permutation-based false discovery rate [FDR] < 0.01, S0 = 0.3). (D) WB analysis for STING and IFI16 in cytoplasmic (CYT) and nuclear (NUC) extracts of BRCA1 mut and BRCA1 WT cells (n = 3). The signal obtained for each protein was normalized to that of housekeeping β-actin and lamin B in CYT and NUC, respectively. Data are presented as mean ± SEM. p values were calculated by an unpaired t test. (E) Pre-ranked gene set enrichment analysis using the difference in the number of enhancers between BRCA1 mut and BRCA1 WT cell lines as a ranking factor. Pathways enriched in BRCA1 WT are in red. All of the others were found in the BRCA1 mut . Right panels display the three most enriched pathways in BRCA1 mut . (F) Association of differentially expressed genes and the presence of enhancers in the BRCA1 mut versus the BRCA1 WT cells by RNA-seq, Hi-C, and H3K27ac ChIP-seq. Genes in red are those implicated in the DS/IFN pathway. (G) WB analysis for pSTAT1 and total STAT1 in the cytoplasmic (C) and nuclear (N) fractions of BRCA1 mut and BRCA1 WT cells treated with liposomes or poly(I:C)- or poly(dA:dT)-loaded liposomes. β-Actin and lamin B were used as protein loading controls in C and N, respectively. (H) RT-PCR analysis of CCL5 , CXCL9 , IFNA1 , TNFA , CXCL10 , and IFNB1 in BRCA1 mut and BRCA1 WT cells treated with liposomes or poly(I:C)- or poly(dA:dT)-loaded liposomes (n = 3). Data are presented as mean ± SEM. p values were calculated by an unpaired t test. (I)WB analysis of BRCA1, STING, IFI16, NF-κB, pIRF3, pSTAT1, and total STAT1 in the C and N fractions of OVCAR5 BRCA1 kd and BRCA1 WT cells treated with liposomes or poly(I:C)- or poly(dA:dT)-loaded liposomes. (J)Immunofluorescence (IF) analysis of pSTAT1 (green), γH2AX (red), and DAPI (blue) in BRCA1 kd and BRCA1 WT cells 48 h after irradiation (10 Gy). Scale bars, 25 mm (10 μm in insets). See also and and .

Article Snippet: anti-human IFI16, clone 1G7, 1:500 , Santa Cruz Biotechnology , Cat#sc-8023; RRID: AB_627775.

Techniques: Western Blot, Expressing, RNA Sequencing, Hi-C, ChIP-sequencing, Liposomes, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Irradiation

Journal: Cell reports

Article Title: Cell-autonomous inflammation of BRCA1 -deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING

doi: 10.1016/j.celrep.2021.109412

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-human IFI16, clone 1G7, 1:500 , Santa Cruz Biotechnology , Cat#sc-8023; RRID: AB_627775.

Techniques: Blocking Assay, Staining, Marker, Control, Recombinant, shRNA, Luciferase, MTT Cell Proliferation, cDNA Synthesis, Extraction, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Software

Journal: Cells

Article Title: IFI16 Induced by Direct Interaction between Esophageal Squamous Cell Carcinomas and Macrophages Promotes Tumor Progression via Secretion of IL-1α.

doi: 10.3390/cells12222603

Figure Lengend Snippet: Figure 1. Direct co-culture with macrophages upregulates the expression level of IFI16 and promotes NF-κB signaling in ESCC cell lines. (A) qRT-PCR results showing that upregulated mRNA levels of IFI16 were observed in co-cultured ESCC cells compared to mono-cultured ESCC cells. GAPDH was quantified as an internal control. (B) Upregulated protein levels of IFI16 and promoted phospho- rylation of NF-κB in co-cultured ESCC cells compared to mono-cultured ESCC cells, shown using Western blotting. The internal control for Western blotting was β-actin. The expression levels were quantified using ImageJ software, and the relative value was set as 1.00 for mono-cultured ESCC cells. Mono, mono-cultured; Co, co-cultured; UND, undetected. Data are presented as the mean ± SEM of triplicate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: ESCC cells were transfected with small interfering RNA (siRNA) targeting human IFI16 (siIFI16; 20 nM; sc-35633; Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Co-Culture Assay, Expressing, Quantitative RT-PCR, Cell Culture, Control, Western Blot, Software

Journal: Cells

Article Title: IFI16 Induced by Direct Interaction between Esophageal Squamous Cell Carcinomas and Macrophages Promotes Tumor Progression via Secretion of IL-1α.

doi: 10.3390/cells12222603

Figure Lengend Snippet: Figure 2. Knockdown of IFI16 suppresses malignant phenotypes via Erk and NF-κB signaling in ESCC cell lines. (A) qRT-PCR was applied to confirm the knockdown of IFI16 mRNA in ESCC cells. GAPDH was quantified as an internal control. (B) Western blotting was applied to confirm the knockdown of IFI16 at the protein level and to evaluate the effect of IFI16 silencing on the phosphorylation levels of Erk and NF-κB in ESCC cells. The internal control for Western blotting was β-actin. The expression levels were quantified using ImageJ software, and the relative value was set as 1.00 for siNC-transfected ESCC cells. (C–E) An MTS assay or transwell migration assay revealed suppressed survival (C), growth (D), and migration (E) following IFI16 knockdown in ESCC cells. Data are presented as the mean ± SEM of triplicate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. siNC, negative control of siRNA; siIFI16, siRNA against IFI16.

Article Snippet: ESCC cells were transfected with small interfering RNA (siRNA) targeting human IFI16 (siIFI16; 20 nM; sc-35633; Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Knockdown, Quantitative RT-PCR, Control, Western Blot, Phospho-proteomics, Expressing, Software, Transfection, MTS Assay, Transwell Migration Assay, Migration, Negative Control

Journal: Cells

Article Title: IFI16 Induced by Direct Interaction between Esophageal Squamous Cell Carcinomas and Macrophages Promotes Tumor Progression via Secretion of IL-1α.

doi: 10.3390/cells12222603

Figure Lengend Snippet: Figure 3. IFI16 regulates the secretion of IL-1α from ESCC cell lines, which plays an important role in their malignant phenotypes. (A) The cytokine array between the culture supernatant of TE-11 cells transfected with siNC and TE-11 cells transfected with siIFI16 revealed suppressed expression of IL- 1α by silencing IFI16. The positive and negative control spots were also shown. The expression levels were quantified using ImageJ software, and the relative value was set as 1.00 for siNC-transfected TE-11 cells. (B) qRT-PCR was applied to confirm the suppression of IL1A mRNA by IFI16 knockdown in ESCC cells. GAPDH was quantified as an internal control. (C) ELISA was applied to confirm the suppressed secretion of IL-1α by IFI16 knockdown in ESCC cells. (D–F) An MTS assay or transwell migration assay was performed between mono-cultured ESCC cells, co-cultured ESCC cells, co- cultured ESCC cells with a control goat IgG antibody, and co-cultured ESCC cells with an anti-IL-1α neutralizing antibody to evaluate survival (D), growth (E), and migration (F). These phenotypes of ESCC cells after co-culture with macrophages were abrogated by the use of anti-IL-1α neutralizing antibodies. Data are presented as the mean ± SEM of triplicate experiments. N/S, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. siNC, negative control of siRNA; siIFI16, siRNA against IFI16; UND, undetected; Control IgG, normal goat IgG control; anti-IL-1α, anti-IL-1α neutralizing antibody.

Article Snippet: ESCC cells were transfected with small interfering RNA (siRNA) targeting human IFI16 (siIFI16; 20 nM; sc-35633; Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Transfection, Expressing, Negative Control, Software, Quantitative RT-PCR, Knockdown, Control, Enzyme-linked Immunosorbent Assay, MTS Assay, Transwell Migration Assay, Cell Culture, Migration, Co-Culture Assay

Journal: Cells

Article Title: IFI16 Induced by Direct Interaction between Esophageal Squamous Cell Carcinomas and Macrophages Promotes Tumor Progression via Secretion of IL-1α.

doi: 10.3390/cells12222603

Figure Lengend Snippet: Figure 5. Patients with ESCC who exhibit a high expression of IFI16 tend to have a poor prognosis in terms of disease-free survival. (A) Immunohistochemical staining for IFI16 was performed in surgically resected ESCC tissues. Representative images for the invasive front of ESCC tissues are shown. Scale bar in ×40 images: 200 µm; Scale bar in ×400 images: 20 µm. (B) The survival curve for overall survival, cause-specific survival, and disease-free survival was plotted with the Kaplan–Meier method. The data were analyzed with the log-rank test.

Article Snippet: ESCC cells were transfected with small interfering RNA (siRNA) targeting human IFI16 (siIFI16; 20 nM; sc-35633; Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cells

Article Title: IFI16 Induced by Direct Interaction between Esophageal Squamous Cell Carcinomas and Macrophages Promotes Tumor Progression via Secretion of IL-1α.

doi: 10.3390/cells12222603

Figure Lengend Snippet: Figure 6. Schematic summary of our findings on the role of IFI16 in the ESCC microenvironment. IFI16 was upregulated in ESCC cells which directly interacted with macrophages. Upregulation of IFI16 led to promoted secretion of IL-1α. IL-1α enhanced malignant phenotypes of ESCC cells, especially migration via Erk and NF-κB signaling.

Article Snippet: ESCC cells were transfected with small interfering RNA (siRNA) targeting human IFI16 (siIFI16; 20 nM; sc-35633; Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Migration

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: IFI16 partners with KAP1 to silence the EBV lytic state. (A and B) HH514-16 BL cells were transfected with control scrambled siRNA (Sc) or siRNA targeting IFI16, exposed to NaB (sodium butyrate; lytic trigger) after 18 h, harvested after another 24 h, and immunoblotted with indicated antibodies (A) or subjected to RT-qPCR analysis of EBV lytic genes of all kinetic classes (BZLF1, immediate early; BMRF1, early; and BFRF3, late) using the ΔΔCT method after normalization to 18S rRNA (B). Error bars, SEM of three technical replicates and two biological replicates; **, P < 0.01. (C) Mutu I BL cells were transfected with control scrambled siRNA (Sc) or siRNA targeting IFI16, exposed to NaB (left) or TGF-β1 (middle) after 18 h, harvested after another 24 h, and immunoblotted with indicated antibodies. (D) HH514-16 BL cells were transfected with empty vector (EV) or IFI16 plasmid, exposed to NaB after 18 h, and harvested after another 24 h before performing immunoblotting with indicated antibodies. (E and F) HH514-16 BL cells were transfected with siRNA targeting IFI16 (versus scrambled RNA as control) (E) or IFI16 plasmid (versus empty vector [EV] as control) (F), exposed to NaB after 20 h, and harvested after another 36 h (F, left) or 48 h (E and F, right) for qPCR analysis of cell-associated viral genomes (left) or released viral genomes following DNase treatment (right). (G) HH514-16 BL cells were transfected with empty vector (EV), IFI16 plasmid, control scrambled siRNA, or siRNA targeting KAP1, exposed to NaB after 18 h, and harvested after another 24 h before immunoblotting with indicated antibodies. Numbers indicate the relative amounts of ZEBRA protein after normalization to β-actin levels. These experiments were performed at least twice.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Transfection, Control, Quantitative RT-PCR, Plasmid Preparation, Western Blot

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: KAP1 and IFI16 coenriched with H3K9me3 and at the BZLF1 promoter, preferentially in latent cells. (A) HH514-16 BL cells were left untreated or exposed to NaB. After 24 h, lysates were immunoprecipitated (IP) with anti-KAP1 antibody versus control IgG and immunoblotted with indicated antibodies. Input represents 5% of the sample. (B) HH514-16 BL cells were treated with or without NaB for 24 h followed by ChIP-re-ChIP. Chromatin was precipitated using rabbit anti-KAP1 or a control rabbit IgG antibody (top) and subjected to the second round of ChIP using mouse anti-IFI16 or a control mouse IgG antibody (bottom). Extracted DNA was analyzed by qPCR using primers spanning the known KAP1 enrichment site on the BZLF1 promoter and normalized to input. (C) HH514-16 BL cells were treated with or without NaB for 6 h followed by ChIP using an anti-H3K9me3 antibody or control IgG. (D) HH514-16 BL cells were transfected with control scrambled siRNA (Sc) or siRNA targeting IFI16 for 18 h (and harvested for immunoblotting in E) or exposed to NaB, harvested after 12 and 24 h, and chromatin was immunoprecipitated with an anti-H3K9me3 antibody or control IgG. Primer sets targeting BZLF1, BMRF1, and BFRF3 promoters were used for qPCR in C and D. Error bars, SEM of the means of three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Immunoprecipitation, Control, Transfection, Western Blot

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: IFI16 was adjacent to KAP1 but not SZF1 in latent cells. HH514-16 BL cells were not induced or induced with NaB for 24 h before performing a proximity ligation assay (PLA) using antibodies targeting KAP1, IFI16, and SZF1. EBV seropositive human reference serum followed by APC-conjugated goat anti-human IgG was used to stain lytic cells (A, B, and D). Negative-control staining in the absence of anti-KAP1, anti-IFI16, or both antibodies is shown in (C). Green foci, in situ interactions between KAP1 and IFI16. The experiment was performed at least 3 times. Three representative fields from two independent experiments are shown in A, B, and D.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Proximity Ligation Assay, Staining, Negative Control, In Situ

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: IFI16 message and protein abundance rapidly decrease with lytic cycle activation in EBV+ BL cells. (A and B) HH514-16 (EBV+ BL; A), CLIX-FZ (doxycycline-inducible EBV+ BL; B), and EBV− B lymphoma (BJAB; C) cells were exposed to lytic triggers (NaB in A and C; doxycycline in B) for different durations followed by immunoblotting with indicated antibodies. (D to F) HH514-16 cells were induced with NaB (D), TSA (E), or VPA as mock induction control (F) for different durations followed by RT-qPCR analysis to determine the relative amounts of IFI16 message after normalization to 18S rRNA using the ΔΔCT method. Error bars, SEM of three technical replicates and two biological replicates; *, P < 0.05; **, P < 0.01; NS, not significant.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Quantitative Proteomics, Activation Assay, Western Blot, Control, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: Reduced transcription contributes to a drop in abundance of IFI16 messages after lytic cycle activation. HH514-16 BL cells were left untreated or induced with NaB for 10 h, before performing BrU-PCR. Nascent RNA was precipitated with an anti-BrdU antibody followed by RT-qPCR analysis with two primer sets (number 1 and number 2) to determine the relative amounts of newly synthesized IFI16 message (A) and BZLF1 message (B) after normalization to 18S rRNA. Error bars, SEM of three technical replicates and two biological replicates; **, P < 0.01; ***, P < 0.001; NS, not significant.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Activation Assay, Quantitative RT-PCR, Synthesized

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: Model of IFI16’s partnership with the constitutive heterochromatin machinery (HCM) that blocked EBV latent-to-lytic transition. N-terminal KRAB domain of the KRAB-ZFP SZF1 interacted with the corepressor KAP1 while C-terminal zinc fingers bind viral DNA. KAP1 recruited histone-lysine methyltransferases (HMT), the deacetylase complex NuRD-HDAC, and the heterochromatin amplification factor HP1 to silence target genes via H3K9me3 marks. Shown also is the proposed involvement of the DNA sensor IFI16 which directly contacts KAP1 but not SZF1.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Zinc-Fingers, Histone Deacetylase Assay, Amplification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication.

doi: 10.1073/pnas.1311669110

Figure Lengend Snippet: Fig. 2. HIV-derived ssDNA induces IFN responses through a pathway dependent on IFI16, STING, TBK1, and IRF3/7. (A–D) BMDMs from mice with homo- zygous deletions of the genes encoding (A) Tmem173 (i.e., STING) (n = 3), (B) TBK1 (n = 2), (C) IRF3 (n = 2), or (D) IRF3/IRF7 (n = 2) and their respective controls were transfected with 2 μg/mL of ssDNA1 or dsDNA and were incubated for 6 h. RNA was isolated and analyzed for induction of IFN-β by RT-qPCR. (E) Western blot of IFI16, STING, and β-actin on extracts from THP1-derived cells stably transduced with lentiviral shRNAs [control (Ctrl), IFI16, and STING]. (F–I) RNA was isolated and analyzed for induction of IFN-β (F and H) and IFN-λ1 (G and I) by RT-qPCR. ssDNA1 or dsDNA (2 μg/mL) was transfected into THP1 control and STING shRNA-knockdown cells (n = 3) (F and G) or into THP1 control and IFI16 shRNA-knockdown cells (H and I) followed by 6 h of incubation (n = 4). (J) Culture supernatants from THP1 control and IFI16 shRNA-knockdown cells treated for 16 h were analyzed for CXCL10 secretion by ELISA (n = 3). (K) Culture supernatants from THP1 control, IFI16, and STING shRNA-knockdown cells were used to stimulate parental THP1 cells for 50 min. Cell lysates were subjected to Western blotting and probed with anti–P-STAT2 antibody. (L) Total IRF3 in nuclear extracts from THP1 control and IFI16 shRNA-knockdown cells treated for 2 h with 2 μg/mL of ssDNA1 or dsDNA. Levels of IRF3 in the nucleus were detected by Western blotting. Regulator of chromosome condensation 1 (RCC1) was used as loading control. A–D and F–K are representative datasets from one of multiple independent experiments performed in duplicate (mean ± SEM). *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test.

Article Snippet: For visualization of IFI16 after transfection with DNA at the indicated times, cells werefixed and permeabilizedwithmethanol at−20 °C for 5 min and were labeled with antibodies against IFI16 (63) or STING (IMG6485A; Imgenex) or with DAPI.

Techniques: Derivative Assay, Transfection, Incubation, Isolation, Quantitative RT-PCR, Western Blot, Stable Transfection, Transduction, Control, shRNA, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication.

doi: 10.1073/pnas.1311669110

Figure Lengend Snippet: Fig. 3. Immunostimulatory ssDNA colocalizes with IFI16 and interacts with the IFI16 HINb domain. (A) Confocal microscopy images of hMDMs transfected for 1 h with FITC-labeled ssDNA1 and ssDNA2 after protein staining for IFI16. Insets illustrate closeup of foci formations. (Scale bar, 10 um.) (B) Quantification of DNA- IFI16 colocalization in hMDMs from two different donors transfected for 1 h with ssDNA1 or ssDNA2. (C) Fluorescence polarization assays for FAM-labeled ssDNA1 upon binding to the IFI16 HINb domain. The binding isotherms between the IFI16 HINb domain and the FAM-labeled DNAs are graphed as shown. The apparent Kd values derived from the fluorescence polarization assays are dsDNA, 1.299 ± 0.404 μM; ssDNA1, 1.198 ± 0.122 μM; ssDNA1 closed, 0.605 ± 0.071 μM; and ssDNA1 open, 9.878 ± 2.225 μM. (D) THP1 cells were transfected with 2 μg/mL of ssDNA1 for 2 h. Cytoplasmic lysates were subjected to immunoprecipitation using beads coupled to the indicated antibodies. (Upper) Coprecipitated ssDNA1 was amplified by qPCR and quantified by comparison with a standard curve using ssDNA1. (Lower) Western blotting was used to quantify the amount of endogenous IFI16 bound to the beads. ***P < 0.001; Student’s t test.

Article Snippet: For visualization of IFI16 after transfection with DNA at the indicated times, cells werefixed and permeabilizedwithmethanol at−20 °C for 5 min and were labeled with antibodies against IFI16 (63) or STING (IMG6485A; Imgenex) or with DAPI.

Techniques: Confocal Microscopy, Transfection, Labeling, Staining, Fluorescence, Binding Assay, Derivative Assay, Immunoprecipitation, Comparison, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication.

doi: 10.1073/pnas.1311669110

Figure Lengend Snippet: Fig. 4. Trex1 counteracts IFI16-dependent induction of IFN expression by stem- rich DNA. (A) Western blot for IFI16, Trex1, and β-actin in THP1 control, Trex1, and Trex1/IFI16 shRNA-knockdown cells. (B and C) THP1 Trex1-knockdown cells were transfected with ssDNA1, ssDNA2, ssDNA3, dsDNA, or Poly(dA:dT) (2 μg/mL). RNA was isolated 6 h after treatment and analyzed for IFN-β (B) and IFN-λ (C) by RT-qPCR (n = 2). (D) Control shRNA, Trex1 shRNA, and Trex1/IFI16 shRNA knockdown THP1 cells were stimulated for 6 h with 2 μg/mL of ssDNA1 or dsDNA. RNA was isolated and analyzed for IFN-β by RT-qPCR (n = 3). Data are shown as one dataset representative of multiple independent experiments performed in duplicate (mean ± SEM). *P < 0.05; **P < 0.01; Student’s t test.

Article Snippet: For visualization of IFI16 after transfection with DNA at the indicated times, cells werefixed and permeabilizedwithmethanol at−20 °C for 5 min and were labeled with antibodies against IFI16 (63) or STING (IMG6485A; Imgenex) or with DAPI.

Techniques: Expressing, Western Blot, Control, shRNA, Knockdown, Transfection, Isolation, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication.

doi: 10.1073/pnas.1311669110

Figure Lengend Snippet: Fig. 5. IFI16 has an important role in controlling HIV-1 and lentiviral vec- tors. (A) THP1 control and IFI16- knockdown cells were infected with HIV-1 Bal. HIV-1 RT DNA products were measured by PCR on HIV-1 Gag DNA 12 h postinfection. (B) Cellular RNA was isolated 12 or 24 h after infection and IFIT1 levels were ana- lyzed by RT-qPCR. (C and D) Cells were infected with HIV-1 Bal and culture supernatants were harvested at the indicated time points and an- alyzed for levels of CXCL10 (C) and HIV-1 p24 (D). (E) THP1 control, IFI16, STING, cGAS, and DDX41 knockdown cells were infected with HIV-1 Bal for 96 h, and p24 levels in the su- pernatant were measured by ELISA. (F–K) THP1 control, IFI16, DDX41, and cGAS knockdown cells were infected with VSV-G pseudotype eGFP lentivi- ruses expressing either proficient or deficient viral integrase. (F and G) eGPF-positive cells were evaluated by flow cytometry 96 h after infection. (H–K) CXCL10 levels in the superna- tant were measured on samples har- vested 24 h after infection. (L) THP1 cells were treated with AZT (5 μM), EFV (5 μM), or MVC (5 μM) and infected with IPLVs for 24 h. Levels of CXCL10 in the supernatants were measured by ELISA. Results in A–L are shown as one representative dataset from two to three independent ex- periments performed in triplicate (mean ± SEM). (M) hMDMs were infected with lentivirus pseudo- particles for 2 or 4 h, and viral DNA was visualized by specific FISH probes [viral DNA (vDNA); green] and was costained with anti-IFI16–specific antibodies (IFI16; red). (Scale bar, 5 μm.) The mock sample was fixed after 4 h of incubation. Data from two other donors are shown in Fig. S7. DIC, differential interference contrast. (N) THP1 cells were treated with entry- proficient (+VSV-G) or entry-deficient (−VSV-G) IPLVs for 4 h. Cytoplasmic lysates were subjected to immunopre- cipitation using anti-IFI16–coupled beads. Coprecipitated viral DNA was amplified by PCRs targeting WPRE and cGFP in the IPLV genome and was quantified by comparison with a standard curve. *P < 0.05; **P < 0.01; Student’s t test.

Article Snippet: For visualization of IFI16 after transfection with DNA at the indicated times, cells werefixed and permeabilizedwithmethanol at−20 °C for 5 min and were labeled with antibodies against IFI16 (63) or STING (IMG6485A; Imgenex) or with DAPI.

Techniques: Control, Knockdown, Infection, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Cytometry, Incubation, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication.

doi: 10.1073/pnas.1311669110

Figure Lengend Snippet: Fig. 6. IFI16 is essential for control of HIV-1 infection in primary human macro- phages. (A) Levels of HIV-1 Gag DNA in hMDMs infected with HIV-1 Bal for 12 h were measured by qPCR (n = 2). (B) Sum- mary of experimental setup in hMDMs. Primary monocytes were differentiated into macrophages and treated with siRNA (165 nM) before HIV-1 Bal infection (MOI of 1 calculated on Tzmbl titration and X- Gal straining). (C) Levels of IFI16 knock- down by a scramble and specific siRNA were measured by RT-qPCR on RNA harvested on the day of viral infection. Results represent four independent siRNA transfections made in cells from both donors (mean ± SEM). (D and E) hMDMs from donors A (D) and B (E) treated with control or IFI16 siRNA were infected with HIV-1 Bal (MOI of 1.0). Cultures were har- vested at the indicated time points, and p24 levels were measured by ELISA (n = 4; two-way ANOVA with Bonferroni multiple comparison). (F) Representative images of hMDMs from donor A at day 4 after viral infection. Arrows indicate cells exhibiting apoptotic morphology.

Article Snippet: For visualization of IFI16 after transfection with DNA at the indicated times, cells werefixed and permeabilizedwithmethanol at−20 °C for 5 min and were labeled with antibodies against IFI16 (63) or STING (IMG6485A; Imgenex) or with DAPI.

Techniques: Control, Infection, Titration, Knockdown, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cellular and Molecular Life Sciences

Article Title: Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E 2 -dependent growth of ER-positive breast cancer

doi: 10.1007/s00018-022-04333-y

Figure Lengend Snippet: IFI16 mediates the type I IFN-induced upregulation of aromatase in SGBS cells. a SGBS cells were transfected with the indicated siRNAs. Twenty-four hours after transfection, the cells were treated with 10 ng/ml IFNβ for 48 h. Expression level of aromatase was measured by qPCR. * P < 0.05 vs siControl with vehicle, # P < 0.05 as indicated ( n = 3). The mRNA levels of ISGs are shown as control in Fig. S3. b SGBS cells were transfected with control siRNA or IFI16 siRNA. Twenty-four hours after transfection, the cells were treated with 10 ng/ml IFNβ for 48 h. Expression levels of protein were analyzed by western blotting (top). Band intensities of aromatase and IFI16 in blots were quantified using ImageJ and normalized to that of Actin band (bottom). c – e SGBS cells were transfected with empty vector (0) or the indicated amount of Myc-IFI16 for 48 h. Expression level of aromatase was analyzed by western blotting ( c , top). Band intensities of aromatase and IFI16 in blots were quantified using ImageJ and normalized to that of Actin band ( c , bottom). The concentration of estradiol in the culture medium was measured by ELISA ( d ). Expression levels of the PI.4, PI.3, or PII-specific aromatase transcripts were measured by qPCR ( e ). * P < 0.05 vs empty vector (0) ( n = 3)

Article Snippet: To generate IFI16 KO cell lines, SGBS cells were transfected with 1 μg of IFI16 CRISPR/Cas9 KO plasmid (sc-416568, Santa Cruz Biotechnology) or a control CRISPR/Cas9 plasmid (sc-418922) using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s protocol.

Techniques: Transfection, Expressing, Control, Western Blot, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E 2 -dependent growth of ER-positive breast cancer

doi: 10.1007/s00018-022-04333-y

Figure Lengend Snippet: IFI16 activates aromatase PI.3/PII promoter through DNA binding together with HIF1α and PRMT2. a Schematic representation of aromatase PI.3 and PII promoters and primers used for ChIP experiments. b SGBS cells were treated with 10 ng/ml IFNβ (left) or transfected with Myc-IFI16 (right) for 48 h. Cells were fixed and subjected to ChIP assay using anti-IFI16 or anti-Myc antibodies. Immunoprecipitated DNA fragments were amplified by PCR analysis using the indicated primers. c SGBS cells were transfected with Myc-HIF1α and treated with 10 ng/ml IFNβ for 48 h. Cells were fixed and subjected to ChIP assay using anti-Myc antibody, followed by qPCR analysis with the indicated primers. * P < 0.05 vs vehicle ( n = 3). d SGBS cells were transfected with HA-PRMT2 and treated with 10 ng/ml IFNβ for 48 h. Cells were fixed and subjected to ChIP assay using anti-HA or anti-H3R8Me2a antibodies, followed by qPCR analysis with the indicated primers. * P < 0.05 vs vehicle ( n = 3). e SGBS cells were transfected with Myc-HIF1α. Whole cell lysates were immunoprecipitated (IP) using IgG or anti-IFI16 antibodies and probed with the indicated antibodies by western blotting (WB). f SGBS cells were treated with 10 ng/ml IFNβ for 48 h. Interactions of IFI16-HIF1α and IFI16-PRMT2 were visualized with red dots by in situ proximity ligation assay. As a negative control, a single staining with the anti-IFI16, anti-HIF1α, or anti-PRMT2 antibodies was performed. DAPI was used to stain the nuclei (left). The fluorescence (Fl) intensity per cell was quantified from at least 100 cells using ImageJ software (right). Data are presented as the mean ± SEM. * P < 0.05 ( n = 3). Bar represents 10 μm. g SGBS cells were transfected with the indicated siRNAs (left) or siRNA together with HA-PRMT2 (right), and treated with 10 ng/ml IFNβ for 48 h. Cells were fixed and subjected to ChIP assay using anti-IFI16 or anti-HA antibodies, followed by qPCR analysis with the indicated primers. * P < 0.05 vs vehicle, # P < 0.05 vs siControl ( n = 3). h SGBS cells were transfected with indicated siRNAs and treated with 10 ng/ml IFNβ for 48 h. Expression level of aromatase was measured by qPCR. * P < 0.05 vs vehicle, # P < 0.05 vs siControl treated with IFNβ ( n = 3). The mRNA levels of IFI16, PRMT2, and HIF1α are shown as control in Fig. S4. i Schematic illustration for binding of HIF1α-IFI16-PRMT2 complex to the HRE on aromatase PI.3/PII promoter that activates aromatase transcription in preadipocytes

Article Snippet: To generate IFI16 KO cell lines, SGBS cells were transfected with 1 μg of IFI16 CRISPR/Cas9 KO plasmid (sc-416568, Santa Cruz Biotechnology) or a control CRISPR/Cas9 plasmid (sc-418922) using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s protocol.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Amplification, Western Blot, In Situ, Proximity Ligation Assay, Negative Control, Staining, Fluorescence, Software, Expressing, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E 2 -dependent growth of ER-positive breast cancer

doi: 10.1007/s00018-022-04333-y

Figure Lengend Snippet: E 2 -dependent growth of MCF7 is decreased in conditioned media obtained from the IFI16 KO SGBS preadipocytes. a Expression level of IFI16 in SGBS control or IFI16 KO cells was analyzed by western blotting (top). SGBS control or IFI16 KO cells were treated with 10 ng/ml IFNs for 48 h. Expression levels of mRNA and protein of aromatase were analyzed by qPCR (middle) and western blotting (bottom), respectively. * P < 0.05 vs vehicle, # P < 0.05 vs control treated with IFN ( n = 4). b SGBS control or IFI16 KO cells were treated with 10 ng/ml IFNβ for 48 h. The concentration of E 2 in the culture supernatants was measured by ELISA. * P < 0.05 vs vehicle, # P < 0.05 vs control treated with IFNβ ( n = 3). c, d THP1 and MCF7 cells were cocultured as described in Fig. S2a. SGBS control or IFI16 KO cells were incubated with conditioned medium (CM) obtained from the THP1 and MCF7 coculture for 48 h. Expression levels of mRNA and protein of aromatase were analyzed by qPCR (top) and western blotting (bottom), respectively ( c ). The concentrations of E 2 in the culture medium were measured by ELISA ( d ). * P < 0.05 vs DMEM/F12, # P < 0.05 vs control with coculture CM ( n = 3). e THP1 cells pre-treated with 100 ng/ml PMA for 48 h, and SGBS cells were seeded in the bottom chamber of the transwell, as indicated in the scheme. MCF7 cells were seeded in the top chamber of the transwell insert. The number of viable MCF7 cells was counted using a hemocytometer. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni posttest. *** P < 0.001 vs MCF7 cocultured with SGBS control with THP1 cells (filled square) ( n = 4) (left). Total RNAs were obtained from the MCF7 cells at the day 5 and subjected to qPCR analysis. * P < 0.05 vs MCF7 monoculture, # P < 0.05 vs MCF7 cocultured with SGBS control with THP1 cells (filled square) ( n = 3) (right)

Article Snippet: To generate IFI16 KO cell lines, SGBS cells were transfected with 1 μg of IFI16 CRISPR/Cas9 KO plasmid (sc-416568, Santa Cruz Biotechnology) or a control CRISPR/Cas9 plasmid (sc-418922) using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s protocol.

Techniques: Expressing, Control, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Cellular and Molecular Life Sciences

Article Title: Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E 2 -dependent growth of ER-positive breast cancer

doi: 10.1007/s00018-022-04333-y

Figure Lengend Snippet: Adipocyte-specific knockdown of Ifi204 attenuates the type I IFN-induced upregulation of aromatase and E 2 production. a Schematic presentation of the domain structure of human IFI16 and mouse Ifi204 proteins. Both proteins comprise a pyrin domain (PYD) involved in protein–protein interaction, and two hematopoietic interferon-inducible nuclear (Hin) domains, HinA and HinB, which are involved in DNA binding (left top). Primary preadipocytes isolated from the inguinal mammary gland of Ifi204-AKO and Ifi204 f/f mice were treated with vehicle (−), or 1 or 10 ng/ml IFNβ for 48 h. Expression levels of aromatase and Ifi204 were measured by western blotting (left bottom) and qPCR (right). The concentration of E 2 in the culture supernatants was measured by ELISA (right). * P < 0.05 vs vehicle, # P < 0.05 vs Ifi204 f/f ( n = 3). b Schematic representation of the E0771 and BMDM coculture experiments (left top). BMDMs were seeded in the bottom chamber of a transwell and E0771 cells were seeded in the top chamber and allowed to incubate for additional 48 h. Primary preadipocytes were incubated with DMEM (M) or conditioned medium (CM) obtained from the E0771 and BMDM coculture for 48 h. Expression levels of aromatase and Ifi204 were measured by western blotting (left bottom) and qPCR (right). The concentration of E 2 in the culture supernatants was measured by ELISA (right). * P < 0.05 vs DMEM, # P < 0.05 vs Ifi204 f/f ( n = 3). c Mouse BMDMs and primary preadipocytes were seeded in the bottom chamber of the transwell, as indicated in the scheme. E0771 cells were seeded in the top chamber of the transwell insert. The number of viable E0771 cells was counted using a hemocytometer. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni posttest. *** P < 0.001 vs E0771 monoculture, ### P < 0.001 vs E0771 cocultured with Ifi204 f/f preadipocytes with BMDMs (filled square) ( n = 4) (left). Total RNAs were obtained from the E0771 cells at the day 5 and subjected to qPCR analysis. * P < 0.05 vs E0771 monoculture, # P < 0.05 vs E0771 cocultured with Ifi204 f/f preadipocytes with BMDMs (filled square) ( n = 3) (right)

Article Snippet: To generate IFI16 KO cell lines, SGBS cells were transfected with 1 μg of IFI16 CRISPR/Cas9 KO plasmid (sc-416568, Santa Cruz Biotechnology) or a control CRISPR/Cas9 plasmid (sc-418922) using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s protocol.

Techniques: Knockdown, Binding Assay, Isolation, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Cellular and Molecular Life Sciences

Article Title: Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E 2 -dependent growth of ER-positive breast cancer

doi: 10.1007/s00018-022-04333-y

Figure Lengend Snippet: IFI16, HIF1α, and PRMT2 are physically associated in cancer-associated adipocytes of mouse allograft tumors and human BC specimens. a Allograft tumor tissue sections from HFD-fed mice were subjected to proximity ligation assay (PLA) using anti-Ifi204 and HIF1α antibodies (left), and anti-Ifi204 and PRMT2 antibodies (right), respectively. Arrows indicate PLA signals detected in the nucleus of adipocytes adjacent to mammary tumor cells. Quantification of the PLA signals are shown in Fig. S8. Bar represents 50 μm. b Luminal-type BC tissue sections were subjected to PLA using anti-IFI16 and HIF1α antibodies (top), and anti-IFI16 and PRMT2 antibodies (bottom), respectively. Cancer-associated adipocytes (CAAs) and BC cells were analyzed from the same specimens. Arrows indicate PLA signals detected in the nucleus of CAA. The number of cells with PLA-positive signal was counted from ten BC specimens (right). Data are presented as the mean ± SD. *** P < 0.001. Bar represents 50 μm. c Schematic model for the role of type I IFNs in aromatase activation and E 2 -dependent growth of ER-positive BC cells. The level of type I IFNs are elevated through the interaction between immune cells and BC cells, which induces the expression of IFI16 in preadipocytes. IFI16 then binds to the HRE site on the aromatase PI.3/PII promoter together with HIF1α and PRMT2, leading to the activation of aromatase transcription. Abundant estradiol in tumor microenvironment promotes growth of ER-positive BC cells

Article Snippet: To generate IFI16 KO cell lines, SGBS cells were transfected with 1 μg of IFI16 CRISPR/Cas9 KO plasmid (sc-416568, Santa Cruz Biotechnology) or a control CRISPR/Cas9 plasmid (sc-418922) using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s protocol.

Techniques: Proximity Ligation Assay, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: IFI16 Protein Mediates the Anti-inflammatory Actions of the Type-I Interferons through Suppression of Activation of Caspase-1 by Inflammasomes

doi: 10.1371/journal.pone.0027040

Figure Lengend Snippet: ( A ) Puromycin-resistant THP-1 cells either infected with control lentivirus (lanes 1 an2) or the virus expressing the shIFI16 RNA (lanes 3–8; cell populations from three different wells) were either left untreated (lanes 1, 3, 5, 7) or treated with IFN-α for 14 h. After treatment, total cell extracts containing increased amounts of proteins (∼100 µg/lane) were analyzed for the constitutive and induced levels of IFI16 and actin proteins. A long exposure was taken to detect the signal in all lanes. ( B ) Control THP-1 cells (lanes 1 and 2) or cell population from well # 9 (lanes 3 and 4) as shown in the panel (a) were either left untreated (lanes 1 and 3) or treated with IFN-α for 14 h (lanes 2 and 4). After the treatment, total RNA was analyzed for the steady-state levels of mRNA for the indicated genes. ( C ) Control THP-1 cells (lanes 1–4) or cells infected with virus expressing the shIFI16 mRNA (lanes 5–8; population # 9) were either left untreated (lanes 1 and 5) or treated with IFN-β (1,000 u/ml; lanes 2 and 6), LPS (100 ng/ml; lanes 3 and 7), or dsRNA (10 µg/ml; lanes 4 and 8) for 14 h. After the treatment, total cell lysates containing equal amounts of protein (∼100 µg/lane) were analyzed by immunoblotting using specific antibodies to the indicated proteins. FC, indicates the fold change in the levels of the activated caspase-1 (the p20 band) with respect to the control (lane 1). ( D ) Control THP-1 cells or cells infected with virus expressing the shIFI16 mRNA (population # 9) were either left untreated (white columns) or treated with IFN-β (1,000 u/ml; columns 2–5 and 7–10) for 14 h. After the treatment, total RNA levels were analyzed for the indicated genes by the quantitative TaqMan real-time PCR. The ratio of the test gene to actin mRNA was calculated in units (one unit being the ratio of the test gene to actin mRNA). Results are mean values of triplicate experiments and error bars represent standard deviation.

Article Snippet: THP-1 cells were either infected with control lentiviral particles (sc-108080; from Santa Cruz Biotech, Santa Cruz,, CA) or the lentiviral particles expressing shIFI16 RNA (sc-35633-V, Santa Cruz Biotech) in a six well plate as suggested by the supplier.

Techniques: Infection, Control, Virus, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: IFI16 was overexpressed in human PAAD and correlated with poor survival of the patients. (A) Expression data of inflammasome-related proteins were retrieved from the human database GEPIA. A comparison of expression between normal pancreatic tissues and PAAD tissues was done. Genes with a significantly different expression between normal and tumor tissues are shown in red. (B) The data on overall survival and disease-free survival of PAAD patients were retrieved. Only NLRP1, among the genes with significantly different expression, showed a negative correlation with patients’ overall survival. Overall survival (C) but not disease-free survival (D) was adversely correlated with the expression of IFI16 in PAAD. We further extracted expression data of IFI16 from the human GEO database, including GDS4336 (E) and GDS4103 (F) . IFI16 was significantly overexpressed in the tumor tissues of PAAD compared with that in the non-tumor adjacent normal pancreas. (G) Pearson correlation between the expression of IFI16 and IL-1β was analyzed, which showed a positive correlation in human PAAD. * p < 0.05.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Expressing, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: Overexpression of IFI16 promoted tumor growth in an experimental model of PAAD. (A) The IFI16-overexpressing PAAD cell line was established by transfecting the CRISPR activation plasmid of IFI16 into the Panc-2 cells. Cells were selected with a culture medium containing 1 μg/ml of puromycin, until a stable IFI16-overexpressing clone was established. Overexpression of IFI16 was validated with immunoblotting. (B) Panc-2 of a stable clone expressing the luciferase reporter was established. Panc-2 cells were transfected with the PGL3 vector expressing firefly luciferase and selected by neomycin (50 μg/ml). Approximately 20 μg Matrigel matrix, containing 1 × 10 8 Panc-2 cells, was orthotopically injected into the pancreas of mice. The luciferase signal intensity was measured by intraperitoneal injection of luciferin (30 mg/kg) and quantification under a live animal imager once per week. Overexpression of IFI16 significantly accelerated the orthotopic growth of the pancreatic tumor. (C) At the end of the study, the mice were sacrificed, and the pancreas along with the spleen was dissected out. The tumor weight was measured, and tumor size was calibrated by the diameters of the tumor. Overexpression of IFI16 potently increased the tumor size and weight in the experimental PAAD model. The black arrow shows an obvious surface tumor nodule found in the pancreas. (D) The dissected tumor was then digested in 0.8 mg/ml of collagenase IV for 30 min at 37°C with gentle shaking. The immune cells were enriched with Ficoll methods. The profile of immune cells in the tumor microenvironment was measured with flow cytometry. Overexpression of IFI16 significantly increased the population of TAMs but not dendritic cells, T helper cells, or cytotoxic T cells. (E) The F4/80 + CD11b + TAMs were collected using a cell sorter, and expressions of HIF-1α, CCL2, and PECAM1 were measured with qRT-PCR. TAMs from IFI16-overexpressing tumors exhibited a significantly high expression of TAM markers, including HIF-1α, CCL2, and PECAM1. * p < 0.05.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Over Expression, CRISPR, Activation Assay, Plasmid Preparation, Western Blot, Stable Transfection, Expressing, Luciferase, Transfection, Injection, Gentle, Flow Cytometry, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: Depletion of TAMs attenuated IFI16-induced tumor growth of PAAD. (A) Flowchart of TAM depletion treatment. To minimize early recruitment of TAMs, mice received a single injection of liposome PBS (as sham control) or liposome clodronate, 3 days before orthotopic implantation of tumor cells, and received subsequent injection twice per week after implantation at a dose of 15 mg/kg. (B) The tumor was dissected out, and TAMs were enriched with Ficoll methods. The enriched cells were stained with an antibody against F4/80 and subjected to flow cytometric analysis. Treatment with liposome clodronate could potently remove TAMs from mice with orthotopic PAAD tumors. (C) Panc-2 of a stable clone expressing the luciferase reporter was established. Panc-2 cells were transfected with the PGL3 vector expressing firefly luciferase and selected by neomycin (50 μg/ml). Approximately 20 μl Matrigel matrix, containing 1 × 10 8 Panc-2 cells, was orthotopically injected into the pancreas of mice. The luciferase signal intensity was measured by intraperitoneal injection of luciferin (30 mg/kg) and quantification under a live animal imager once per week. Depletion of TAMs attenuated the tumor growth of PAAD induced by IFI16 overexpression. (D) At the end of the study, the mice were sacrificed, and the pancreas along with the spleen was dissected out. The tumor weight was measured, and tumor size was calibrated by the diameters of the tumor. Depletion of TAMs abolished the IFI16 overexpression-induced increase in tumor size and weight in the experimental PAAD model. The black arrow shows an obvious surface tumor nodule found on the pancreas. * p < 0.05 and *** p < 0.001.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Injection, Control, Staining, Stable Transfection, Expressing, Luciferase, Transfection, Plasmid Preparation, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: Conditional medium from IFI16-overexpressing PAAD cells increased the TAM population. (A) The culture medium of wild-type and IFI16-overexpressing PAAD cells was collected. BMDMs were cultured with 30% of the aforementioned conditional medium for 7 days. Cells were then collected and stained with antibodies against CD11b and F4/80 and subjected to flow cytometric analysis. BMDMs cultured with IFI16-overexpressing PAAD cells showed a higher level of CD11b + F4/80 + cells. Total RNA was extracted, and the expressions of HIF-1α, CCL2, and PECAM1 were analyzed with qRT-PCR. BMDMs cultured with conditional medium from IFI16-overexpressing PAAD cells showed significantly higher expressions of HIF-1α, CCL2, and PECAM1. (B) The Panc-2 cells with or without IFI16 overexpression were cultured on the apical side of the 0.8-μM-pore size Transwell, while BMDMs were seeded on the receiving chamber with the non-attached surface, supplemented with 10 ng/ml of M-CSF. This co-culture was maintained for 7 days to allow all possible non-contact interaction between the Panc-2 cells and BMDMs. BMDMs were then collected for analysis of the TAM population with flow cytometry and qPCR. Co-culture of Panc-2 cells with IFI16 overexpression significantly increased the population of TAMs from the BMDM culture compared to the co-culture of vector-expressing Panc-2 cells. (C) Added to the culture medium was 10 μM of BrdU 4 h prior to sample collection by trypsinization. BrdU-incorporated CD11b + F4/80 + cells were stained with anti-BrdU antibody and detected with a flow cytometer. BMDMs cultured with the conditional medium from IFI16-overexpressing PAAD cells showed significantly higher incorporation of BrdU into the DNA. (D) Approximately 2 × 10 5 BMDMs cultured with the conditional medium from Panc-2 cells were seeded at the apical side of the Transwell insert with serum-free medium. A culture medium supplemented with chemotaxis MCP-1 (10 ng/ml) was added into the receiving chambers and cultured for 3 h. Cells remaining at the apical side of inserts were scraped away, and cells at the basal side of the membrane were collected by trypsinization. The cells were then collected and stained with 50 μM calcein AM and quantified with a fluorescence microplate reader. BMDMs cultured with the conditional medium from IFI16-overexpressing PAAD cells showed significantly higher motility. (E) Intraperitoneally injected into the mice was 10 mg/kg of BrdU 24 h prior to sample collection by enriching TAMs from the dissected tumor by Ficoll methods. BrdU-incorporated CD11b + F4/80 + cells were stained with an anti-BrdU antibody and detected with a flow cytometer. TAMs from tumors with IFI16 overexpression showed significantly high incorporation into the DNA. (F) BMDMs were isolated from the femurs and cultured into macrophages in a medium containing 10 μg/ml of M-CSF for 7 days. Cells were then stained with 100 μM PKH26PCL for labeling. Labeled cells were then intraperitoneally injected into the mice bearing PAAD tumors with or without IFI16 overexpression and allowed circulation for 24 h. The tumor was then dissected out, and the number of PKH26PCL-labeled cells infiltrated into the tumor was measured with a flow cytometer. IFI16-overexpressing tumors showed more cell infiltration into the tumor microenvironment. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Cell Culture, Staining, Quantitative RT-PCR, Over Expression, Pore Size, Co-Culture Assay, Flow Cytometry, Plasmid Preparation, Expressing, Chemotaxis Assay, Membrane, Fluorescence, Injection, Isolation, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: IL-1β is responsible for the IFI16-induced TAM profile changes in PAAD. (A) Protein expression of Panc-2 cells at intracellular and extracellular levels was measured with immunoblotting. IFI16 overexpression induced further activation of the inflammasome, as evidenced by the cleavage of intracellular pro-caspase-1 and pro-IL-1β, as well as the extracellular expression of cleaved IL-1β and caspase-1 in the culture medium. (B) IL-1β levels were quantified with ELISA. IFI16 overexpression significantly increased IL-1β production and secretion in Panc-2 cells in the presence or absence of poly dA:dT. Supplementation with an IL-1β-neutralizing antibody mitigated the function of secreted IL-1β in the conditional medium from Panc-2 cells. This method was adopted from a published protocol, with minor modifications. For the blocking experiments, the BMDMs were preincubated with 10 μg/ml of the IL-1β-neutralizing antibody, for 15 min before the functional studies. (C) Cells were then collected and stained with antibodies against CD11b and F4/80 and subjected to flow cytometry analysis. Neutralization of IL-1β significantly blocked the increase in TAM population cultured in the conditioned medium from IFI16-overexpressing Panc-2 cells. (D) Added to the culture medium was 10 μM of BrdU 4 h prior to sample collection by trypsinization. BrdU-incorporated CD11b + F4/80 + cells were stained with anti-BrdU antibody and detected using a flow cytometer. Neutralization of IL-1β significantly blocked the increase in BrdU incorporation into the DNA of BMDMs cultured in conditioned medium from IFI16-overexpressing Panc-2 cells. (E) Approximately 2 × 10 5 BMDMs cultured with the conditioned medium from Panc-2 cells were seeded at the apical side of Transwell inserts with serum-free medium. Culture medium supplemented with chemotaxis MCP-1 (10 ng/ml) was added to the receiving chambers, followed by culture for 3 h. Cells remaining on the apical side of the inserts were scraped away, and cells at the basal side of the membrane were collected by trypsinization. The cells were then collected and stained with 50 μM calcein AM and quantified using a fluorescence microplate reader. Neutralization of IL-1β significantly blocked the increased motility of the BMDMs cultured in the conditioned medium from IFI16-overexpressing Panc-2 cells. * p < 0.05.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Expressing, Western Blot, Over Expression, Activation Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Functional Assay, Staining, Flow Cytometry, Neutralization, Cell Culture, BrdU Incorporation Assay, Chemotaxis Assay, Membrane, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment

doi: 10.3389/fcell.2021.640786

Figure Lengend Snippet: Suppression of IFI16 improves gemcitabine sensitivity in PAAD tumors. (A) Flowchart of gemcitabine treatment. Approximately 20 μl Matrigel matrix, containing 1 × 10 8 Panc-2 cells, was orthotopically injected into the pancreas of mice. Mice were then orally administered gemcitabine at a dose of 100 mg/kg or vehicle every day. (B) The IFI16-knockdown PAAD cell line was established by transfecting the shRNA plasmid of IFI16 into the Panc-2 cells. Cells were selected with a culture medium containing 1 μg/ml of puromycin, until a stable IFI16-knockdown clone was established. Knockdown of IFI16 was validated with immunoblotting, which showed that RNA interference using shRNA against IFI16 can significantly attenuate gemcitabine-induced IFI16 upregulation. (C) The luciferase signal intensity was measured by intraperitoneal injection of luciferin (30 mg/kg) and quantification under a live animal imager once per week. Knockdown of IFI16 could significantly improve the suppression of tumor growth by gemcitabine. (D) At the end of the study, the mice were sacrificed, and the pancreas along with the spleen was dissected out. The tumor weight was measured, and tumor size was calibrated by the diameters of the tumor. Knockdown of IFI16 in gemcitabine-treated mice further reduced the size and weight of the tumor in the experimental PAAD model. The black arrow shows an obvious surface tumor nodule found on the pancreas. (E) The tumor was dissected out, and TAMs were enriched by Ficoll methods. The enriched cells were stained with an antibody against F4/80 and subjected to flow cytometry analysis. Knockdown of IFI16 could attenuate the infiltration of TAMs induced by gemcitabine in the tumor microenvironment of PAAD. (F) The major M1/M2/TAM markers were quantified using qPCR. The ratio of expression of IFN-γ and TGF-β was calculated to represent the M1/M2 ratio in the tumor microenvironment. Gemcitabine had no significant effect on the polarization of macrophages toward either phenotype but potently activated the expression of TAM markers HIF-1α, CCL2, and PECAM1. (G) Expression of IFI16 was measured by immunoblotting in different PAAD cell lines. Expression of IFI16 was highest in BxPC3 cells, then SW1990 cells, and then Panc-2 cells, while Panc-1 cells expressed the lowest level of IFI16. (H) Expression of IFI16 was forcefully activated and knocked down in SW1990 cells. (I) BMDMs were co-cultured with SW1990 cells with or without IFI16 knockdown in the presence or absence of 100 nM gemcitabine for 7 days. The activation of BMDMs was then examined by flow cytometry. Knockdown of IFI16 reduced co-cultured BMDM activation in the presence or absence of gemcitabine. (J) BMDMs were co-cultured with SW1990 cells with or without IFI16 overexpression in the presence or absence of 100 nM gemcitabine for 7 days. The activation of BMDMs was then examined by flow cytometry. Overexpression of IFI16 further increased co-cultured BMDM activation in the presence or absence of gemcitabine. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The CRISPR-cas activation plasmid and IFI16 shRNA were purchased from Santa Cruz (United States).

Techniques: Injection, Knockdown, shRNA, Plasmid Preparation, Western Blot, Luciferase, Staining, Flow Cytometry, Expressing, Cell Culture, Activation Assay, Over Expression

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Heat map of upregulated genes in GO term innate immune response. Reads of the most differentially expressed genes (specified on the right of the heat map) between AAV2-infected (A1 to A3) and mock-infected NHF cells (M1 to M3). The dendrogram (left side of the heat map) illustrates the unsupervised clustering of the genes. The selected GOI ( IFI16 ) is highlighted in red.

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Infection

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Post-transcriptional silencing of IFI16 increases AAV2 transduction efficiency independent of the vector genome structure. NHF cells were transfected with no, scr control, or IFI16 targeting siRNAs. At 40 hpt, cells were either mock-infected or infected with rAAVeGFP (MOI 4,000) or scAAVeGFP (MOI 2,000). ( A and D ) At 24 hpi, cells were counted using a fluorescence microscope. ( B and E ) Graphs show mean and SD of the relative cell count of GFP-positive NHF cells from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001). ( C and F ) Knock-down of IFI16 was confirmed on protein level.

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Transduction, Plasmid Preparation, Transfection, Control, Infection, Fluorescence, Microscopy, Cell Counting, Knockdown

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Role of interferon signaling. 2fTGH Jak1 -/- cells were transfected with no, scr control, or IFI16 targeting siRNAs. At 40 hpt, cells were either mock-infected or infected with rAAVeGFP (MOI 1,000). At 24 hpi, transduced cells were counted by fluorescence microscopy. ( A ) Graph shows mean and SD of the relative cell count of GFP positive 2fTGH Jak1 -/- cells from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001). ( B ) Knock down of IFI16 was confirmed on protein level.

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Transfection, Control, Infection, Fluorescence, Microscopy, Cell Counting, Knockdown

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Effect of STING on AAV2 vector-mediated transduction. NHF cells were transfected with no, scr control, STING, or IFI16 targeting siRNAs. At 40 hpt, cells were mock-infected or infected with either rAAVeGFP (MOI 6,000) or scAAVeGFP (MOI 4,000). At 24 hpi, transduced cells were counted by fluorescence microscopy. ( A ) Graphs show mean and SD of the relative cell count of GFP-positive NHF cells from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001). ( B ) Knock down of IFI16 and STING was confirmed on protein level.

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Plasmid Preparation, Transduction, Transfection, Control, Infection, Fluorescence, Microscopy, Cell Counting, Knockdown

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Exogenous complementation of IFI16 in U2OS IFI16 -/- cells. U2OS IFI16 -/- were either untransduced, transduced with lentiviral vectors expressing GFP (MOI 5), or transduced with lentiviral vectors expressing IFI16 fused to monomeric GFP (MOI 5) in the presence of polybrene. After 72 hours, the cells were infected with rAAV2mCherry (MOI 500). ( A ) mCherry expression was assessed by RT-qPCR using specific primers for mCherry. ( B ) Exogenous complementation of IFI16 was confirmed on transcript level. Graphs show mean and SD of the relative gene expression from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001).

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Transduction, Expressing, Infection, Quantitative RT-PCR, Gene Expression

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Multicolor IF combined with FISH. NHF cells were infected with AAV2 (MOI 20,000). After 24 h, the cells were fixed and processed for multicolor IF analysis combined with FISH and CLSM. IFI16 was detected by direct labeling of the antibody to ATTO-390 (blue). Nucleoli were visualized using an antibody against fibrillarin (red). Capsids were detected using an antibody against intact AAV2 capsids (green). AAV2 DNA (gray) was detected by linking the amine-modified DNA to AF647. ( A ) Nucleolar localization of IFI16 and AAV2 capsids. ( B ) Nucleolar localization of IFI16 and AAV2 DNA. ( C ) Nucleolar localization of IFI16, AAV2 DNA, and, conditionally, intact AAV2 capsids.

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Infection, Labeling, Modification

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Post-transcriptional silencing of IFI16 increases AAV2 rep but not cap expression. ( A ) NHF and ( B ) U2OS cells were transfected with scr control or IFI16 targeting siRNAs, respectively. At 40 hpt, cells were infected with AAV2 (NHF; MOI 4,000, U2OS; MOI 2,000). At 24 hpi, total RNA was extracted and subjected to RT-qPCR using specific primers for the Rep helicase domain ( rep ), cap gene ( cap ), or IFI16 . Graphs show mean and SD of the relative gene expression from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001).

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Expressing, Transfection, Control, Infection, Quantitative RT-PCR, Gene Expression

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Post-transcriptional silencing of IFI16 increases AAV2 genome replication in the presence of AdV5. NHF cells were transfected with scr control or IFI16 targeting siRNAs, respectively. At 40 hpt, cells were either infected with ( A ) AAV2 (MOI 2,000), ( B ) AdV5 (MOI 5), or ( C ) co-infected with AAV2 (MOI 2,000) and AdV5 (MOI 5). After 24 h, total DNA was isolated and subjected to quantitative PCR using specific primers for AAV2 or AdV5, respectively. ( D ) Knock down of IFI16 was confirmed on transcript level. Graphs show mean and SD of the relative genome copy numbers or the relative gene expression, respectively, from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001).

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Transfection, Control, Infection, Isolation, Real-time Polymerase Chain Reaction, Knockdown, Gene Expression

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: Post-transcriptional silencing of IFI16 increases vector-mediated GFP expression. NHF and U2OS cells were transfected with scr control, GFP control, or IFI16 targeting siRNAs. At 40 hpt, cells were infected either with ( A and B ) rAAVeGFP (NHF; MOI 4,000, U2OS; MOI 2,000) or ( C and D ) scAAVeGFP (NHF; MOI 2,000, U2OS; MOI 1,000). At 24 hpi, total RNA was extracted and subjected to RT-qPCR using specific primers for GFP or IFI16 . Graphs show mean and SD of the relative gene expression from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001).

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Plasmid Preparation, Expressing, Transfection, Control, Infection, Quantitative RT-PCR, Gene Expression

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: IFI16 inhibits AAV2 gene expression in an Sp1-dependent manner. U2OS IFI16 -/- and U2OS wt cells were either untransduced (U2OS IFI16 -/- or U2OS wt, respectively), transduced with lentiviral vectors expressing GFP (MOI 5; U2OS IFI16 -/- + GFP ctrl.), or transduced with lentiviral vectors expressing IFI16 fused to monomeric GFP (MOI 5; U2OS IFI16 -/- + IFI16_GFP). After 72 hours, the cells were infected with AAV2 (MOI 20,000) and 24h later subjected to ChIP assays using an anti-Sp1 antibody and primers for the ( A ) p5 or ( B ) p19 promoter regions, respectively. ( C ) Interaction of IFI16 and Sp1 was assessed by co-immunoprecipitation in U2OS wt and U2OS IFI16 -/- cells. Graphs show mean and SD of the relative promoter occupancy from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001).

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Gene Expression, Transduction, Expressing, Infection, Immunoprecipitation

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: IFI16 inhibits AAV2 vector-mediated gene expression in an Sp1-dependent manner. U2OS IFI16 -/- cells or the parental cell line U2OS wt were infected with ( A ) rAAVeGFP (MOI 20,000) or ( B ) scAAVeGFP (MOI 20,000), and at 24 hpi, the cells were subjected to ChIP assays using an anti-Sp1 antibody and primers for CMV promoter regions. Graph shows mean and SD of the relative promoter occupancy from triplicate experiments. P -values were calculated using an unpaired Student’s t -test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001).

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Plasmid Preparation, Gene Expression, Infection

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: RT-qPCR primers used in this study

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques:

Journal: Journal of Virology

Article Title: Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way

doi: 10.1128/jvi.00110-24

Figure Lengend Snippet: siRNAs used in this study

Article Snippet: Lentiviral vectors expressing GFP (TR30021V) or IFI16 fused to monomeric GFP (RC202193L2V) were obtained from OriGene (Rockville, USA).

Techniques: Sequencing, Control