lucia reporter construct  (InvivoGen)

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A is methylated and suppressed in pancreatic cancer. (A) Global DNA methylation analysis (Illumina Human Methylation 450K array) in 20 human pancreatic cancer and 11 normal tissues (Stanford University Medical Center, USA). Heatmap of methylation beta values for the top 7242 differentially regulated loci in cancer vs control ( P < .05, FC ≥ 1.5). (B) Venn diagram showing 2717 unique methylation sites identified in our study. (C) Heatmap of methylation beta values across the HNF4A locus. The HNF4A promoter area with increased DNA methylation (high beta values) corresponds to probes covering the proximal promoter area (from −200 nt to −3 nt, TSS200). (D) HNF4A expression as assessed by RT-qPCR, in pancreatic cancer and control tissues. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to control tissues (set as 1). (E) Immunohistochemical analysis for HNF4A in normal (N) and pancreatic cancer tissues (red, HNF4A; blue, nuclei staining). Scale bar: 1 mm and 100 μm (left and right panel, respectively).

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Methylation, DNA Methylation Assay, Control, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Verification of HNF4A CpG methylation sites through bisulfite sequencing. (A) HNF4A expression as assessed by RT-qPCR, in 8 pancreatic cancer cell lines. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to the low HNF4A expressing cell line, MIA PaCa-2 (set as 1). (B) Diagram illustrating the generation of HNF4A isoforms through transcriptional regelation by P1 and P2 promoters and alternative splicing. Different primers were designed to recognize the 4 subgroups of the 12 HNF4A isoforms. (C) HNF4A P1a isoforms expression in pancreatic and liver (SNU-475 and Hep-3B) cancer cell lines as assessed by RT-qPCR. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to the high HNF4A expressing cell line, Hep-3B (set as 1). (D) Diagram illustrating the experimental design for the evaluation of HNF4A methylation through bisulfite sequencing. (E) Heatmaps of the methylation ratio across the HNF4A locus, at the single CpG site level, for untreated (Cont) or 5-AZA-CdR–treated MIA PaCa-2 cells (5-Aza). Cells were treated with 5-Aza (1μM), for 48 h. Analysis was performed in 40 CpG sites spanning from a distal locus upstream of +1 position to exon 3.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: CpG Methylation Assay, Methylation Sequencing, Expressing, Quantitative RT-PCR, Alternative Splicing, Methylation

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: DNA methylation at the proximal promoter area regulates HNF4A transcription in pancreatic cancer. (A) HNF4A expression restoration following pancreatic cancer cell treatment with 5-AZA-CdR (5-Aza). Low HNF4A-expressing cells (MIA PaCa-2 and PANC-1) were treated with different concentrations (1 and 2 μM) for 48 or 96 h. HNF4A expression was assessed through RT-qPCR, normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to MIA PaCa-2 untreated cells (set as 1). (B and C) HNF4A isoforms expression in MIA PaCa-2 and BxPC-3, respectively. Cells were treated with different concentrations (1 and 2 μM) of 5-Aza, for 48 h. RT-qPCR data were normalized to GAPDH and β-actin levels. (D) HNF4A expression in high (Capan-1 and HPAF-II) HNF4A-expressing pancreatic cancer cell lines. Cells were treated with different concentrations (1 and 2 μM) of 5-Aza, for 48 h. HNF4A expression was assessed through RT-qPCR, normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to untreated cells (set as 1). (E–G) Pearson’s correlation analyses between HNF4A site-specific DNA methylation and gene expression, in human pancreatic cancer tissues. Correlations in TSS200 (proximal promoter area, from −200 nt to −3 nt), distal promoter, and the gene body. (H) Pearson’s correlation analyses between HNF4A site-specific DNA methylation (TSS200 area) and gene expression, in pancreatic cancer cell lines. DNA methylation is expressed in normalized beta values and gene expression assessed by RT-qPCR is expressed in comparison to MIA PaCa-2 cells (set as 1). (I) Methylation of the HNF4A promoter area using the pCpGfree-basic- Lucia reporter plasmid. Upper panel : Diagram illustrating the HNF4A cloned promoter fragment containing 8 CpG sites, highlighted in red on the sequence (represented as lollipops). M.SssI and M.HpaII enzymes were used to methylate the HNF4A promoter region. Lower panel : HEK293T cells were transiently transfected with unmethylated, M.SssI or M.HpaII methylated reporter constructs and luciferase activity was measured in cell supernatants at 48 and 72 h after transfection. The cells were co-transfected with pCMV-Cypridina Luc Vector which secretes a variant of Cypridina luciferase and was used for normalization. Results were expressed as mean ± SEM of Lucia / Cypridina activity compared to the unmethylated (Un) respective control (set as 1). Asterisks denote statistically significant differences, ∗∗∗ P < .001, Student’s t -test.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Comparison, Methylation, Plasmid Preparation, Clone Assay, Sequencing, Transfection, Construct, Luciferase, Activity Assay, Variant Assay, Control

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss-of-function and gain-of-function studies in pancreatic cancer cell lines. (A–C) Stable HNF4A knockdown was achieved by means of 2 different shRNAs (shHNF4A_1 and shHNF4A_2) in Capan-1 and HPAF-II, through lentiviral transduction. Cells transduced with shGFP were used as the control. (A) HNF4A protein levels were determined through western blot analysis and loading was assessed using an antibody against CREB. (B and C) Cell growth was assessed by the MTT and CellTiter-Glo luminescent cell viability assay. Data were expressed as mean ± SEM (the respective control cells, at day 2, were set as 100%). (D–F) HNF4A was stably overexpressed, through lentiviral transduction (HNF4A) in PANC-1 and MIA PaCa-2 cells. Cells transduced with the empty retroviral vector tagged with GFP (EV-GFP) were used as the control. (D) HNF4A protein levels were determined through western blot analysis and loading was assessed using an antibody against CREB. (E and F) Cell growth was assessed by the MTT and CellTiter-Glo luminescent cell viability assay. Data were expressed as mean ± SEM (the respective control cells, at day 2, were set as 100%). (G and H) Anchorage-independent cell growth was assessed by soft agar assays for 5 days. Data were expressed as the mean number of colonies ± SEM (respective control cells set as 100). Representative images were acquired at a 10× and 4× magnification, respectively, using an Evos microscope. (I) Spheroid formation assays using the ultra-low attachment 96-well plate method for PANC-1 (1000 cells/well) and the hanging drop method for MIA PaCa-2 (30,000 cells/20 μL drop). Representative images were acquired on day 7, at a 10× magnification, using an Evos microscope. Asterisks denote statistically significant differences, ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, Student’s t -test.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Knockdown, Transduction, Control, Western Blot, Cell Viability Assay, Stable Transfection, Retroviral, Plasmid Preparation, Microscopy

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss is an early event and promotes PDAC growth in vivo. (A–C) Effect of HNF4A on in vivo xenograft tumour growth. HNF4A was stably overexpressed, through lentiviral transduction (HNF4A) in MIA PaCa-2 and PANC-1 cells. Cells transduced with the empty retroviral vector tagged with GFP (GFP) were used as the control. Stable HNF4A knockdown was achieved by means of shRNA in HPAF-II cells, through lentiviral transduction and cells transduced with shControl were used as the control. Cells were injected subcutaneously in NOD-SCID mice and tumor growth was monitored for a total period of 4 weeks. Tumor volumes were calculated by the equation V (mm 3 ) = a × b 2 /2, where a is the largest diameter and b is the perpendicular diameter. (D) Representative images of tumours extracted from mice at the end of the experiment. (E) HNF4A is suppressed at early stages of pancreatic cancer growth in the KPC ( LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre ) mouse model. Tissues extracted from different stages of pancreatic cancer development were subjected to HNF4A immunohistochemical analysis (brown, HNF4A; blue, haematoxylin). PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma. For PANC-1, N = 3 mice/group. For MIA PaCa-2 and HPAF-II, N = 8 mice/group. Asterisks denote statistically significant differences, ∗ P < .05, ∗∗ P < .01, Student’s t -test.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: In Vivo, Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Control, Knockdown, shRNA, Injection, Immunohistochemical staining

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Discovery cohort of 168 pancreatic cancer patients reveals that HNF4A loss is an early event and correlates with poor overall survival. HNF4A expression was assessed by immunohistochemical analysis, in 168 pancreatic cancer and 38 normal (uninvolved) tissues (QMC: Queen’s Medical Centre, Nottingham, UK). (A) Staining and scoring of tissues was performed in Histopathology Department of QMC and results were expressed as mean ± SEM compared to normal tissues (set as 1). (B) Assessment of HNF4A staining in 168 pancreatic cancer tissues according to their tumor stage. Results were expressed as mean ± SEM compared to normal tissues (set as 1). (C) Survival analysis in 153 patients divided into low, intermediate (interm), and high HNF4A expression subgroups. Survival estimates were generated using the Kaplan-Meier method and compared using log-rank tests.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Expressing, Immunohistochemical staining, Staining, Histopathology, Generated

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Effect of HNF4A Expression on Overall Survival was Assessed in 153 Patients (QMC Cohort), Using the Univariate and Multivariate Cox Proportional Hazard Analyses

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Expressing

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss indicates an increased risk of death in a validation cohort of 145 pancreatic cancer patients. HNF4A expression was assessed by immunohistochemical analysis, in 145 pancreatic cancer tissues (UCLA, USA). (A) Staining and scoring of the HNF4A immunostained tissues was performed in the Department of Pathology at UCLA Medical Center. Assessment of HNF4A staining in pancreatic cancer tissues according to their tumor stage. (B) Survival analysis in patients divided into low, intermediate (interm), and high HNF4A expression subgroups. Survival estimates were generated using the Kaplan-Meier method and compared using log-rank tests.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Expressing, Immunohistochemical staining, Staining, Generated

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Effect of HNF4A Expression on Overall Survival Was Assessed in 145 Patients (UCLA Cohort), Using the Univariate and Multivariate Cox Proportional Hazard Modelling

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Expressing

Journal: bioRxiv

Article Title: Induction of transposable element expression is central to innate sensing

doi: 10.1101/2021.09.10.457789

Figure Lengend Snippet: LPS-elicited physiological depletion of SUV39H1 promotes a type-I IFN response in myeloid cells independently of direct regulation of ISG loci, see also Figure S3. (A) Western blot of SUV39H1 in RAW264.7 macrophages transduced with empty lentivector or with a SUV39H1 overexpression (OE) lentiviral vector. (B) Left, RAW264.7 macrophages transduced with lentivirus control (n=5, closed circles) or SUV39H1 overexpressing lentivirus (n=5, open circles) were stimulated for 8 hours with the indicated concentrations of LPS and the IFN-I response measured was measured by ISG54-driven luciferase assay. Right, flow cytometric analysis of intracellular expression of the ISG, VIPERIN, by APC-control and SUV39H1 overexpressing RAW264.7 marophages stimulated as in left panel. (C) Left, RAW264.7 macrophages transduced with lentivirus as in B and stimulated with the STING ligand 2’3’cGAMP for 8hrs and the IFN-I response measured by ISG54-driven luciferase assay as in B. Right, flow cytometric analysis of ISG, VIPERIN, expression in untreated and 2’3’cGAMP transfected cells after 8hrs. (D) Western blot analysis of innate signal transduction pathways elicited in control and SUV39H1 overexpressing RAW264.7 macrophages by LPS stimulation for the indicated times. (E) Volcano plot depicting differential gene expression in WT versus Suv39h1 KO BMDCs at two hours of LPS stimulation. anti-viral genes with an adjusted P value <0.05 and FC ≥ 2 are indicated in red. (F) Gene set enrichment analysis (GSEA) comparing expression of anti-viral genes from mRNAseq data between the following groups: untreated Suv39h1 KO x Ifnar +/+ (n=4) versus Suv39h1 WT x Ifnar +/+ (n=3) BMDCs (Blue); untreated Ifnar —/— Suv39h1 KO (n=3) versus Ifnar —/— x Suv39h1 WT BMDCs (n=3) (gold). (D) Western blots of the indicated total proteins and phosphor-proteins during LPS treatment of BMDCs with the following genotypes from left to right: Ifnar +/+ x Suv39h1 WT , Ifnar +/+ x Suv39h1 KO , Ifnar —/— x Suv39h1 WT and Ifnar —/— x Suv39h1 KO. Western blots represent one of three experiments where similar results were obtained. In B and C, statistical significance was determined by two-way ANOVA and Bonferonni ad-hoc analysis: * P <0.05, ** P <0.001, *** P <0.001, **** P <0.0001. In (E), significance cutoff for differential gene expression analysis was defined as an adjusted P -value < 0.05 and a fold change ≥ 2. WT, n=3 mice per group; KO, n=4. In (F), statistical significance was GSEA; Suv39h1 WT x Ifnar +/+ (n=3) , Suv39h1 KO x Ifnar +/+ (n=4) , Suv39h1 WT x Ifnar —/— (n=3), and Suv39h1 KO x Ifnar —/— (n=3).

Article Snippet: RAW264.7-ISG-Lucia cell lines (all from Invivogen) deleted for Tmem173 (rawl-kostg ), Mb21d1 (rawl-kocgas ), Mda5 (rawl-komda5) , and Rig-I (rawl-korigi ) carried a Lucia luciferase reporter construct under control of an IFN-inducible ISG54 promoter (Invivogen) and were cultured in DMEM containing 10% Serum (Fischer Scientific), 1% Penicilin/Streptomycin (Fischer Scientific) and Zeocin (1μg/ml; Invivogen).

Techniques: Western Blot, Transduction, Over Expression, Plasmid Preparation, Control, Luciferase, Expressing, Transfection, Gene Expression

Journal: bioRxiv

Article Title: Induction of transposable element expression is central to innate sensing

doi: 10.1101/2021.09.10.457789

Figure Lengend Snippet: Loss of Suv39h1 amplifies the LPS-induced anti-viral response independently of direct regulation of gene loci, related to . (A) Ifnb1 expression from mRNAseq data of Suv39h1 WT and KO BMDCs that were treated with LPS for the indicated times. Data are displayed as FPKM. (B) ELISA quantification of Type-I IFN (IFNb) protein levels in conditioned supernatants from Suv39h1WT (n=4) or Suv39h1KO (n=5) BMDCs treated with LPS for the indicated times. (C) Western blots of the indicated proteins and phosphor-proteins in Suv39h1 WT and KO BMDCs that were treated with LPS (100ng/ml) for the specified durations. (D) Heatmap displaying semi-supervised clustering (Pearson correlation) of anti-viral (ISG) and inflammatory gene expression in WT and Suv39h1KO BMDCs stimulated with LPS for the indicated times. Data are from the same mRNA-seq data set used for TE analysis in . (E) Flow cytometric data quantifying SIGLEC1 (ISG) and CD86 (NFkB-regulated) surface expression on mDCs transduced with nontargeting or one of three different SUV39H1-targeting lentiviral shRNA constructs (n=4 patient samples per condition). (F) Western blot of SUV39H1, demonstrating targeted depletion by three different lentiviral shRNAs in freshly cultivated human monocyte-derived DCs (mDCs). (G) qPCR data exhibiting the time course of Suv39h1 mRNA expression during LPS treatment in RAW macrophages transduced with the designated lentiviral shRNAs (n=2). (H) Western blot of SUV39H1, SETDB1, and gp96 (loading control) to assess shRNA-mediated knockdown in untreated RAW264.7 macrophages transduced with the indicated lentiviral shRNA particle. (I) ISG54-Luciferase assay data comparing luciferase concentration in conditioned supernatants of untreated RAW264.7 in which Suv39h1 was depleted by lentiviral encoded shRNA ( shSuv39h1 , n=4) or not (shGFP, n=4). (J) Left, heatmap showing z-scores of ISG mRNA expression (FPKM) in untreated Suv39h1 WT (n=3) or Suv39h1 KO (n=4) BMDCs, and right, the corresponding H3K9me3 peak status from H3K9me3 ChIPseq data (WT, n=3; KO, n=3): red, H3K9me3 peak positive, grey, no H3K9me3 peak detected. Several genes along with Ifnb1 are designated in bold to the right as exemplars of ISGs with a grey or red box indicating H3K9me3 peak status. Statistical differences in panels A, B, and E were determined by two-way ANOVA, followed by Bonferroni’s ad hoc analysis for individual comparison between groups:* P <0.05, ** P <0.001, *** P <0.001, **** P <0.0001. In I, a two-tailed student’s t-test was performed to determine statistical significance ( P < 0.05).

Article Snippet: RAW264.7-ISG-Lucia cell lines (all from Invivogen) deleted for Tmem173 (rawl-kostg ), Mb21d1 (rawl-kocgas ), Mda5 (rawl-komda5) , and Rig-I (rawl-korigi ) carried a Lucia luciferase reporter construct under control of an IFN-inducible ISG54 promoter (Invivogen) and were cultured in DMEM containing 10% Serum (Fischer Scientific), 1% Penicilin/Streptomycin (Fischer Scientific) and Zeocin (1μg/ml; Invivogen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Gene Expression, Transduction, shRNA, Construct, Derivative Assay, Control, Knockdown, Luciferase, Concentration Assay, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Induction of transposable element expression is central to innate sensing

doi: 10.1101/2021.09.10.457789

Figure Lengend Snippet: LPS elicits cGAS-dependent but not MAVS-dependent ISG expression, see also Figure S4. (A) Western blots of the indicated proteins and phosphor-proteins in WT and Cgas —/— BMDCs treated as follows: LPS treatment was for 4 hours; cGAMP (250-500ng/ml), 2 hours. (B) The indicated ISGs indicated in bold italics on the left were measured by mRNAseq of steady-state and LPS-stimulated (4hrs, 100ng/ml) WT, Myd88 —/— , Trif —/— BMDCs or by RT-qPCR of WT and Mavs —/— BMDCs treated with STING inhibitor (STINGi, H-151) or vehicle control (DMSO) at steady state or stimulated with the indicated LPS dose (33 or 100ng/ml). As a positive control for inhibition of the cGAS/STING pathway, WT BMDCs were treated with STINGi or DMSO and then transfected with 1µg/ml HT-DNA for 5 hours. ISG expression was calculated relative b-Actin by the delta Ct method. (C) Scatter plot of ISG54-Luciferase assay of conditioned supernatants from WT, Myd88 —/— , or Trif —/— RAW264.7 macrophges stimulated with the indicated concentration of LPS. (D) Scatter plot of ISG54-Luciferase assay of conditioned supernatants from WT, Cgas —/— , Mavs —/— , and Cgas —/— x Mavs —/— RAW264.7 macrophages that were stimulated with the indicated concentration of LPS. (E) The indicated LINE1families indicated in bold on the left were measured by mRNAseq of steady-state and LPS-stimulated (4hrs, 100ng/ml) WT, Myd88 —/— , Trif —/— BMDCs or by RT-qPCR of WT and Mavs —/— BMDCs treated with STING inhibitor (STINGi, H-151) or vehicle control (DMSO) at steady state or stimulated with the indicated LPS (100ng/ml) for 4hrs. As a positive control for inhibition of the cGAS/STING pathway. (F) ISG54-driven luciferase assay as in C and D. WT and Cgas —/— RAW264.7 macrophages in which Suv39h1 was left intact (shCtrl) or depleted ( shSuv39h1 ) with lentiviral-derived shRNA were treated with the indicated concentrations of LPS for 8. WT ( shCtrl , n=8; shSuv39h1 , n=8), Cgas —/— ( shCtrl , n=8; shSuv39h1 , n=8). (G) ISG54-driven luciferase assay as in C,D, and F. Cgas +/+ or Cgas —/— RAW cells that were transduced with APC control or SUV39H1 overexpression lentivectors and stimulated with the indicated concentrations of LPS for 8hrs (n=10 per group). B-G, Statistical significance was determined by two-way ANOVA with Bonferonni’s ad hoc analysis for individual comparisons:* P <0.05, ** P <0.001, *** P <0.001, **** P <0.0001.

Article Snippet: RAW264.7-ISG-Lucia cell lines (all from Invivogen) deleted for Tmem173 (rawl-kostg ), Mb21d1 (rawl-kocgas ), Mda5 (rawl-komda5) , and Rig-I (rawl-korigi ) carried a Lucia luciferase reporter construct under control of an IFN-inducible ISG54 promoter (Invivogen) and were cultured in DMEM containing 10% Serum (Fischer Scientific), 1% Penicilin/Streptomycin (Fischer Scientific) and Zeocin (1μg/ml; Invivogen).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Positive Control, Inhibition, Transfection, Luciferase, Concentration Assay, Derivative Assay, shRNA, Transduction, Over Expression

Journal: bioRxiv

Article Title: Induction of transposable element expression is central to innate sensing

doi: 10.1101/2021.09.10.457789

Figure Lengend Snippet: The cGAS pathway of DNA sensing, but not the RNA-sensing MAVS pathway is required for an optimal IFN-I/anti-viral response following stimulation with LPS, related to . (A) ELISA quantification of IFNb protein concentration in conditioned supernatants from Cgas +/+ (n=3) and Cgas —/— (n=3) BMDCs stimulated with LPS or with transfected HT-DNA, 2’3’cGAMP. (B) Compiled flow cytometric data showing the percentage of Viperin + or CCL5 + CD11c + CD11b + MHC-II + splenic Cgas +/+ (n=3) and Cgas —/— (n=3) DCs stimulated ex vivo with 10-fold dilutions of LPS (top, 100ng/ml highest concentration) or bottom, with 2’3’cGAMP. (C) Western blot of the indicated total and phosphor-proteins following stimulation of WT and Mavs —/— BMDCs with transfected poly(I:C) (5hrs) or infection with IAV/PR8 (7hrs). (D) RT-qPCR data of the ISGs, 5hrs after transfection of poly(I:C) in WT and Mavs —/— BMDCs. ISG expression was calculated relative to b-Actin by the delta Ct method. (E) Western blot displaying protein expression of MAVS, cGAS, and gp96 after CRISPR-Cas9-targeted deletion of Mavs in WT and Cgas —/— RAW264.7 macrophages. (F) Functional validation of CRISPR-Cas9-targeted Mavs deletion in RAW264.7 macrophages displayed in E; top, transfection of poly(I:C), 2µg/ml; bottom, transfection of HT-DNA, 1µg/ml. (G) ELISA quantification of IFNB concentration in conditioned supernatants of untreated or LPS (100ng/ml) treated RAW264.7 macrophages that were deleted for Ifnar1/2 by CRISPR-Cas9 and the transduced with control lentiviral particles (shGFP) or shRNA lentiviral particles targeting Suv39h1 (shSuv39h1). (H, I) Functional validation of CRISPR-Cas9 targeted deletion of Ifnar in RAW264.7 macrophges. (C) ISG54-driven luciferase assay measuring responsiveness of Ifnar +/+ Cgas +/+ , Ifnar +/+ Cgas —/— , Ifnar —/— Cgas +/+ , and Ifnar —/— Cgas —/— cells to overnight stimulation with titrated recombinant IFNB protein.Error bars represent standard deviation and bar graphs represent the mean. Statistical significance was determined with two-way ANOVA and Bonferonni’s ad hoc analysis for individual comparisons. A, B, D, and G, statistical significance was determined with two-way ANOVA and Bonferonni’s ad hoc analysis for individual comparisons.

Article Snippet: RAW264.7-ISG-Lucia cell lines (all from Invivogen) deleted for Tmem173 (rawl-kostg ), Mb21d1 (rawl-kocgas ), Mda5 (rawl-komda5) , and Rig-I (rawl-korigi ) carried a Lucia luciferase reporter construct under control of an IFN-inducible ISG54 promoter (Invivogen) and were cultured in DMEM containing 10% Serum (Fischer Scientific), 1% Penicilin/Streptomycin (Fischer Scientific) and Zeocin (1μg/ml; Invivogen).

Techniques: Enzyme-linked Immunosorbent Assay, Protein Concentration, Transfection, Ex Vivo, Concentration Assay, Western Blot, Infection, Quantitative RT-PCR, Expressing, CRISPR, Functional Assay, Biomarker Discovery, Transduction, Control, shRNA, Luciferase, Recombinant, Standard Deviation

Journal: bioRxiv

Article Title: Induction of transposable element expression is central to innate sensing

doi: 10.1101/2021.09.10.457789

Figure Lengend Snippet: LINE1 is an endogenous cGAS PAMP, see also Figure S5. (A) qPCR of digitonin-extracted, RNAse-treated cytosolic extracts from untreated or LPS stimulated (100ng/ml, 4hrs) WT BMDCs that were pretreated with an RTI cocktail or DMSO vehicle control. (B) High-throughput sequencing of DNA extracted from the following fractions from LPS-treated BMDCs expressing Cgas gfp/gfp : cytosolic input fraction, cytosolic cGAS, nuclear input fraction, and nuclear cGAS. The pie charts display the enrichment in each fraction visualized as the distribution of the sum of the read densities (FPKM≥1) per unique element belonging to the indicated DNA species. N=2 for each sample. (C) Hypergeometric statistical comparison of read density proportions in panel B between input and the respective cGAS-IP within each cellular fraction (indicated in bold on right). Red color denotes —log 10 ( P value); grey, no statistical difference. (D) Correlation between the number of individual LINE1 elements within each subfamily expressed as mRNA and detected in the top 10% of fold changes in cytosolic cGAS-bound DNA. Dot size indicates the DNA density (FPKM) of cGAS-bound LINE1 families and the color scale indicates the average sequence divergence from consensus of the elements detected on cGAS. Less divergence indicates evolutionarily younger elements. (E) Scattered dot plots exhibiting FPKM of cGAS-bound unique LINE1 element DNA (FPKM ≥ 1 and FC ≥ 2 over input) in untreated and treated Suv39h1 WT xCgas egfp/egfp and Suv39h1 KO xCgas egfp/egfp BMDCs. Red bars represent median values; box ends represent quartiles. Statistical analysis was performed with one-way ANOVA followed by Bonferroni’s ad hoc analysis for individual comparison between samples. (F) Scattered dot plots as in E except FPKM values represent density of unique SINE element DNA bound to cGAS. Statistical analysis was performed as in E. (G) qPCR of LINE1, ERVK, and genomic Gapdh DNA bound to cytosolic cGAS immunoprecipitated with anti-GFP magnetic beads from BMDC cytosolic fractions as in B and E (filled green bars). Controls include control-bead IP from Cgas egfp/egfp (hashed green bars), GFP-bead (filled grey bars) and control-bead IP (hashed grey bars) from Cgas +/+ BMDCs. (H) ISG54-Luciferase assay of resting and LPS-stimulated (8hrs) WT RAW264.7 cells expressing mutant d(dead)Cas9 KRAB fusion protein and control or LINE1-targeted gRNAs. (I) Representative volcano plot comparing differentially expressed (DE) LINE1 elements in gRNA_1 versus Ctrl cells stimulated with LPS. (I) Bar chart representing the log 2 fold-change between DE LINE1 elements (Adjusted P value <0.05) in Ctrl (“Down in gRNA”) and gRNA-expressing cells (“Up in gRNA”) at steady state or after LPS treatment. (K) Above, GSEA of the effect of the specified gRNA versus control on anti-viral gene expression in resting and LPS-stimulated cells. Line color indicates the specific comparison. Below, gene rank plots. Each bar represents an anti-viral gene and its position along the x-axis indicates relative enrichment in the indicated group: left indicates enrichment in control cells; right, LINE1-targeted gRNA expressing cells. bar color corresponds to the same color used to specify the specific comparison above. P value to the right indicates statistical significance of enrichment of anti-viral gene expression in the control group versus LINE1-targeted gRNA-expressing cells. NES, normalized enrichment score. A, E, F, and H, statistical significance was determined by 2-way ANOVA, and Bonferonni’s ad hoc analysis was used for individual comparisons. A, E, F, H: * P <0.05, ** P <0.001, *** P <0.001, **** P <0.0001.

Article Snippet: RAW264.7-ISG-Lucia cell lines (all from Invivogen) deleted for Tmem173 (rawl-kostg ), Mb21d1 (rawl-kocgas ), Mda5 (rawl-komda5) , and Rig-I (rawl-korigi ) carried a Lucia luciferase reporter construct under control of an IFN-inducible ISG54 promoter (Invivogen) and were cultured in DMEM containing 10% Serum (Fischer Scientific), 1% Penicilin/Streptomycin (Fischer Scientific) and Zeocin (1μg/ml; Invivogen).

Techniques: Control, Next-Generation Sequencing, Expressing, Comparison, Sequencing, Immunoprecipitation, Magnetic Beads, Luciferase, Mutagenesis, Gene Expression

Journal: bioRxiv

Article Title: Induction of transposable element expression is central to innate sensing

doi: 10.1101/2021.09.10.457789

Figure Lengend Snippet: Immunoprecipitation of endogenous cGAS from nuclear and cytosolic extracts followed by NGS reveals significant changes in cytosolic cGAS-DNA binding activity, related to . (A) Flow cytometric quantification of expression of the ISG VIPERIN in untreated or 8hr LPS-stimulated WT BMDCS that were pretreated with DMSO vehicle or an RTI cocktail. (B) Western blot revealing WT cGAS and the eGFP knock-in cGAS (KI) allele from whole cell lysate (WCE), cytosolic (C) and nuclear (N) fractions, as well as immunoprecipitation of cross-linked cytosolic and nuclear cGAS species. RAB6 and H3 serve as cytosolic and nuclear loading controls, respectively. Arrowheads indicate lanes displaying immunprecipitated eGFP-cGAS. (C) qPCR of the indicated amplicons in digitonin-extracted RNAse-treated cytosolic DNA and pellet fractions from untreated BMDCs. (D) qPCR of indicated DNA species that bound to an overexpressed eGFP-tagged cGAS contstruct in WT (shControl, n=4) and Suv39h1 (n=4) knockdown RAW cells following LPS stimulation. (E) Pie chart depicting the relative proportions of raw reads in the indicated DNA species from NGS of cytosolic cGAS-IP. (F) Left, comparison of the fold changes in LPS versus untreated BMDCs of TE binding density on endogenous cGAS in cytosolic and nuclear fractions. Right, variance describing the fold changes in the binding density of the indicated TE class enriched on nuclear and cytosolic cGAS following LPS treatment (n=2 per condition). An F-test was used for statistical comparison of variances. (G) Flow cytometric plots displaying lentiviral-based expression levels of the indicated GFP-cGAS or SFFV-BFP control constructs used for experiments in Figure S5H, I. (H) ISG54-driven luciferase assay quantification of baseline IFN-I production by Cgas —/— RAW cells following lentiviral reconstitution with different cGAS constructs—full-length WT (cGAS-FL, n=12), catalytically-dead (cGAS CD, n=7), DNA binding mutant (cGAS-DBM, n=7), SFFV (empty vector control). (I) ISG54-driven luciferase assay quantification of IFN-I production by Cgas —/— RAW cells following lentiviral reconstitution with different cGAS constructs—full-length WT (cGAS-FL, n=12), catalytically-dead (cGAS-CD, n=7), DNA binding mutant (cGAS-DBM, n=7), SFFV (empty vector control)—and titrated stimulation with HT-DNA, 2’3’cGAMP, or LPS. Baseline values were subtracted from treated values to compare the response between groups reconstituted with distinct cGAS constructs. (J) Distribution of the cytosolic cGAS-IP read density sums (FPKM≥1) among all classes of DNA species in Suv39h1KO BMDCs. (K) Hypergeometric statistical comparison of the Suv39h1 KO BMDC cytosolic cGAS-IP read density sums in panel E with WT cytosolic cGAS IP . (L) Violin plot exhibiting the difference in the number of cytosolic cGAS-bound LINE1 elements by family between Suv39h1 KO and Suv39h1 WT BMDCs. (M) Inhibition of cell surface myeloid marker CD11b on RAW264.7 using the dCas9-KRAB transcriptional inhibitor system with two different gRNAs directed to the promoter region of the Itgax locus. Top, flow cytometric dot plots of CD11b (y-axis) and MHC Class II (MHC-II, x-axis); bottom, quantification of flow cytometric data for percentage of CD11b and MHC-II expressing cells and the MFI of each marker (n=3 per condition). (N) In silico prediction of the number of LINE1 gRNA targets (y-axis) according to the number of allowable mismatches between gRNA and target sequences (color intensity). Above, pie charts exhibiting enrichment of gRNA target sequences in LINE1 families. The most highly enriched family is denoted at the top of each pie chart and the corresponding gRNA number is listed below. (O) Scatter dot plot displaying the number of gRNA target sequences upstream of ISGs (blue circles) versus targeted LINE1 elements (red circles) . (P) ISG54-Luciferase assay of IFN-I and ISG induction in resting and LPS-stimulated (8hrs) Cgas —/— RAW264.7 cells expressing mutant dCas9 KRAB fusion protein and the indicated gRNA (Ctrl, gRNA_1, or gRNA_2; n=4 per group). Statistical significance was determined by 2-way ANOVA and Bonferonni’s ad hoc analysis for individual comparisons:* P <0.05, ** P <0.001, *** P <0.001, **** P <0.0001.

Article Snippet: RAW264.7-ISG-Lucia cell lines (all from Invivogen) deleted for Tmem173 (rawl-kostg ), Mb21d1 (rawl-kocgas ), Mda5 (rawl-komda5) , and Rig-I (rawl-korigi ) carried a Lucia luciferase reporter construct under control of an IFN-inducible ISG54 promoter (Invivogen) and were cultured in DMEM containing 10% Serum (Fischer Scientific), 1% Penicilin/Streptomycin (Fischer Scientific) and Zeocin (1μg/ml; Invivogen).

Techniques: Immunoprecipitation, Binding Assay, Activity Assay, Expressing, Western Blot, Knock-In, Knockdown, Comparison, Control, Construct, Luciferase, Mutagenesis, Plasmid Preparation, Inhibition, Marker, In Silico