Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: DIRAS family expression is downregulated in ovarian cancers and cancer cell lines. (A) Normal ovaries and tumor tissue microarrays with 122 cases represented were analyzed using immunohistochemistry with anti-DIRAS1 and anti-DIRAS2 antibodies and scored 0 to 3. Examples from the tumor array representing the score 0 (no expression), 1 (low expression), 2 (moderate expression) and 3 (high expression) are shown with 10x magnification, and enlarged 40x magnification insets for each antibody. Bar: 100 µm. (B) The fraction of ovarian cancers with DIRAS family expression. (C) The correlation between disease-free survival and DIRAS family member expression. Each circle represents one patient sample. The median disease-free survival time is listed on top. (D) The fraction of ovarian cancers with DIRAS2 expression. (E) The correlation between disease-free survival and overall survival by staining score of DIRAS1. Each circle represents one patient sample. Asterisk denotes significant difference (*p<0.05 or **p<0.01). (F) Kaplan-Meier overall survival analysis of DIRAS1 expression by staining score. (G) Kaplan-Meier overall survival analysis of DIRAS2 expression by staining score. Statistical significance was determined by Mantel-Cox Log-rank analysis of the overall survival. (H) DIRAS1 and DIRAS2 protein expression levels were determined in normal ovarian epithelial scrapings (NOE), 11 ovarian cancer primary tumors and 14 ovarian cancer cell lines using western blot analysis.

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: Re-expression of DIRAS1 or DIRAS2 inhibits ovarian cancer cell growth in vitro. (A and B) Two distinct subclones of OVCAR8-DIRAS1 or OVCAR8-DIRAS2 cells were seeded at 400 cells/well and doxycycline (1 µg/mL) was added to the media every 48 h for 6 d to induce gene expression. Colonies were allowed to grow for 2 wk and then stained with Coomassie Brilliant Blue. Percentage of growth inhibition was determined for 3 independent experiments all performed with triplicate samples. (C) OVCAR8-Praental cells were seeded at 400 cells/well and doxycycline (1 µg/mL) was added to the media every 48 h for 6 d. Colonies were allowed to grow for 2 wk and then stained with Coomassie Brilliant Blue. Percentage of growth inhibition was determined for 3 independent experiments all performed with triplicate samples. (D–I) Similar experiments were carried out in SKOv3-IP-inducible lines and Hey-A8-inducible lines or their parental control cells, seeded at 800 and 600 cells per well, respectively. Asterisk denotes significant difference (*p<0.05 or **p<0.01).

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, In Vitro, Staining, Inhibition

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: Re-expression of DIRAS family members induces autophagy. OVCAR8-inducible cells were treated with or without doxycycline for 18–48 h with or without the presence of chloroquine (5 µM). (A) Cell lysate was collected and western blot analysis was performed to examine the conversion of LC3-I to LC3-II, SQSTM1/p62 levels, and expression of DIRAS1 or DIRAS2. ACTB/β-actin was used as a loading control. Upon re-expression of DIRAS1 or DIRAS2 we observed decreased SQSTM1 levels and increased conversion to LC3-II, which was further enhanced by the addition of chloroquine. (B and C) Immunofluorescence staining of DIRAS1 or DIRAS2 (green) and LC3B (red) was performed to document the formation of autophagosomes. Scale bar: 10 µm. (D and E) Fluorescence microscopy of OVCAR8-DIRAS1 or OVCAR8-DIRAS2 ovarian cancer cells with or without doxycycline treatment (1 µg/mL), for 18–30 h to induce gene expression, 24 h post infection with GFP-mCherry-LC3B-expressing lentivirus to visualize autophagic flux. Yellow puncta indicate the presence of GFP and mCherry signal. Red puncta indicate the fusion of the autophagosome with the lysosome and quenching of GFP. Scale bar: 10 µm. Average puncta counts were determined for each time point and condition using ImageJ and plotted with GraphPad Prism. Data were collected over 3 independent experiments and more than 50 cells were analyzed per condition. Asterisk denotes significant difference as determined by Student t test (*p<0.05 or **p<0.01). (F) TEM images of induced or noninduced OVCAR8-DIRAS1 or DIRAS2 cells. Red arrows indicate typical double-membrane autophagosomes. Scale bars: 5 µm and 5 nm for close-up image. Data were collected over 3 independent experiments. Asterisk denotes significant difference (*p<0.05 or **p<0.01).

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy, Infection

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: DIRAS1 and DIRAS2 growth inhibition is dependent upon functional autophagy. Using CRISPR/Cas9 double-nickase control plasmid or ATG7 sgRNA, OVCAR8-DIRAS1 and OVCAR8-DIRAS2 inducible cells were generated from single-cell clones. (A and B) Western blot analysis revealed that control cells induced autophagy upon re-expression of DIRAS1 or DIRAS2 whereas ATG7−/− cells did not. Knockout efficiency was observed by western blot analysis of ATG7. (C and D) Clonogenic assays were performed with these sublines, documenting that growth inhibition seen by re-expressing DIRAS1 or DIRAS2 is dependent upon functional autophagy. Experiments were completed in triplicate. Asterisk denotes significant difference (*p<0.05 or **p<0.01).

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Inhibition, Functional Assay, CRISPR, Plasmid Preparation, Generated, Clone Assay, Western Blot, Expressing, Knock-Out

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: DIRAS1 and DIRAS2 induce autophagy by inhibiting PI3K and regulating the FOXO3 transcription factor. (A) OVCAR8-inducible cells were treated with or without doxycycline for 24 h to induce DIRAS1 or DIRAS2 gene expression. Cell lysate was collected and western blot analysis was performed. Re-expression of DIRAS1 and DIRAS2 resulted in a significant decrease in p-AKT (S473) and p-MAPK/ERK (42/44). p-MTOR (S2488), a downstream target of AKT and key inhibitor of autophagy, was also decreased following re-expression of DIRAS1 or DIRAS2. (B) ImageJ quantification was performed for 3 independent western blot analyses to determine the relative expression of p-AKT, p-MAPK/ERK and p-MTOR following re-expression of DIRAS1 or DIRAS2 (*p<0.05; **p<0.01). (C) Cell lysate was collected and western blot analysis was performed to examine the nuclear localization of FOXO3 or TFEB. PARP and TUBA/α-tubulin were used as nuclear and cytoplasm extraction controls. The numbers below the blots correspond to ImageJ densitometry of the blot. (D) Immunofluorescence staining of FOXO3 and TFEB nuclear localization following re-expression of DIRAS1 or DIRAS2. Scale bars: 20 µm. (E and F) OVCAR8-inducible cell lines were treated with or without doxycycline for 24 h to induce gene expression of DIRAS1 or DIRAS2. mRNA expression of LC3B, ULK1, RAB7, GABARAP, BECN1, and LAMP1 was measured by qRT-PCR. (E) Re-expression of DIRAS1 in OVCAR8 ovarian cancer cells increased mRNA expression of ULK1, RAB7, GABARAP, BECN1 and LAMP1. (F) Re-expression of DIRAS2 in OVCAR8 increases mRNA expression of LC3B, ULK1, RAB7, GABARAP, and BECN1.

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: Transient expression of murine DIRAS1 and DIRAS2 inhibit murine ovarian cancer cell clonogenic growth and induce autophagy. Long-term clonogenic growth was assessed following transient overexpression of murine DIRAS1 and DIRAS2 in ID8 (A), IG10 (B), TBR2 (C) and TKOOV10 (D) murine ovarian cancer cell lines. Selection was performed with G418 for 2 wk until colonies reached at least 50 cells in size. Columns represent the mean and bars represent the S.D. The experiment was performed at least 3 independent times. Asterisk denotes significant difference (**p<0.01). ID8 (A), IG10 (B), TBR2 (C) and TKOOV10 (D) murine ovarian cancer cells were seeded at 0.3 × 106 cells per well and transfected with empty vector, DIRAS1- or DIRAS2-encoding plasmid DNA and treated with or without chloroquine (5 µm) for 24 h prior to lysing and immunoblotting as indicated. Densitometry was measured with ImageJ and experiments were performed at least 3 times. Autophagic flux was determined by the ratio of LC3-II:LC3-I and normalized to the loading control, ACTB/β-actin.

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, Over Expression, Selection, Transfection, Plasmid Preparation, Western Blot

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: Murine DIRAS1 and DIRAS2 are essential for starvation-induced autophagy. Mouse ovarian cancer cells were transfected with control, murine Diras1 or murine Diras2 siRNA for 72 h prior to autophagy induction by serum starvation for 4–16 h. (A) Western-blot analysis was performed as indicated and documented a decrease in serum starvation-induced autophagic flux following knockdown of DIRAS1 or DIRAS2. (B) Immunofluorescence staining of IG10, TBR2, and TKOOV10 cells for LC3 puncta following autophagy induction by serum starvation for 16 h was performed. Quantification of puncta/cell was calculated for at least 100 cells per experiment, for 3 experiments. Columns indicate the mean and bars represent the S.D. Significance denoted by the asterisk (*p<0.05, **p<0.01). (C) qRT-PCR analysis of murine Diras1 and murine Diras2 mRNA expression following knockdown with siRNA. Columns indicate the mean and bars represent the s.d. Significance denoted by the asterisk (*p<0.05, **p<0.01).

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Transfection, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality

doi: 10.1101/2023.10.03.560559

Figure Lengend Snippet: (a) Schematic representation of the proposed cellular response to mtDox damage. mtDNA damage caused by mtDox leads to mitochondrial herniation followed by release of mitochondrial contents into the cytoplasm, sensed through RIG-I innate immune pathway. Functional WRNIP1 transports activated RIG-I to MAVS which stimulates immune response activation, leading to nuclear recognition of damage. (b) Digitonin based extraction followed by qPCR measurement of mtDNA released into the cytoplasm over time following 8 μM mtDox treatment (n = 4, * p < 0.0011, ** p < 0.013). (c) Digitonin based extraction followed by RT-qPCR measurement of mtRNA released into the cytoplasm over time (n = 4, * p < 0.013, ** p < 0.037). (d) Western blot of RIG-I-FLAG co-immunoprecipitation displaying time-course interaction of RIG-I with WRNIP1 following mtDox treatment. Anti-FLAG M2 magnetic beads were used to pull down RIG-I-FLAG transfected lysates, and blot was counter-stained with antibodies for FLAG and WRNIP1 (n = 3). (e) Time-course Western blot for phosphorylated IRF3 (p-IRF3) indicating downstream innate immune activation. Performed with parent (WT) and WRNIP1 knockout (KO) cells lines (n = 3). (f) Clonogenic analysis of relative survival following mtDox treatment between LacZ, cGAS, and MAVS knockout (n = 4). All p -values determined using unpaired t -test. Data represented as mean ± s.d.

Article Snippet: HCT116 TP53 (-/-) cells were transfected with a FLAG-tagged RIG-I expression vector purchased from Origene (RC217615) using Lipofectamine 3000 in six 150 mm dishes plated to 90% confluency the day before.

Techniques: Functional Assay, Activation Assay, Extraction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Magnetic Beads, Transfection, Staining, Knock-Out

Journal: Oncotarget

Article Title: Bisphenol A induces cell cycle arrest in primary and prostate cancer cells through EGFR/ERK/p53 signaling pathway activation

doi: 10.18632/oncotarget.23360

Figure Lengend Snippet: In (A) , LNCaP cells were left untreated or treated for the indicated times with 50 μM Bisphenol A (BPA) in absence or presence of 10 μM Bicalutamide (Bic), 10 μM ICI 182,780 (ICI) and 20 μM PD 98,059 (PD). Phosphorylation of p53 on residue Ser15 (p-Ser15-p53) was analyzed in lysate proteins using the appropriate antibody. Filters were stripped and re-probed with the anti-p53 antibody and α-Tubulin antibody, as loading control. In (B) , LNCaP cells were stimulated or unstimulated for 5 minutes with 50 μM Bisphenol A (BPA) in absence or presence of 5 μM ZD 1839 (ZD), 10 μM Bicalutamide (Bic) and 10 μM ICI 182,780 (ICI). Lysate proteins were immune-precipitated with EGFR antibody or non specific mouse immunoglobulin (Ctrl Ab) as control. Proteins in immune complexes were detected by Western blot using appropriate antibodies against indicated proteins. In (C) , ERK inhibition rescues the BPA-treated LNCaP cell ability to form colonies. LNCaP cells were seed in 6 well plates and stimulated or unstimulated with 50 μM Bisphenol A (BPA) in absence or presence of 20 μM PD 98,059. After 12 days the cells were fixed, stained and counted as reported in Materials and Methods.

Article Snippet: Anti-p-Ser15-p53 (catalog MAB1839) was from R&D Systems.

Techniques: Phospho-proteomics, Residue, Control, Western Blot, Inhibition, Staining

Journal: Oncotarget

Article Title: Bisphenol A induces cell cycle arrest in primary and prostate cancer cells through EGFR/ERK/p53 signaling pathway activation

doi: 10.18632/oncotarget.23360

Figure Lengend Snippet: Bisphenol A stimulates EGFR phosphorylation and triggers EGFR/AR/ERβ complex assembly. BPA treatment induces ERK activity and the consequent phosphorylation of p53 on residue Ser15 and hence p53 stabilization. This results in increased p27 and p21 expression levels and cyclin D1 protein down regulation, which lead to cell cycle arrest.

Article Snippet: Anti-p-Ser15-p53 (catalog MAB1839) was from R&D Systems.

Techniques: Phospho-proteomics, Activity Assay, Residue, Expressing

Journal: Nature microbiology

Article Title: Systems-based Analysis of RIG-I-dependent Signaling Identifies KHSRP as an Inhibitor of RIG-I Receptor Activation

doi: 10.1038/nmicrobiol.2017.22

Figure Lengend Snippet: a, Schematic representation of the RIG-I signaling pathway (left) and genome-wide siRNA screens (right) are shown. b , HEK293T ISRE-luciferase cells were transfected with indicated siRNA followed by infection with either wild type or delNS1 IAV. 24hr post infection, luciferase activities were analyzed. Results are the mean ± s.d. of three biological replicates. *** P < 0.001 (Student's t -test). Data is representation of three independent experiments. c, Primary results of genome-wide screens for positive (left panel) and negative (right panel) regulators of RIG-I signaling. ISRE-activity score values were calculated as described in RNAi Screening Analysis in Discussion. Negative controls (depicted by red circles) were assigned an activity score of 1, while positive controls (depicted by black circles) were scaled to a value 0.1. Screen hits are indicated as open circles. RSA analysis is used to generate P values; * P < 0.05, ** P < 0.01, *** P < 0.001. d, Network integration of candidate RIG-I pathway regulators (permutation test p<0.001). Circles indicate protein interactions identified by GeneGo analysis. Hexagon and square shapes indicate AP-MS bait and prey interactions, respectively. Confirmed negative regulators (pink), high confidence positive regulators based on RSA cutoff (orange; p<0.01), and canonical RIG-I regulators (red and blue) are also shown. An enlarged sub network of RIG-I pathway regulators is encircled by dashed line (right). AP-MS interactions between RNAi hits and canonical RIG-I bait proteins are indicated (red edges). This sub-network is expanded using GeneGo to the first neighbor interactors of RNAi hits (indicated by blue edges; right). e, Functional enrichment of RIG-I network proteins using gene ontology resources. Nodes represent enriched functions for an annotated ontology term, and the node size indicates the number of genes that fall into that term. The pie charts embedded within the nodes represent the percentage of RIG-I positive regulators (green) and negative regulators (red) for that term. Nodes are clustered into sub-networks that encompass a representative description for the annotations.

Article Snippet: The following siRNAs were used: KHSRP_5 (Qiagen; SI00300587), KHSRP_3 (Qiagen; SI00054691), PIN_6 (Qiagen; SI02662667), DDX58_6 (Qiagen; SI03019646), and influenza A NP (5'-GGAUCUUAVUUCUUCGGAG-3') .

Techniques: Genome Wide, Luciferase, Transfection, Infection, Activity Assay, Functional Assay

Journal: Nature microbiology

Article Title: Systems-based Analysis of RIG-I-dependent Signaling Identifies KHSRP as an Inhibitor of RIG-I Receptor Activation

doi: 10.1038/nmicrobiol.2017.22

Figure Lengend Snippet: a, Confirmed RIG-I negative regulators were depleted by siRNA in wild type or CRISPR IRF9 (cIRF9) knockout HEK293T cells, followed by infection with wild type IAV at multiplicity of infection (MOI) of 2. RIG-I pathway activation was assessed by ISG54 mRNA levels using RT-qPCR. The heat map represents mean values of experimental duplicates calculated as ISG54 fold induction over the value of the non-targeting siRNA control. b, cDNAs encoding confirmed negative regulators were ectopically expressed in ISRE-luciferase HEK293T cells followed by delNS1 IAV infection and assaying for luciferase activity (see ). Among those factors, the expression of 13 genes resulted in a repression of reporter activity at least by 50% compared to the activity of the RevGFP negative control. Results are the mean ± s.d. of four biological replicates. Data shown here is a representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t -test) c, cDNAs in b were ectopically co-expressed in ISRE-luciferase HEK293Ts with canonical RIG-I signaling members, RIG-I 2CARD, MAVS, and TBK1. The concentration of the selected cDNAs were fixed, while canonical RIG-I signaling component cDNAs were titrated over 5 dilutions. Reporter activities were used to devise a linear regression for each candidate regulator (see ), and the statistical comparison (PRISM) between each antagonist cDNA slope and the slope of RevGFP control is indicated as a P value, and was used to determine whether a cDNA expression blocked (blue), or did not impact (activity-yellow), ISRE-reporter activity induced by transfection of indicated pathway signaling members (P<0.05; see ). A schematic is shown for pathway mapping and localization of identified groups (right). Results are representative of two independent experiments and samples were run in biological quadruplicate.

Article Snippet: The following siRNAs were used: KHSRP_5 (Qiagen; SI00300587), KHSRP_3 (Qiagen; SI00054691), PIN_6 (Qiagen; SI02662667), DDX58_6 (Qiagen; SI03019646), and influenza A NP (5'-GGAUCUUAVUUCUUCGGAG-3') .

Techniques: CRISPR, Knock-Out, Infection, Activation Assay, Quantitative RT-PCR, Luciferase, Activity Assay, Expressing, Negative Control, Concentration Assay, Transfection

Journal: Nature microbiology

Article Title: Systems-based Analysis of RIG-I-dependent Signaling Identifies KHSRP as an Inhibitor of RIG-I Receptor Activation

doi: 10.1038/nmicrobiol.2017.22

Figure Lengend Snippet: a, HEK293T were transfected with the indicated siRNAs and subsequently infected with delNS1 IAV at MOI of 0.7, MOI of 1.4, or 40 hemagglutination units (HAU/mL) SeV, as indicated. ISG54 expression, analyzed by RT-qPCR (left), and knockdown efficiency of KHSRP, verified by western blot analysis (right), are shown. b, Primary normal human bronchial-tracheal epithelial cells (NHBE) were subjected to analogous analysis as in a . c, Wild type or CRISPR-mediated IRF9 knock out (cIRF9) HEK293T cells depleted of KHSRP were assessed for ISG54 induction relative to non-targeting control (nCTL; left) or IRF3 phosphorylation status (western; right) upon delNS1 infection. Error bars represent means ± s.d., biological triplicates. Data on ISG54 induction is a representative of three independent experiments. *** P < 0.001 (Student's t -test). Western analysis on the phospho-IRF3 status has been subjected to two independent experiments. d, HEK293T cells transfected with a cDNA encoding KHSRP or RevGFP control were infected with delNS1 IAV, and ISG54 mRNA levels were determined. The values indicated are relative to the value of RevGFP control (left). A dose range of KHSRP or negative control (LacZ) cDNAs (1.5ug, 0.9ug, 0.3ug) were transfected, followed by infection with delNS1 IAV at MOI 0.7 for 12 hours. Western blot analysis on the lysates was conducted with the indicated antibodies (right). e , wild type or CRISPR-mediated KHSRP knock out (cKHSRP) cells were either transfected with mock or Flag-tagged RIG-I cDNA, and subsequently infected with 40 HAU/mL SeV for 12 hours. ISG54 induction (left) and western blot analysis (right) are shown. Error bars represent means ± s.d., biological triplicates. Data is a representative of two independent experiments. *** P < 0.001 (Student's t -test).

Article Snippet: The following siRNAs were used: KHSRP_5 (Qiagen; SI00300587), KHSRP_3 (Qiagen; SI00054691), PIN_6 (Qiagen; SI02662667), DDX58_6 (Qiagen; SI03019646), and influenza A NP (5'-GGAUCUUAVUUCUUCGGAG-3') .

Techniques: Transfection, Infection, Expressing, Quantitative RT-PCR, Western Blot, CRISPR, Knock-Out, Negative Control

Journal: Nature microbiology

Article Title: Systems-based Analysis of RIG-I-dependent Signaling Identifies KHSRP as an Inhibitor of RIG-I Receptor Activation

doi: 10.1038/nmicrobiol.2017.22

Figure Lengend Snippet: a , Non-targeting control, or KHSRP siRNAs, were transfected into HEK293Ts and then stimulated by transfection of in vitro -transcribed SeVDI vRNA for 6 hours. ISG54 mRNA induction was accessed by RT-qPCR (left). HEK293Ts, pre-stimulated with 1000U/mL IFNβ for 16 hours to induce RIG-I protein levels, were challenged with SeV (40 HAU/mL) for 20 hours (top right panel), or transfected with SeVDI vRNA for 6 hours (bottom right panel), and subjected to immunoprecipitation of endogenous RIG-I. Immunoprecipitants were probed for KHSRP. Inputs represent 1% (top right) and 10% (bottom right) of binding reaction, respectively. b, V5-tagged KHSRP and HA-tagged RIG-I full-length (FL) or truncation mutants [helicase (H), regulatory domain (RD) and helicase plus RD (HRD)] were co-expressed in HEK293Ts for 36 hours followed by immunoprecipitation of HA-RIG-I and probed for V5-KHSRP association. Where indicated, HA-RIG-I (FL) and V5-KHSRP co-expressing cells were also subjected to SeVDI vRNA transfection for 5 hours prior to immunoprecipitation. Schematic of RIG-I domains are shown at the bottom. * ns denotes a non-specific band. c, HA-RIG-I (FL) and FLAG-KHSRP full-length (FL) or domain mutants (KH12, KH34, KH34C, KH1234 and CTD) were co-expressed and subjected to co-immunoprecipitation analysis, as indicated. Schematic of KHSRP domain mutants are shown at the bottom. Arrows on the blot depicts FLAG-KHSRP-CTD. d , cKHSRP cells, reconstituted with either KHSRP FL, truncation mutants (KH12 and KH1234), or RevGFP control, were infected with 0.7 MOI of delNS1 IAV (left) or 40 HAU SeV (right) for 24 hours. ISG54 mRNA expression relative to RevGFP reconstitution was determined. Error bars represent means ± s.d., biological triplicates. Data is a representative of three independent experiments. ** P <0.01, *** P < 0.001 (Student's t -test). The reconstituted cells were also analyzed for KHSRP (FL and truncation mutants) expression (bottom).

Article Snippet: The following siRNAs were used: KHSRP_5 (Qiagen; SI00300587), KHSRP_3 (Qiagen; SI00054691), PIN_6 (Qiagen; SI02662667), DDX58_6 (Qiagen; SI03019646), and influenza A NP (5'-GGAUCUUAVUUCUUCGGAG-3') .

Techniques: Transfection, In Vitro, Quantitative RT-PCR, Immunoprecipitation, Binding Assay, Expressing, Infection

Journal: Nature microbiology

Article Title: Systems-based Analysis of RIG-I-dependent Signaling Identifies KHSRP as an Inhibitor of RIG-I Receptor Activation

doi: 10.1038/nmicrobiol.2017.22

Figure Lengend Snippet: a, Whole cell lysates prepared from wild type or cKHSRP cells were subjected to a limited trypsin digestion. A commassie stain of mock treated and anti-RIG-I blot of both untreated and trypsin digested lysates are shown. b, Wild type or cKHSRP cells transfected with SeVDI vRNA for 5 hrs were subjected to immunoprecipitation against endogenous RIG-I, followed by blotting for Serine 8 phosphorylated RIG-I (left). These cell lines were also transfected with FLAG-RIG-I in the absence of SeVDI RNA and subjected to immunoprecipitation against FLAG (right). Where indicated, cells were treated with 100 nM calculinA for 45 mins before harvesting. c, cIRF9 cells transfected with KHSRP or non-targeting control (nCTL) siRNAs were infected by SeV (40 HAU/ml) for 18 hours and subjected to CLIP assay (see Methods for details). SeVDI vRNAs in both input and anti-RIG-I or normal IgG IPs were quantified by RT-qPCR. In the IPs, the relative abundance of SeVDI RNA was calculated from the value of associating SeVDI vRNAs, relative to the value of normal IgG in siKHSRP lysates. To calculate levels of vRNA in the input, SeVDI vRNA abundance was calculated relative to the input value of siKHSRP. KHSRP and RIG-I expression in the input, and the level of RIG-I in the IPs, are shown by western blot analysis (bottom). d , cKHSRP cells reconstituted with full length (FL), truncated mutants KH12 or KH1234 KHSRP, or RevGFP were treated with 1000U/mL IFNβ for 15 hours and subsequently transfected with 1ug SeVDI RNA for 5 hours. Samples were prepared and relative abundance was calculated as in c , but are represented relative to the SeVDI RNA value of RevGFP for IPs and input. For c–d , error bars represent means ± s.d. four technical replicates. Three independent experiments were conducted and a representative is shown here. *** P < 0.001 (Student's t -test). Reconstitution of FL KHSRP, KHSRP truncated mutants, and the RIG-I expression in the inputs and IPs were also analyzed by western (bottom).

Article Snippet: The following siRNAs were used: KHSRP_5 (Qiagen; SI00300587), KHSRP_3 (Qiagen; SI00054691), PIN_6 (Qiagen; SI02662667), DDX58_6 (Qiagen; SI03019646), and influenza A NP (5'-GGAUCUUAVUUCUUCGGAG-3') .

Techniques: Staining, Transfection, Immunoprecipitation, Infection, Quantitative RT-PCR, Expressing, Western Blot

Journal: Nature microbiology

Article Title: Systems-based Analysis of RIG-I-dependent Signaling Identifies KHSRP as an Inhibitor of RIG-I Receptor Activation

doi: 10.1038/nmicrobiol.2017.22

Figure Lengend Snippet: a, KHSRP, NP IAV, and nCTL siRNAs were transfected into HEK293Ts. After 36 hours, the cells were infected with wild type H1N1 PR/8/34 IAV (MOI of 0.1) for one hour. At the indicated time points, viral titer in the supernatants was measured using plaque assays. Titer values for each siRNA time point were normalized to the 0 hpi titer. Error bars represent means ± s.d. biological triplicate. A representative of six independent experiments is presented here (left). Wild type or cKHSRP HEK293Ts were infected with MHV-68 luciferase reporter virus (MOI 0.5) for the indicated time points and luciferase activity was measured to assess MHV replication (right). Error bars represent means ± s.d., biological triplicates. Data shown here is a representative of at least two independent experiments. ns indicates not significant, * P < 0.05, (Student's t -test). b , Wild type 5–6 week old C57BL/6 male mice were given 100ug KHSRP, non-targeting PPMOs, or PBS (5 mice per treatment) for two consecutive days. Subsequently they were infected with 500 pfu H1N1 PR/8/34 IAV intranasally. At day 6, lungs were harvested and homogenized for viral plaque assays to measure titers. Error bars represent means ± s.d., of five mice. Data shown here is a representative of two independent experiments. *** P < 0.001 (Student's t -test). KHSRP knockdown was also confirmed by western in the lung homogenates (right). c , Model for RIG-I regulation by KHSRP. In a basal state, KHSRP is bound to the RIG-I through the latter’s RD domain, and maintains an inactive closed conformation of RIG-I accompanied by high level of Serine 8 phosphorylation, which results in a dimished interaction with ligand. Upon introduction of activating levels of PAMPs, KHSRP dissociates with RIG-I, resulting in a conformational change of the receptor, induction of dephosphorylation, and activation of the signaling cascade.

Article Snippet: The following siRNAs were used: KHSRP_5 (Qiagen; SI00300587), KHSRP_3 (Qiagen; SI00054691), PIN_6 (Qiagen; SI02662667), DDX58_6 (Qiagen; SI03019646), and influenza A NP (5'-GGAUCUUAVUUCUUCGGAG-3') .

Techniques: Transfection, Infection, Luciferase, Activity Assay, Western Blot, De-Phosphorylation Assay, Activation Assay